ES2730173T3 - Adjustable Promoter - Google Patents

Abstract

A method of producing a protein of interest (PDI) by culturing a line of recombinant eukaryotic cells comprising an expression construct comprising an adjustable promoter and a nucleic acid molecule encoding a PDI under the transcriptional control of said promoter, comprising the steps of: a) culturing the cell line with a basal carbon source that represses the promoter, wherein the basal carbon source is a suitable carbon source for cell growth, b) cultivating the line of cells without a supplemental carbon source, or with a limited amount thereof, that depresses the promoter to induce the production of the PDI at a transcription rate of at least 20%, compared to the cell's native pGAP promoter, and c ) produce and retrieve the POI; wherein the promoter is selected from the group consisting of: (i) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3 ), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6); and (ii) a sequence comprising a nucleotide sequence of at least 200 bp and having at least 80% sequence identity with any one of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6); or with a part of any of the above with a length of at least 200 bp.

Description

DESCRIPTION

Adjustable Promoter

The invention relates to adjustable promoters and a method for producing a protein of interest in a eukaryotic cell culture under the control of an adjustable promoter.

Background

Successful production of recombinant proteins with eukaryotic hosts has been achieved. The most notable examples are yeasts, such as Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha, filamentous fungi, such as Aspergillus awamori or Trichoderma reesei, or mammalian cells, such as, for example, CHO cells. Although the production at high rates of some proteins is easily achieved, many other proteins are obtained only at comparatively low levels.

Heterologous expression of a gene in a host organism usually requires a vector that allows stable transformation of the host organism. A vector would provide the gene with a functional promoter adjacent to the 5 'end of the coding sequence. With this, transcription is regulated and initiated by this promoter sequence. Most of the promoters used to date have been derived from genes encoding proteins that are usually present at high concentrations in the cell.

EP0103409A2 describes the use of yeast promoters associated with the expression of specific enzymes in the glycolytic pathway, specifically promoters involved in the expression of pyruvate kinase, triosaphosphate isomerase, phosphoglucose isomerase, phosphoglycerate mutase, hexokinase 1 and 2, glucokinase, phosphofructinase , aldolase and the glycolytic regulation gene.

WO 97/44470 describes yeast promoters from Yarrowia lipolytica for translation elongation factor 1 protein (TEF1) and for S7 ribosomal protein, which are suitable for heterologous expression of proteins in yeasts, and EP1951877A1 describes the use of the P. pastoris TEF1 promoter for the production of heterologous proteins.

WO2005003310 provides methods for the expression of a coding sequence of interest in yeasts using a promoter of glyceraldehyde-3-phosphate dehydrogenase or phosphoglycerate mutase from oil yeast Yarrowia lipolytica.

Promoter sequences derived from genes involved in the methanol metabolic pathway of Pichia pastoris are described in US4808537 and US4855231 (alcohol oxidase AOX1, AOX2) and US6730499B1 (formaldehyde dehydrogenase FLD1). US20080153126A1 includes mutant promoter sequences based on the AOX1 promoter.

The AOX1 promoter is induced only in response to methanol and is repressed by other carbon sources, such as glucose or ethanol. Methanol has the disadvantage that it is not suitable for use in the production of certain products, since it is potentially dangerous due to its toxicity and flammability. Therefore, alternatives to the AOX1 promoter are sought.

US2008299616A1 describes the regulatory sequences of the malate synthase (MLS1) gene for the expression of heterologous genes in P. pastoris, which is repressed in glucose-containing media and is repressed under glucose deprivation conditions or when acetate is present. However, this system is not considered suitable for effective production methods, since the MLS1 promoter is weak and has little activity under conditions of derepression.

Scholer and Schüller (Mol. Cell. Biol., 199414 (6): 3613-3622) describe the control region of the ICL1 isocitrate lyase gene, which is derepressed after cell transfer from fermentative to non-growth conditions. fermentative

WO2008063302A2 describes the use of new inducible promoters derived from the genes of ADH1 (alcohol dehydrogenase), ENO1 (enolasa) and GUT1 of P. pastoris for the expression of heterologous proteins, document CN1966688A describes the sequence of the fatty acid promoter P. pastoris omega 3 dehydrogenase, and WO002007117062A1 describes the self-inducible NPS promoter derived from P. pastoris, which is induced by phosphorus limitation.

WO2008128701A2 describes the use of new promoters, of which the promoter derived from the THI3 gene (thiamine metabolism) of P. pastoris is repressed in a medium containing thiamine, and is suppressed after the loss of thiamine.

US2009325241A1 describes a method of producing ethanol in a yeast cell that employs an xylose inducible promoter (FAS2 promoter).

It is desirable to provide improved recombinant eukaryotic cell lines to produce products of fermentation that can be isolated with high yields. Therefore, an object of the present invention is to provide alternative regulatory elements suitable for recombinant production methods, which are simple and effective.

Summary of the invention

The object is solved by means of the claimed aspects.

The present invention provides a method of producing a protein of interest (PDI) by culturing a line of recombinant eukaryotic cells comprising an expression construct comprising an adjustable promoter and a nucleic acid molecule encoding a PDI under transcriptional control. of said promoter, comprising the steps of:

a) culturing the cell line with a basal carbon source that represses the promoter, in which the basal carbon source is a suitable carbon source for cell growth,

b) culturing the cell line without a supplemental carbon source, or with a limited amount of it, which depresses the promoter to induce the production of the PDI at a transcription rate of at least 20%, compared to the native pGAP promoter of the cell, and

c) produce and retrieve the POI,

in which the promoter is selected from the group consisting of:

(i) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6); and

(ii) a sequence comprising a nucleotide sequence of at least 200 bp and having at least 80% sequence identity with any one of pG1 (SEQ ID NO: 1), pG3 (SEQ ID No: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 ( ^{s Eq} ID NO: 6); or with a part of any of the above with a length of at least 200 bp.

Specifically, the basal carbon source is selected from the group consisting of glucose, glycerol, ethanol, one of its mixtures and a complex nutrient material.

Specifically, the supplemental carbon source is a hexose, such as glucose, fructose, galactose or mannose, a disaccharide, such as sucrose, an alcohol, such as glycerol or ethanol, or one of its mixtures.

Specifically step b) employs a feeding medium that does not provide the supplemental carbon source or provides it in a limited amount, preferably at 0-1 g / l in the culture medium.

Specifically, the limited amount of the supplemental carbon source limits growth to maintain the specific growth rate within the range of 0.02 h-1 to 0.2 h-1, preferably 0.02 h-1 to 0, 15 h-1.

Specifically, the promoter is capable of controlling the transcription of a wild-type eukaryotic cell gene, and said gene is selected from the group consisting of G1 (SEQ ID NO: 7), G3 (SEQ ID NO: 8), G4 (SEQ ID NO: 9), G6 (SEQ ID NO: 10), G7 (SEQ ID NO: 11) and G8 (SEQ ID NO: 12), or one of its functionally active variants comprising a nucleotide sequence of at at least 200 bp and a sequence identity of at least 80% with any of the promoter sequences or with one of its parts with a length of at least 200 bp. Specifically, the promoter is pG1 (SEQ ID NO: 1) or one of its functionally active variants.

Specifically, said functionally active variants are selected from the group consisting of homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or in any or both of the distal ends of the sequence, and analogues derived from species other than Pichia pastoris.

Specifically, the promoter is selected from the group consisting of pG1 (SEQ ID NO: 1), pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46).

Specifically, the cell line is selected from the group consisting of mammalian, insect, yeast, filamentous and plant cell lines, preferably a yeast.

Specifically, PDI is a heterologous protein, preferably selected from therapeutic proteins, including antibodies or their fragments, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and proteins or particles similar to vaccines, process enzymes, growth factors, hormones and cytokines, or a metabolite of a POI.

Specifically, the promoter is a functionally active variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO : 5), or pG8 (SEQ ID NO: 6), which is selected from the group consisting of homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, and analogues derived from species other than Pichia pastoris. Specifically, the promoter is selected from the group consisting of pG1 (SEQ ID NO: 1), pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46).

The invention further provides an isolated promoter nucleic acid that is operably linked to a nucleotide sequence encoding a PDI, and said promoter nucleic acid is not natively associated with the nucleotide sequence encoding the PDI, in which the nucleic acid Promoter comprises a nucleic acid sequence selected from the group consisting of:

a) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 ( SEQ ID NO: 6); and

(b) a sequence comprising a nucleotide sequence of at least 200 bp and having at least 80% sequence identity with any one of pG1 (SEQ ID NO: 1), pG3 (SEQ ID No: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (s Eq ID NO: 6); or with a part of any of the above with a length of at least 200 bp;

wherein said nucleic acid comprises a functionally active promoter, which is a promoter adjustable by a carbon source capable of expressing a PDI in a recombinant eukaryotic cell at a transcription rate of at least 20%, compared to the native pGAP promoter of the cell.

Specifically, the promoter is a functionally active variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO : 5), or pG8 (SEQ ID NO: 6), which is selected from the group consisting of homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, and analogues derived from species other than Pichia pastoris. Specifically, the promoter is selected from the group consisting of pG1 (SEQ ID NO: 1), pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46).

The invention further provides a vector comprising the promoter nucleic acid described herein, wherein the nucleotide sequence encoding a PDI is under the transcriptional control of said promoter, and said promoter nucleic acid is not natively associated with the nucleotide sequence encoding the PDI. The invention further provides a recombinant eukaryotic cell comprising the vector described herein.

The invention further provides a method of producing a protein of interest (PDI) using the eukaryotic cell described herein.

A method of producing a protein of interest (PDI) is provided herein by culturing a line of recombinant eukaryotic cells comprising an expression construct comprising an adjustable promoter and a nucleic acid molecule encoding a PDI under control. transcriptional of said promoter, comprising the steps of:

a) cultivate the cell line with a basal carbon source that represses the promoter,

b) culturing the cell line without a supplemental carbon source, or with a limited amount of it, which depresses the promoter to induce the production of the PDI at a transcription rate of at least 20%, compared to the native pGAP promoter of the cell, and

c) produce and recover the POI.

Said culture steps specifically comprise culturing the cell line in the presence of said carbon sources, therefore in a culture medium comprising said carbon sources, or in step b) also in the absence of a supplementary carbon source.

Said induction of the production of PDI refers specifically to the induction of transcription, which specifically includes a subsequent translation and an optional expression of said PDI.

Said transcription rate refers specifically to the amount of transcripts obtained after fully inducing said promoter. Said promoter is considered depressed and fully induced if the culture conditions provide approximately maximum induction, for example, at glucose concentrations less than 0.4 g / l, preferably less than 0.04 g / l, specifically lower than 0.02 g / l. The fully induced promoter preferably shows a transcription rate of at least 15%, preferably at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100 % or an even higher transcription rate of at least 150% or at least 200%, compared to the native pGAP promoter. The transcription rate can be determined, for example, by the amount of transcripts of an indicator gene, such as eGFP, as described in the examples section below, which shows a relatively high transcription rate of the pG1 promoter of at least 50 %, compared to the cell's native pGAP promoter, after culturing a clone in solution. Alternatively, the transcription rate can be determined by means of the transcription power in a microarray, in which the microarray data shows the difference in the level of expression between the repressed and depressed state and a high intensity signal in the fully induced state, compared to the native pGAP promoter. These microarray data specifically show a transcription rate greater than 200% for pG1, greater than 30% for pG3 and pG4, greater than 60% for pG6, greater than 30% for pG7, greater than 20% for pG8, and each value is compared with the native pGAP. It was found that the prior art MLS1 or ICL1 promoters were too weak and therefore not suitable for the purpose described herein.

Said native pGAP promoter is specifically active in said recombinant eukaryotic cell in a manner similar to that in a native eukaryotic cell of the same species or strain, which includes the unmodified (non-recombinant) or recombinant eukaryotic cell. It is commonly understood that said native pGAP promoter is an endogenous promoter, therefore homologous to the eukaryotic cell, and acts as a standard or reference promoter for comparative purposes.

For example, a P. pastoris native pGAP promoter is the unmodified endogenous promoter sequence of P. pastoris, as used to control GAPDH expression in P. pastoris, for example having the sequence shown in Figure 13 , which is the sequence of the P. pastoris native pGAP promoter (GS115) (SEQ ID NO: 13). If P. pastoris is used as a host to produce a PDI described herein, the transcription rate or potency of the promoter described herein is compared with said native P. pastoris pGAP promoter. As another example, a S. cerevisiae native pGAP promoter is the unmodified endogenous promoter sequence of S. cerevisiae, as used to control GAPDH expression in S. cerevisiae. If S. cerevisiae is used as a host to produce a PDI described herein, the transcription rate or potency of the promoter described herein is compared with said native S. cerevisiae pGAP promoter.

Therefore, the relative transcription rate or potency of a promoter provided herein is usually compared to the native pGAP promoter of a cell of the same species or strain that is used as a host to produce a PDI.

According to a specific embodiment, the basal carbon source is different from the supplemental carbon source, for example, it is quantitatively and / or qualitatively different. The quantitative difference may provide different conditions to repress or suppress promoter activity.

According to another specific embodiment, the sources of basal and supplementary carbon comprise the same type of molecules or carbohydrates, preferably in different concentrations. According to another specific embodiment, the carbon source is a mixture of two or more different carbon sources.

Any suitable organic carbon used for the cultivation of eukaryotic cells can be used. According to a specific embodiment, the carbon source is a hexose, such as glucose, fructose, galactose or mannose, a disaccharide, such as sucrose, an alcohol, such as glycerol or ethanol, or a mixture thereof.

According to a specifically preferred embodiment, the basal carbon source is selected from the group consisting of glucose, glycerol, ethanol, one of its mixtures and a complex nutrient material. According to a preferred embodiment, the basal carbon source is glycerol.

According to another specific embodiment, the supplemental carbon source is a hexose, such as glucose, fructose, galactose and mannose, a disaccharide, such as sucrose, an alcohol, such as glycerol or ethanol, or one of its mixtures. According to a preferred embodiment, the source of supplemental carbon is glucose.

Specifically, the method may employ glycerol as the source of basal carbon, and glucose as the source of supplemental carbon.

The derepression conditions can be adequately achieved by specific means. Step b) optionally employs a feeding medium that does not provide the supplemental carbon source or provides it in a limited amount.

Specifically, the feeding medium is chemically defined and does not contain methanol.

The feeding medium can be added to the culture medium in liquid form or in another alternative form, such as a solid, for example, as a tablet or other sustained release medium, or a gas, for example, carbon dioxide. According to a preferred embodiment, the limited amount of the supplemental carbon source added to the cell culture medium may even be zero. Preferably, the concentration of the supplementary carbon source in the culture medium is 0-1 g / l, preferably less than 0.6 g / l, more preferably less than 0.3 g / l, more preferably less than 0, 1 g / l, preferably 1-50 mg / l, more preferably 1-10 mg / l, and 1 mg / l or less is specifically preferred, such as below the detection limit, as measured by a suitable conventional assay , for example determined as the residual concentration in the culture medium after consumption by growing the growing cells.

In a preferred method, the limited amount of the supplementary source provides a residual amount in the cell culture that is below the detection limit, as determined in the fermentation broth at the end of a production phase or at the exit of a fermentation process, preferably after collecting the fermentation product.

Preferably, the limited amount of the supplemental carbon source limits growth to maintain the specific growth rate within the range of 0.02 h-1 to 0.2 h-1 preferably from 0.02 h-1 to 0.15 h-1

According to a specific embodiment, the promoter is a Pichia pastoris promoter or one of its functionally active variants.

Hereby, the promoter always refers to the sequences described herein and its functionally active variants. As explained in detail below, said variants include homologues and analogues derived from species other than Pichia pastoris.

The method provided herein may employ a promoter that is a wild-type P. pastoris promoter or one of its functionally active variants, for example, capable of controlling the transcription of a specific gene in a recombinant or wild-type eukaryotic cell. , for example, a wild type promoter for selected genes, and said gene is selected from the group consisting of G1 (SEQ ID NO: 7), such as a gene encoding a glucose transporter (high affinity), G3 ( SEQ ID NO: 8), G4 (SEQ ID NO: 9), such as a gene encoding a mitochondrial aldehyde dehydrogenase, G6 (SEQ ID NO: 10), G7 (SEQ ID NO: 11), such as a gene that encodes a member of the family of major sugar transporter facilitators, or G8 (SEQ ID NO: 12), such as a gene encoding a member of the Gti1_Pac2 superfamily, or one of its functionally active variants.

Specifically, a promoter, or one of its functionally active variants, is provided that would be natively associated with one of said genes in a wild-type yeast cell.

According to a specific embodiment, the cell line is selected from the group consisting of mammalian, insect, yeast, filamentous and plant cell lines, preferably a yeast.

Specifically, the yeast is selected from the group consisting of Pichia, Candida, Torulopsis, Arxula, Hensenula, Yarrowia, Kluyveromyces, Saccharomyces, Komagataella, preferably a methylotrophic yeast. A specifically preferred yeast is Pichia pastoris, Komagataella pastoris, K. phaffii, or K. pseudopastoris. According to another specific embodiment, the promoter is not natively associated with the nucleotide sequence encoding the PDI.

Specifically, the PDI is a eukaryotic protein, preferably a mammalian protein.

A PDI produced according to the method provided herein may be a multimeric protein, preferably a dimer or a tetramer.

According to a specific embodiment, the PDI is a recombinant or heterologous protein, preferably selected from therapeutic proteins, including antibodies or their fragments, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and proteins or particles similar to vaccines, process enzymes, growth factors, hormones and cytokines, or a metabolite of a PDI.

A specific PDI is an antigen binding molecule, such as an antibody, or one of its fragments. Specific PDIs include antibodies, such as monoclonal antibodies (mAb), immunoglobulins (Ig) or class G immunoglobulins (IgG), heavy chain antibodies (HcAb), or their fragments, such as the antigen-binding fragment ( Fab), Fd, single stranded variable fragments (mcFv), or their modified variants, such as, for example, Fv dimers (diabodies), Fv trimers (triabodies), Fv tetramers, or single domain antibodies and antibodies, such as VH or VHH or V-NAR.

According to a specific embodiment, a fermentation product is manufactured using the PDI, a metabolite or a derived from this.

According to a specific embodiment, a method is provided to control the expression of a PDI in a recombinant eukaryotic cell under the transcriptional control of a promoter adjustable by a carbon source having a transcription potency of at least 20%, compared to the promoter. pGAP native to the cell, in which expression is induced under conditions that limit the carbon source. The promoter adjustable by a carbon source preferably has a transcription potency of at least 20%, compared to the native pGAP promoter, and specifically has a transcription potency as described above with respect to the transcription rate, compared with the native pGAP promoter. Thus, the fully induced promoter preferably has a transcription potency of at least 20%, preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100% or a potency. of transcription even greater than at least 150% or at least 200%, compared to the native pGAP promoter, as determined in the eukaryotic cell selected to produce the PDI.

In a preferred embodiment, said promoter employed has a transcriptional activity or a transcriptional potency in the derepressed state that is at least 2 times, more preferably at least 5 times, even more preferably at least 10 times, more preferably at least 20 times, more preferably at least 30, 40, 50, or 100 times greater in the suppressed state, compared to the repressed state.

According to a specific embodiment, a method is provided for producing a PDI in a recombinant eukaryotic cell under the transcriptional control of a promoter adjustable by a carbon source, wherein said promoter has a transcription potency as described above, that is, , at least 20%, compared to the cell's native pGAP promoter. The promoter adjustable by a carbon source preferably has a transcription potency of at least 20%, compared to the reference pGAP promoter, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100% or an even greater transcription potency of at least 150% or at least 200%, compared to the native pGAP promoter. In a preferred embodiment, said promoter employed has a transcriptional activity in the derepressed state that is at least 2 times, more preferably at least 5 times, even more preferably at least 10 times, more preferably at least 20 times, more preferably at least 30 , 40, 50, or 100 times greater in the suppressed state, compared to the repressed state. Suitably, a specific promoter, as described herein, is employed in said method.

In a specifically preferred embodiment, the promoter is an adjustable promoter comprising a nucleic acid sequence selected from the group consisting of:

a) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 ( SEQ ID NO: 6);

b) a sequence that has at least 80% homology with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6);

c) a sequence that hybridizes under stringent conditions with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6); and

d) a fragment or variant derived from a), b) or c),

wherein said promoter is a functionally active promoter, which is a promoter adjustable by a carbon source capable of expressing a PDI in a recombinant eukaryotic cell at a transcription rate of at least 20%, compared to the native pGAP promoter of the cell.

Specifically, the variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6) is a functionally active variant selected from the group consisting of homologues with at least about 80% nucleotide sequence identity, homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, preferably with a nucleotide sequence of at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp, and analogues derived from species other than Pichia pastoris.

Some of the preferred functionally active variants of the promoter described herein are fragments of any of the nucleotide sequences of the promoters pG1, pG3, pG6, pG7 or pG8, preferably fragments that include the 3 'end of a promoter nucleotide sequence , for example, a nucleotide sequence derived from one of the promoter nucleotide sequences having a specific length and a deletion of the 5 'terminal region, for example, a cleavage of the nucleotide sequence at the 5' end, for obtaining a specific length with an amplitude from the 3 'end to a variable 5' end, such as with a nucleotide sequence length of at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at at least 800 bp, at least 900 bp, or at least 1000 bp.

Examples of variants that have proven to be functionally active comprise or consist of said fragments, for example, fragments with a specific length with an amplitude of 200 to 1000 bp, preferably with an amplitude of 250 to 1000 bp, more preferably with an amplitude of 300 to 1000 bp, for example, which include the 3 'terminal sequence. For example, a functionally active variant of pG1 is selected from the group consisting of pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44 ), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46), therefore, a nucleotide sequence with an amplitude of 300-1000 bp, which includes the 3 'terminal sequence to nucleotide 1001.

According to another specific embodiment, an isolated nucleic acid is provided comprising a nucleic acid sequence selected from the group consisting of:

a) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 ( SEQ ID NO: 6),

b) a sequence that has at least 80% homology with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6),

c) a sequence that hybridizes under stringent conditions with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6), and

d) a fragment or variant derived from a), b) or c),

wherein said nucleic acid comprises a functionally active promoter, which is a promoter adjustable by a carbon source capable of expressing a PDI in a recombinant eukaryotic cell at a transcription rate of at least 20%, compared to the native pGAP promoter of the cell.

Specifically, the variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6) is a functionally active variant selected from the group consisting of homologues with at least about 80% nucleotide sequence identity, homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, preferably with a nucleotide sequence of at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp, and analogues derived from species other than Pichia pastoris.

Some of the preferred functionally active variants of the promoter described herein are fragments of any of the nucleotide sequences of the promoters pG1, pG3, pG4, pG6, pG7 or pG8, preferably fragments that include the 3 'end of a nucleotide sequence promoter, for example, a nucleotide sequence derived from one of the promoter nucleotide sequences having a specific length and a deletion of the 5 'terminal region, for example, a cleavage of the nucleotide sequence at the 5' end , to obtain a specific length with an amplitude from the 3 'end to a variable 5' end, such as with a nucleotide sequence length of at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp.

Examples of variants that have proven to be functionally active comprise or consist of said fragments, for example, fragments with a specific length with an amplitude of 200 to 1000 bp, preferably with an amplitude of 250 to 1000 bp, more preferably with an amplitude of 300 to 1000 bp, for example, which include the 3 'terminal sequence. For example, a functionally active variant of pG1 is selected from the group consisting of pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44 ), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46), therefore, a nucleotide sequence with an amplitude of 300-1000 bp, which includes the 3 'terminal sequence to nucleotide 1001.

The promoter adjustable by a carbon source preferably has a transcription potency as described above, preferably at least 20%, compared to the reference pGAP promoter, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least 100% or an even greater transcription potency of at least 150% or at least 200%, compared to the native pGAP promoter. In a preferred embodiment, said promoter employed has a transcriptional activity in the derepressed state that is at least 2 times, more preferably at least 5 times, even more preferably at least 10 times, more preferably at least 20 times, more preferably at least 30 , 40, 50, or 100 times greater in the suppressed state, compared to the repressed state. Suitably, a specific promoter, as described herein, is employed in said method.

In addition, according to another specific embodiment, an expression construct is provided comprising a promoter described herein, operably linked to a nucleotide sequence encoding a PDI under the transcriptional control of said promoter, and said promoter is not natively associated with the PDI coding sequence.

Another specific embodiment relates to a vector comprising the construction described herein.

Another specific embodiment relates to a recombinant eukaryotic cell comprising the construction or vector described herein.

Specifically, the cell is selected from the group consisting of mammalian, insect, yeast, filamentous and plant cell lines, preferably a yeast.

The yeast can be suitably selected from the group consisting of Pichia, Candida, Torulopsis, Arxula, Hensenula, Yarrowia, Kluyveromyces, Saccharomyces, Komagataella, preferably a methylotrophic yeast. Preferably, the yeast is Pichia pastoris, Komagataella pastoris, K. phaffii, or K. pseudopastoris.

According to a specific embodiment, a cell having a higher specific growth rate is used in the presence of an excess of carbon source, relative to limited carbon source conditions.

Another specific embodiment relates to the use of the recombinant eukaryotic cell described herein for the production of the PDI.

According to another specific embodiment, a method is provided for selecting or identifying a promoter adjustable by a carbon source from eukaryotic cells, comprising the steps of:

a) culturing eukaryotic cells in the presence of a carbon source in a discontinuous culture under cell growth conditions,

b) then culturing the cells in a batch feed culture in the presence of a limited amount of a supplemental carbon source,

c) provide samples of the cell culture of stage a) and b), and

d) perform a transcription analysis on said samples to identify an adjustable promoter that shows a higher transcriptional potency in the cells of stage b) than in the cells of stage a).

Said higher transcriptional potency can be determined by the transcription potency in the fully induced state, which is obtained, for example, under conditions of chemostat cultures with glucose limitation, which is at least 2 times, more preferably at least 5 times, even more preferably at least 10 times, more preferably at least 20 times, more preferably at least 30, 40, 50, or 100 times greater in the depressed state, compared to the repressed state.

Preferably, the transcription analysis is quantitative or semi-quantitative, and preferably employs DNA microarrays, RNA sequencing and transcriptome analysis.

Figures

Figure 1: Sequence of the pG1 promoter (SEQ ID NO: 1) of P. pastoris.

Figure 2: Sequence of the pG3 promoter (SEQ ID NO: 2) of P. pastoris.

Figure 3: Sequence of the pG4 promoter (SEQ ID NO: 4) of P. pastoris.

Figure 4: Sequence of the pG6 promoter (SEQ ID NO: 3) of P. pastoris.

Figure 5: Sequence of the pG7 promoter (SEQ ID NO: 5) of P. pastoris.

Figure 6: Sequence of the pG8 promoter (SEQ ID NO: 6) of P. pastoris.

Figure 7: G1 gene coding sequences of the GS115 genome (SEQ ID NO: 7) of P. pastoris.

Figure 8: G3 gene coding sequences of the GS115 genome (SEQ ID NO: 8) of P. pastoris.

Figure 9: G4 gene coding sequences of the GS115 genome (SEQ ID NO: 9) of P. pastoris.

Figure 10: G6 gene coding sequences of the GS115 genome (SEQ ID NO: 10) of P. pastoris.

Figure 11: G7 gene coding sequences of the GS115 genome (SEQ ID NO: 11) of P. pastoris.

Figure 12: G8 gene coding sequences of the GS115 genome (SEQ ID NO: 12) of P. pastoris.

Figure 13: P. pastoris native pGAP promoter sequence (GS115) (SEQ ID NO: 13)

Figure 14: Desrepression properties of the promoter pG1 (circle), pG3 (triangle), pG4 (rhombus) and pG6 (square): the maximum transcription activity is reached for pG1 at approximately 0.04 g of glucose / less, while that the other pG promoters reach it at approximately 4 g / l or less. To compare the relative induction behavior of the different promoters, the data was normalized by dividing each value by the D20 value of the respective promoter construct. Therefore, the data are relative fluorescence values, and the data points in D20 are 1.0.

Figure 15: Functionally active variants of the pG1 promoter sequence; pG1a-f (SEQ ID NO: 41-46) of P. pastoris.

Detailed description of the invention

The specific terms and expressions used throughout the specification have the following meaning.

The term "carbon source," as used herein, means a fermentable carbon substrate, generally a carbohydrate, suitable as an energy source for microorganisms, such as those that are capable of being metabolized by host organisms or lines. Production cells, in particular sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, alcohols, including glycerol, in purified form or provided as raw material, such as a complex nutrient material. The carbon source can be used as described herein as a single carbon source or as a mixture of different carbon sources.

A "basal carbon source," as used herein, is generally a carbon source suitable for cell growth, such as a nutrient for eukaryotic cells. The basal carbon source can be provided in a medium, such as a basal medium or a complex medium, but also in a chemically defined medium containing a source of purified carbon. The basal carbon source is generally provided in an amount to provide cell growth, in particular during the growth phase in a culture process, for example to obtain cell densities of at least 5 g / l of dry cell mass. , preferably at least 10 g / l of dry cell mass, or at least 15 g / l of dry cell mass, for example, showing a viability greater than 90% during conventional subculture stages, preferably greater than 95% .

As described herein, the basal carbon source is generally used in an excess amount, which is understood as an excess that provides energy to increase biomass, for example, during the growth phase of a cell line in a process of discontinuous feeding culture. This excess amount is, in particular, in excess of the limited amount of a supplemental carbon source to achieve a residual concentration in the fermentation broth that is measurable and generally at least 10 times higher, preferably at least 50 times or at least 100 times greater than during the supply of the limited amount of the supplemental carbon source.

The term "chemically defined" with respect to a cell culture medium, such as a feeding medium in a batch feeding process, means a suitable growth medium for in vitro cell culture of a production cell line, in which all chemical components and peptides are known. Generally, a chemically defined medium is completely free of animal derived components and represents a pure and constant cell culture environment.

A "supplementary carbon source", as used herein, is generally a supplementary substrate that facilitates the production of fermentation products by the production cell lines, in particular in the production phase of a culture process. The production phase specifically follows the growth phase, for example, in a process of continuous cultivation, of discontinuous and discontinuous feeding. The supplementary carbon source may specifically be contained in the feed of a batch feed process.

A "limited amount" of a carbon source or a "limited carbon source" is understood herein as the amount of a carbon source necessary to maintain a production cell line in a production phase or a mode of production. Said limited quantity can be used in a batch feeding process, in which the carbon source is contained in a feeding medium and is supplied to the crop at low feed rates for the sustained supply of energy to produce a ^p D ⁱ , at same time as maintains biomass at low growth rates. Generally, a feed medium is added to the fermentation broth during the production phase of a cell culture.

The limited amount of the supplemental carbon source can be determined, for example, by the residual amount of the supplemental carbon source in the cell culture broth, which is below a predetermined threshold or even below the detection limit. , as measured in a conventional test (carbohydrates). The residual amount is generally determined in the fermentation broth after collecting a fermentation product.

The limited amount of a supplementary carbon source can be determined by defining the average feed rate of the supplemental carbon source to the fermenter, for example, as determined by the amount added throughout the entire cultivation process, for example, the phase of discontinuous feed, by cultivation time, to determine an average amount calculated by time. This average feeding rate is kept low to ensure full use of the supplemental carbon source by cell culture, for example, between 0.6 g l-1 h-1 (g carbon source per l fermentation volume initial and h time) and 25 g l-1 h-1, preferably between 1.6 g l-1 h-1 and 20 g l-1 h-1.

The limited amount of a supplemental carbon source can also be determined by measuring the specific growth rate before and during the production process, in which the specific growth rate is kept low during the production process, for example, within a range. predetermined, such as in the range of 0.02 h-1 to 0.20 h-1, preferably between 0.02 h-1 and 0.15 h-1.

The term "cell line" or "cell line", as used herein, refers to an established clone of a particular type of cell that has acquired the ability to proliferate over a prolonged period of time. The term "host cell line" refers to a cell line as used to express an endogenous or recombinant gene or products of a metabolic pathway to produce polypeptides or metabolites of cells mediated by said polypeptides. A "production host cell line" or "production cell line" is commonly understood as a cell line ready for use in a bioreactor to obtain the product of a production process, such as a PDI. The term "eukaryotic host" or "eukaryotic cell line" means any organism or eukaryotic cell that can be cultured to produce a PDI or a metabolite of the host cell. It is understood that the expression does not include human beings.

The term "expression" or the terms "expression system" or "expression module" refers to nucleic acid molecules that contain a desired coding sequence and control sequences in an operable linkage, so that hosts transformed or transfected with These sequences are capable of producing the encoded proteins or metabolites of the host cell. To perform the transformation, the expression system can be included in a vector; however, relevant ^to D ⁿ can also be integrated into the chromosome of the host. The expression may refer to segregated or non-segregated expression products, which include polypeptides or metabolites.

The "expression constructs" or "vectors" used herein are defined as DNA sequences that are necessary for the transcription of cloned recombinant nucleotide sequences, that is, of recombinant genes, and for the translation of their mRNA in an organism. suitable host. Expression vectors usually comprise an origin for autonomous replication in host cells, selectable markers (for example, an amino acid synthesis gene or a gene that confers resistance to antibiotics, such as zeocin, kanamycin, G418 or hygromycin), a series of restriction enzyme cleavage sites, a suitable promoter sequence and a transcription terminator, and these components are operably linked to each other. The terms "plasmid" and "vector", as used herein, include autonomously replicating nucleotide sequences, as well as nucleotide sequences that integrate into the genome.

The term "variant", as used herein, refers to any sequence with a specific homology or analogy. The variant promoter can be derived, for example, from the promoter sequence pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6) by mutagenesis to produce sequences suitable for use as a promoter in recombinant cell lines. These variant promoters can be obtained from a bank of mutant sequences by selecting the members of the bank with predetermined properties. Variant promoters can have the same or even improved properties, for example, improved to induce the production of a PDI, with a greater differential effect under repression and derepression conditions. The variant promoter can also be derived from analogous sequences, for example, from eukaryotic species other than Pichia pastoris or from a genus other than Pichia, such as K. lactis, Z. rouxii, P. stipitis, H. polymorpha. Specifically, the sequences of analog promoters natively associated with genes analogous to the corresponding P. pastoris genes can be used as such, or as sequences of origin to produce their functionally active variants. Specifically:

- a promoter analogous to pG1 is characterized in that it is natively associated with a gene analogous to G1 (high affinity glucose transporter; description in P. pastoris GS115: putative transporter, member of the family of sugar carriers; coding sequence: SEQ ID NO: 7);

- a promoter analogous to pG3 is characterized in that it is natively associated with a gene analogous to G3 (coding sequence: SEQ ID NO: 8);

- a promoter analogous to pG4 is characterized in that it is natively associated with a gene analogous to G4 (P. pastoris GS115: predicted mitochondrial aldehyde dehydrogenase; coding sequence: SEQ ID NO: 9);

- a promoter analogous to pG6 is characterized in that it is natively associated with a gene analogous to G6 (coding sequence: SEQ ID NO: 10);

- a promoter analogous to pG7 is characterized in that it is natively associated with a gene analogous to G7 (P. pastoris GS115: member of the family of major facilitators of sugar transporters; coding sequence: SEQ ID NO: 11);

- a promoter analogous to pG8 is characterized in that it is natively associated with a gene analogous to G8 (P. pastoris GS115: member of the superfamily Gti1_Pac2; coding sequence: SEQ ID NO: 12).

The properties of said sequences of analogous promoters or their functionally active variants can be determined using conventional techniques.

The "functionally active" variant of a nucleotide or promoter sequence, as used herein, means a sequence that arises from the modification of a sequence of origin by insertion, deletion or substitution of one or more nucleotides. within the sequence or at either or both of the distal ends of the sequence, and such modification does not affect (in particular, hinders) the activity of this sequence.

Specifically, the functionally active variant of the promoter sequence described herein is selected from the group consisting of:

- homologs with at least about 80% nucleotide sequence identity,

- homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, preferably with a nucleotide sequence ( that is, comprising or consisting of it) of at least 200 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp , at least 900 bp, or at least 1000 bp, and - analogues derived from species other than Pichia pastoris.

Specifically preferred functionally active variants are those derived from a promoter described herein and / or fragments of the promoter sequence, with a nucleotide sequence (i.e., comprising or consisting of it) of at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 pb.

Some of the preferred functionally active variants of the promoter described herein are fragments of any of the nucleotide sequences of the promoters pG1, pG3, pG4, pG6, pG7 or pG8, preferably fragments that include the 3 'end of a nucleotide sequence promoter, for example, a nucleotide sequence derived from one of the promoter nucleotide sequences having a specific length and a deletion of the 5 'terminal region, for example, a cleavage of the nucleotide sequence at the 5' end , to obtain a specific length with an amplitude from the 3 'end to a variable 5' end, such as with a nucleotide sequence length of at least 200 bp, preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp.

Examples of variants that have proven to be functionally active comprise or consist of said fragments, for example, fragments with a specific length with an amplitude of 200 to 1000 bp, preferably with an amplitude of 250 to 1000 bp, more preferably with an amplitude of 300 to 1000 bp, for example, which include the 3 'terminal sequence. For example, a functionally active variant of pG1 is selected from the group consisting of pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44 ), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46), therefore, a nucleotide sequence with an amplitude of 300-1000 bp, which includes the 3 'terminal sequence to nucleotide 1001.

The term "adjustable" with respect to the promoter as used herein refers to a promoter that is repressed in a eukaryotic cell in the presence of an excess amount of a carbon source (nutrient substrate) in the growth phase. of a discontinuous culture, and is depressed to exert a potent promoter activity in the production phase of a production cell line, for example, after reducing the amount of carbon, such as after introducing a growth-limiting carbon source (nutrient substrate) to a crop according to the discontinuous feeding strategy. In this regard, the term "adjustable" is understood as "Adjustable by a carbon source limit" or "adjustable by a glucose limit", which refers to the release of a promoter by the consumption, reduction, lack or depletion of carbon, or by the limited addition of the carbon source so that it can be easily consumed by the cells.

The functionally active promoter described herein is a relatively potent, adjustable promoter that is silenced or repressed under cell growth conditions (growth phase) and that is activated or depressed under production conditions (production phase) and, therefore, is suitable for inducing the production of PDI in a production cell line by limiting the carbon source. Therefore, the functionally active variant of a promoter has at least said adjustable properties.

The potency of the adjustable promoter described herein refers to its transcription potency, represented by the efficiency of transcription initiation that occurs in this promoter with high or low frequency. The higher the transcription potency, the more frequently transcription will occur in that promoter. The potency of the promoter is important, because it determines how often a specific mRNA sequence is transcribed, and thus gives higher priority to the transcription of some genes over others, which leads to a higher concentration of the transcript. A gene that encodes a necessary protein in large quantities, for example, generally has a relatively potent promoter. RNA polymerase can only perform one transcription task at the same time and, therefore, they must prioritize their work to be effective. Differences in promoter potency are selected to allow this prioritization. Specifically, the adjustable promoter described herein is relatively potent in the fully induced state, which is generally understood as the approximately maximum activity state. The relative potency is usually determined with respect to a standard promoter, such as the respective pGAP promoter of the cell used as the host cell. The frequency of transcription is usually understood as the transcription rate, for example, determined by the amount of a transcript in a suitable assay, for example, RT-PCR or Northern blotting. For example, the transcription potency of a promoter described herein is determined in the host cell that is P. pastoris and compared to the native pGAP promoter of P. pastoris.

The pGAP promoter initiates the expression of the gap gene that encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a constitutive promoter present in any microorganism capable of growing with glucose. GAPDH (EC 1 \ 2 \ 1 \ 12), a key glycolysis enzyme, plays a crucial role in catabolic and anabolic carbohydrate metabolism.

The adjustable promoter described herein exerts a relatively high transcription potency, reflected by a transcription rate or a transcription potency of at least 20%, compared to the native pGAP promoter in the host cell, and sometimes referred to as "homologous pGAP promoter. " Preferably, the transcription rate or potency is at least 20%, in specifically preferred cases at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 100% or even greater, such as at least 150% or at least 200%, compared to the native pGAP promoter, for example, determined in the eukaryotic cell selected as the host cell to produce the PDI.

Specifically preferred is an adjustable promoter having, in the induced state, at least one transcription potency of one of the promoters pG1, pG3, PG4, pG6, pG7 or pG8. The comparative transcription potency employed by the pGAP promoter as a reference can be determined by conventional means, such as measuring the amount of transcripts, for example, using a microarray, or also in a cell culture, such as measuring the amount of the respective products of gene expression in recombinant cells. An example test is illustrated in the examples section.

Specifically, the promoter described herein is regulable by a carbon source, with a differential promoter potency determined in an assay that compares its potency in the presence of glucose and with glucose limitation, showing that it is still repressed at relatively glucose concentrations. high, preferably at concentrations of at least 10 g / l, preferably at least 20 g / l. Specifically, the promoter described herein is fully induced at limited glucose concentrations and glucose threshold concentrations that completely induce the promoter, whose threshold is less than 20 g / l, preferably less than 10 g / l, less than 1 g / l, even less than 0.1 g / l or less than 50 mg / l, preferably with a full transcription potency, for example, of at least 50% of the native homologous pGAP promoter, at glucose concentrations less than 40 mg / l .

Preferably, the power of the differential promoter is determined by the start of PDI production after switching to inductive conditions below a predetermined carbon source threshold, and compared with the power in the repressed state. Usually, it is understood that the transcription potency is the potency in the fully induced state, that is, it shows approximately maximum activities under conditions of derepression. The potency of the differential promoter is determined, for example, according to the efficacy or performance of the production of PDI in a recombinant host cell line under derepression conditions, compared to repression conditions, or also by the amount of a transcribed The adjustable promoter described herein has a preferred differential promoter potency that is at least 2 times, more preferably at least 5 times, even more preferably at least 10 times, more preferably at least 20 times, more preferably at least 30, 40 , 50, or 100 times higher in the derepressed state, compared to the repressed state, which is also understood as induction in number of times. Said difference promoter potency can be determined by an assay, as illustrated by the attached examples.

A prior art promoter (MLS1 promoter or ICL1 promoter) was found to have a significantly lower differential promoter potency than 2 times induction. This prior art promoter is not useful for industrial production of PDI, with a promoter potency of approximately 5% compared to the conventional pGAP promoter. This has been demonstrated in a direct comparison with the promoter described herein. The term "homology" indicates that two or more nucleotide sequences have the same base pairs or base pairs conserved in a corresponding position, to a certain degree, to a degree close to 100%. A homologous sequence generally has at least about 50% nucleotide sequence identity, preferably at least about 60% identity, more preferably at least about 70% identity, more preferably at least about 80% identity, more preferably at least about 90% identity, more preferably at least about 95% identity.

The homologous promoter sequence provided herein preferably has some homology with any of the nucleotide sequences of the pG1, pG3, pG4, pG6, pG7 or pG8 promoters of P. pastoris in at least specific parts of the nucleotide sequence, such such as that which includes the 3 'region of the respective promoter nucleotide sequence, preferably a part with a specific length to the 3' end of the respective promoter nucleotide sequence, such as a part with a length of at least 200 bp , preferably at least 250 bp, preferably at least 300 bp, more preferably at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp , and analogues derived from species other than Pichia pastoris. Specifically, at least these parts are preferably homologous within a range of 300-1000 bp, including the 3 'terminal sequence of the respective promoter nucleotide sequence.

Generally, analogous sequences are derived from other species or strains. It is expressly understood that any of the sequences of analogous promoters provided herein that are derived from species other than Pichia pastoris may comprise a homologous sequence, that is, a sequence with a certain homology as described herein. Therefore, the term "homologue" may also include analogous sequences. On the other hand, it is understood that the sequences described herein also refer to analogous sequences and their counterparts comprising a certain homology.

"Percent (%) identity" with respect to the nucleotide sequence of a gene is defined as the percentage of nucleotides in a sequence ^to D ⁿ candidate that are identical to nucleotides in the DNA sequence after aligning the sequences and introduce gaps, if necessary, to achieve the maximum percentage of sequence identity and without considering conservative substitutions as part of the sequence identity. Alignment with the objective of determining the percentage of nucleotide sequence identity can be achieved in several ways that are known in the art, for example, using a publicly accessible computer program. Those skilled in the art can determine the appropriate parameters for measuring alignment, which include any algorithm necessary to achieve maximum alignment along the full length of the sequences being compared.

The term "mutagenesis", as used herein, refers to a method of providing mutants of a nucleotide sequence, for example, by insertion, deletion and / or substitution of one or more nucleotides, to obtain their variants with at least one change in the coding or non-coding region. Mutagenesis can occur through random, semi-random or site-directed mutation. Generally, large randomized gene banks with high gene diversity are produced, which can be selected according to a specifically desired genotype or phenotype.

The term "protein of interest (PDI)", as used herein, refers to a polypeptide or a protein that is produced by means of recombinant technology in a host cell. More specifically, the protein may be a polypeptide that does not appear in nature in the host cell, that is, a heterologous protein, or may also be native to the host cell, that is, a protein homologous to the host cell, but that it is produced, for example, by transformation with a self-replicating vector containing the nucleic acid sequence encoding the PDI, or after integration by recombinant techniques of one or more copies of the nucleic acid sequence encoding the PDI in the genome of the host cell, or by recombinant modification of one or more regulatory sequences that control the expression of the gene encoding the PDI, for example, of the promoter sequence. In some cases, the term "PDI", as used herein, also refers to any metabolite product of the host cell, which is mediated by recombinantly expressed protein.

The term "recombinant", as used herein, means "prepared by genetic engineering or arising from genetic engineering." Thus, a "recombinant microorganism" comprises at least one "recombinant nucleic acid." A recombinant microorganism specifically comprises an expression vector or a cloning vector, or has been genetically modified to contain a nucleic acid sequence. recombinant A "recombinant protein" is produced by expressing a respective recombinant nucleic acid in a host. A "recombinant promoter" is a genetically modified non-coding nucleotide sequence suitable for use as a functionally active promoter as described herein. Surprisingly, it has been determined that eukaryotic cells are capable of inducing the production of a PDI by limiting the availability of the carbon source. It has been found that carbon deprivation conditions activate the induction of a potent promoter activity that, to date, was not known in the art. The MLS1 promoter of Pichia pastoris, which in US2008299616A1 indicates that it is depressed under sugar limitations, is really a comparatively weak regulatable promoter for the production of a PDI. Therefore, it was surprising that said potent, P. pastoris promoter could be identified and used in eukaryotic production cell lines, in particular for the production of a recombinant PDI.

Although the 9.43 Mpb genomic sequence of P. pastoris strain GS115 has been determined and described in US20110021378A1, the properties of individual sequences, such as promoter sequences, have not been investigated in detail. For example, the sequence of pG4 (SEQ ID NO: 4), as described herein, was identified as a promoter sequence in US20110021378A1, although its adjustable properties and its use under carbon deprivation conditions were unknown. . It was also surprising that said promoter could be used effectively in the method provided herein. Regulated promoters of the prior art, such as those used on an industrial scale in the production of PDI, are derived primarily from the metabolic pathway of methanol and need the addition of methanol to induce the production of PDI, which often does not It is desirable. The method provided herein has the advantage that it can provide increased production by enhanced expression, and has a reduced risk of contamination due to specific promoter regulation, in particular when a chemically defined medium without methanol is employed.

It has been found that the adjustable promoter provided herein exerts its adjustable activity only after the use of a very specific culture medium suitable for establishing conditions of repression and derepression of the promoter. As an example, P. pastoris can be grown successfully under conditions of an industrial production process. First, a discontinuous culture is used on a basal carbon source, such as glycerol, followed by a discontinuous feed with a limited feed from a supplemental carbon source, such as glucose. Samples are taken near the end of the first discontinuous phase and under conditions of limited growth, for example, using a limited amount of the supplemental carbon source. The analysis of the transcriptome with DNA microarrays reveals certain specific genes that are very active on the supplemental carbon source and are weak or inactive in the presence of an excess of carbon, namely, the basal carbon source in an excess amount. At least six promoter sequences were identified as adjustable promoters as described herein, namely pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), and pG8 (SEQ ID NO: 6). The comparable MLS1 or ICL1 promoters of the prior art are weak, with less than 1/10 of the pG1 promoter potency, and no detectable regulation is appreciated.

The characteristics of repressing the expression of recombinant genes on the basal carbon source, and the potent expression on the limited supplementary carbon source, that is, induction by substrate change, can be verified in the fermentation processes.

Nucleotide sequences that can be used as regulatory sequences described herein, which provide improved production of a recombinant protein, can be obtained from a variety of sources. The origin of the promoter described herein is preferably a yeast cell, most preferably a methylotrophic yeast, such as of the genus Pichia or the species P. pastoris, whose promoter can then be used as a sequence of origin to produce suitable variants, by example, mutants or analogs.

It is contemplated that a series of yeast cells, in particular Pichia strains, may be suitable for obtaining the respective promoter sequences that are responsible for the production of proteins under carbon deprivation conditions, or the respective analogs in different species.

The identified P. pastoris promoter variants, which include functionally active variants, such as homologs and the like, can be produced using conventional techniques. The promoter can be modified, for example, to generate promoter variants with altered expression levels and altered regulatory properties.

For example, a promoter bank can be prepared by mutagenesis of the promoter sequences described herein, which can be used as origin molecules, for example, to fine-tune gene expression in eukaryotic cells by analyzing variants for expression. under different fermentation strategies and the selection of suitable variants. A synthetic bank of variants can be used, for example, to select a promoter that meets the requirements to produce a selected POI. These variants may have greater expression efficiency in eukaryotic host cells, and high expression after the decrease of a carbon source.

Differential fermentation strategies can distinguish between a growth phase, such as stage a), as described herein, and a production phase, such as stage b).

Growth and / or production can be carried out suitably in a discontinuous mode, a discontinuous feeding mode or a continuous mode. Any suitable bioreactor can be used, which includes a discontinuous, continuous, discontinuous, stirred tank or air lift reactor.

It is advantageous to provide the fermentation process at pilot or industrial scale. The industrial scale of the process preferably employs a volume of at least 10 l, specifically at least 50 l, preferably at least 1 m3, preferably at least 10 m3, most preferably at least 100 m3

Industrial scale production conditions that refer, for example, to a discontinuous feed culture in reactor volumes of 100 to 10 m3 or greater, which employ typical process times of several days, or continuous processes in volumes are preferred of the fermenter of approximately 50-1000 the greater, with dilution rates of approximately 0.02-0.15 h-1.

Suitable culture techniques may include cultivation in a bioreactor that begins with a discontinuous phase, followed by an exponential discontinuous feeding phase cut at a high specific growth rate, followed by a discontinuous feeding phase at a low specific growth rate. . Another suitable culture technique may include a discontinuous phase, followed by a continuous culture phase at a low dilution rate. A preferred embodiment includes a batch culture to provide a biomass, followed by a batch feed culture to achieve high yields of PDI production.

For example, the cell line can be cultured in step a) as described herein on glycerol or glucose to obtain a biomass.

According to the method provided herein, it is preferred to grow the host cell line in a bioreactor under growth conditions to obtain a cell density of at least 1 g / l of dry cell weight, more preferably at least 10 g / l of dry weight of cells, preferably at least 20 g / l dry weight of cells. It is advantageous to provide such biomass production yields on a pilot or industrial scale.

A growth medium that allows the accumulation of biomass, specifically a basal growth medium, generally comprises a carbon source, a nitrogen source, a sulfur source and a phosphate source. Generally, said medium further comprises trace elements and vitamins, and may further comprise amino acids, peptone or yeast extract.

Preferred nitrogen sources include NH ⁴ H ² PO ⁴ , or NH ³ or (NH ⁴ ) ² SO ⁴ .

Preferred sulfur sources include MgSO ⁴ , or (NH ⁴ ) ² SO ⁴ or K ² SO ⁴ .

Preferred phosphate sources include NH ⁴ H ² PO ⁴ , or H ³ PO ⁴ or NaH ² PO ⁴ , KH ² PO ⁴ , Na ² HPO ⁴ or K ² HPO ⁴ .

Other typical components of the medium include KCl, CaCh, and trace elements such as: Fe, Co, Cu, Ni, Zn, Mo, Mn, I, B.

Preferably, the medium is supplemented with vitamin B ⁷ .

A typical growth medium for P. pastoris comprises glycerol or glucose, NH ⁴ H ² PO ⁴ , MgSO ⁴ , KCl, CaCl ² , biotin, and trace elements.

A production medium with only a limited amount of a supplemental carbon source is specifically employed in the production phase.

Preferably, the host cell line is cultured in a mineral medium with a suitable carbon source, thereby further simplifying the isolation process. An example of a preferred mineral medium is a medium containing a usable carbon source (for example, glucose, glycerol or methanol), salts containing macroelements (potassium, magnesium, calcium, ammonium, chloride, sulfate, phosphate) and trace elements ( salts of copper, iodide, manganese, molybdate, cobalt, zinc and iron, and boric acid), and optionally vitamins or amino acids, for example, to complement auxotrophies.

The cells are cultured under suitable conditions to carry out the expression of the desired PDI, which can be purified from the cells or the culture medium, depending on the nature of the expression system and the expressed protein, for example, if the Protein is fused with a signal peptide and if the protein is soluble or is bound to a membrane. As those skilled in the art will understand, culture conditions vary according to factors that include the type of host cell and the specific expression vector employed.

The induction of PDI production by the promoter provided herein is preferably controlled by culturing the cells on a source of supplemental carbon in a limited amount as the sole source of carbon and energy. The cells grow very slowly under conditions of carbon limitation, but produce high yields of the PDI under the control of the adjustable promoter.

The difference in promoter activity specifically is at least 2 times, more preferably at least 5 times, even more preferably at least 10 times, more preferably at least 20 times, more preferably at least 30, 40, 50, or 100 times higher in the suppressed state, compared to the repressed state.

By selecting the appropriate promoter sequence, optionally in combination with other preferred regulatory sequences, it is possible to provide, under comparable conditions, at least the same activity or an increase in at least about 1.5, or at least about 2 times , or at least about 5 times, 10 times or at least up to about 15 in activity, as represented by promoter activity or transcription potency, or regulated by promoter potency relative to a GAP promoter which is homologous with the production cell, a native pGAP, or isolated from P. pastoris. A typical production medium comprises a supplemental carbon source and also NH ⁴ H ² PO ⁴ , MgSO ⁴ , KCl, CaCl ² , biotin, and trace elements.

For example, the supply of the additional carbon source added to the fermentation may comprise a carbon source with up to 50% by weight of fermentable sugars. The low feeding rate of the supplementary medium will limit the effects of the inhibition of the product on cell growth, and thus a high yield of the product will be possible based on the substrate supply.

The fermentation is preferably carried out at a pH ranging from 3 to 7.5.

Typical fermentation times are approximately 24 to 120 hours with temperatures in the range of 20 ° C to 35 ° C, preferably 22-30 ° C.

In general, the recombinant organisms or nucleic acids mentioned herein can be produced by recombination techniques well known to those skilled in the art. Accordingly, conventional recombinant molecular biology, microbiology and DNA techniques can be employed within the art. These techniques are fully explained in the bibliography. See, for example, Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual (1982).

According to a preferred embodiment, a recombinant construct is obtained by coupling the promoter and the relevant genes in a vector. These genes can be stably integrated into the genome of the host cell by transforming the host cell using these vectors.

Expression vectors may include, but are not limited to cloning vectors, modified cloning vectors and specifically designed plasmids. The preferred expression vector as used in the method provided herein may be any expression vector suitable for the expression of a recombinant gene in a host cell, and is selected depending on the host organism. The recombinant expression vector can be any vector that is capable of replicating or integrating into the genome of the host organism, also called the host vector.

According to a preferred embodiment, plasmids derived from pPUZZLE are used as the vector.

Appropriate expression vectors generally comprise other regulatory sequences suitable for expressing a DNA encoding a PDI in a eukaryotic host cell. Examples of regulatory sequences include operators, enhancers, ribosome binding sites and sequences that control the start and end of transcription and translation. The regulatory sequences may be operably linked to the DNA sequence to be expressed.

To allow the expression of a recombinant nucleotide sequence in a host cell, the expression vector may provide the promoter described herein adjacent to the 5 'end of the coding sequence, for example, upstream of a signal peptide gene. With this, transcription is regulated and initiated by this promoter sequence.

A signal peptide can be a heterologous signal peptide or a hybrid of a heterologous and native signal peptide, and specifically can be heterologous or homologous with the host organism that produces the protein. The function of the signal peptide is to allow the PDI to be secreted to enter the endoplasmic reticulum. It is usually a short peptide chain (with a length of 3-60 amino acids) that directs the transport of a protein outside the plasma membrane, thereby making the heterologous protein easy to separate and purify. Some signal peptides are cleaved from the protein by signal peptidases after the proteins are transported. Examples of signal peptides are signal sequences of the prepro peptide of the alpha mating factor of S. cerevisiae and the signal peptide of the acid phosphatase gene (PHO1) of P. pastoris.

It is understood that a promoter sequence is operably linked to a coding sequence if the promoter controls the transcription of the coding sequence. If a promoter sequence is not natively associated with the coding sequence, its transcription is not controlled by the promoter in native (wild-type) cells or the sequences are recombined with different contiguous sequences.

To demonstrate the function of the relevant sequences, expression vectors comprising one or more regulatory elements can be constructed to drive the expression of a POI, and the expressed performance is compared with constructions with conventional regulatory elements. A detailed description of the experimental procedure can be found in the following examples. The identified genes can be amplified by PCR from P. pastoris using specific nucleotide primers, cloned into an expression vector and transformed into a eukaryotic cell line, for example, using a yeast vector and a P. pastoris strain for High-level production of several different IDPs. To calculate the effect of the promoter provided herein on the amount of recombinant PDI produced in this manner, the eukaryotic cell line can be cultured in shake flask experiments and batch-fed fermentations or in chemostat, compared to strains comprising a conventional promoter not regulable by a carbon source, such as, for example, the conventional pGAP promoter in the respective cell. In particular, the choice of the promoter has a great impact on the production of the recombinant protein.

Preferred transformation methods for the uptake of the recombinant DNA fragment by the microorganism include chemical transformation, electroporation or transformation with protoplasts. The transformants described herein can be obtained by introducing said vector DNA, for example, plasmid DNA, into a host and selecting the transformants that express the relevant protein or the metabolite of the host cell in high yield.

The PDI can be produced using the recombinant host cell line by culturing a transformant thus obtained in an appropriate medium, isolating the expressed metabolite or product from the culture, and optionally purifying it by a suitable method.

The transformants described herein can be obtained by introducing said vector DNA, for example, plasmid DNA, into a host and selecting the transformants that express the PDI or the metabolite of the host cell in high yield. The host cells are treated to allow them to incorporate a foreign DNA by methods that are conventionally employed for the transformation of eukaryotic cells, such as the method of electrical pulses, the method of protoplasts, the method of lithium acetate and modifications of these methods P. pastoris is preferably transformed by electroporation.

The preferred host cell line according to the invention maintains the genetic properties employed herein, and the level of production remains high, for example, at least at a level of | jg, even after about 20 generations of culture, preferably at least 30 generations, more preferably at least 40 generations, most preferably at least 50 generations. The stable recombinant host cell is considered a great advantage when used for industrial scale production.

Several different strategies for the production of the PDI are preferred according to the method provided herein. The substances can be expressed, processed and optionally segregated by transforming a eukaryotic host cell with an expression vector that carries a recombinant DNA encoding a relevant protein and at least one of the regulatory elements as described above, the preparation of a culture of the transformed cell, the growth of the culture, the induction of transcription and the production of the PDI, and the recovery of the product of the fermentation process.

The PDI is preferably expressed using conditions to produce yields of at least 1 mg / l, preferably at least 10 mg / l, preferably at least 100 mg / l, most preferably at least 1 g / l.

The host cell described herein is preferably tested for performance or expression ability by the following assay: ELISA, activity assay, HPLC, or other suitable assays.

It is understood that the methods described herein may also include culturing said recombinant host cells under conditions that allow expression of the PDI, preferably in a secreted form or as an intracellular product. A recombinantly produced PDI or a metabolite of the host cell can then be isolated from the cell culture medium and subsequently purified by techniques well known to those skilled in the art.

The PDI produced as described herein can generally be isolated and purified using current techniques, which include increasing the concentration of the desired POI and / or decreasing the concentration of at least one impurity.

If the PDI is secreted from the cells, it can be isolated and purified from the culture medium using current techniques. The secretion of the recombinant expression products from the host cells in general is advantageous for reasons that include facilitating the purification process, since the products are recovered from the culture supernatant and not from the complex mixture of proteins that is produced when yeast cells they break to release intracellular proteins.

Cultured transformant cells can also be sonically or mechanically, enzymatically or chemically broken to obtain a cell extract containing the desired PDI, from which the PDI is isolated and purified. As isolation and purification methods to obtain a recombinant protein or polypeptide product, methods, such as methods that employ differences in solubility, such as desalting and solvent precipitation, methods that employ differences in molecular weight, such as ultrafiltration, can be used. and gel electrophoresis, methods that employ differences in electric charge, such as ion exchange chromatography, methods that employ specific affinity, such as affinity chromatography, methods that employ differences in hydrophobicity, such as high performance liquid chromatography in reverse phase, and methods that employ differences in the isoelectric point, such as isoelectric focus.

The highly purified product is essentially free of contaminating proteins, and preferably has a purity of at least 90%, more preferably at least 95%, or even at least 98%, up to 100%. The purified products can be obtained by purification of the cell culture supernatant or from cell debris.

As isolation and purification methods, the following conventional methods are preferred: cell rupture (if the PDI is obtained intracellularly), cell separation (waste) and washing by microfiltration or tangential flow filter ("Tangential Flow Filter" ", TFF) or centrifugation, purification of the PDI by precipitation or heat treatment, activation of the PDI by enzymatic digestion, purification of the PDI by chromatography, such as ion exchange (" ion exchange ", IEX), hydrophobic interaction chromatography ("hydrophobic interaction chromatography", HIC), affinity chromatography, molecular exclusion chromatography (SEC) or HPLC chromatography, precipitation or concentration of the PDI and washing by ultrafiltration steps.

The isolated and purified PDI can be identified by conventional methods, such as Western blotting, HPLC, activity assay, or ELISA.

The PDI can be any eukaryotic, prokaryotic or synthetic polypeptide. It can be a secreted protein or an intracellular protein. The recombinant production of functional homologues, equivalent functional variants, derivatives and biologically active fragments of natural proteins is also provided herein. Functional homologues are preferably identical or corresponding and have the functional characteristics of a sequence.

A PDI mentioned herein may be a product homologous with the eukaryotic host cell, or heterologous, preferably for therapeutic, prophylactic, diagnostic, analytical or industrial use.

The PDI is preferably a heterologous recombinant protein or polypeptide produced in a eukaryotic cell, preferably a yeast cell, preferably as secreted proteins. Examples of preferably produced proteins are immunoglobulins, immunoglobulin fragments, aprotinin, tissue factor pathway inhibitor or other protease inhibitors, and insulin or insulin precursors, insulin analogs, growth hormones, interleukins, plasminogen activator tissue, transforming growth factor aob, glucagon, glucagon-like peptide 1 ("glucagon-like peptide 1", GLP-1), glucagon-like peptide 2 (GLP-2), GRPP, factor VII, factor VIII, factor XIII , growth factor 1 derived from platelets, serum albumin, enzymes, such as lipases or proteases, or a functional homologue, functional equivalent variant, derivative and biologically active fragment with a function similar to the native protein. The PDI can be structurally similar to the native protein and can be derived from the native protein by adding one or more amino acids to either or both of the C- and N-terminal ends or to the side chain of the native protein, replacing one or more amino acids at one or a number of different sites in the native amino acid sequence, the deletion of one or more amino acids at either or both ends of the native protein or at one or more sites in the amino acid sequence , or the insertion of one or more amino acids in one or more sites of the native amino acid sequence. These modifications are well known for several of the proteins mentioned above.

A PDI can also be selected from substrates, enzymes, inhibitors or cofactors that provide the biochemical reactions in the host cell, in order to obtain the product of said biochemical reaction or a cascade of several reactions, for example, to obtain a metabolite of the host cell Examples of products may be vitamins, such as riboflavin, organic acids and alcohols, which can be obtained with higher yields after expression of a recombinant protein or a PDI as described herein. In general, the host cell, which expresses a recombinant product, can be any eukaryotic cell suitable for the recombinant expression of a PDI.

Examples of preferred mammalian cells are BHK, CHO cells (CHO-DG44, CHO-DUXB11, CHO-DUKX, CHO-K1, CHOK1SV, CHO-S), HeLa, HEK293, MDCK, NIH3T3, NS0, PER.C6, SP2 / 0 and VERO.

Examples of preferred yeast cells used as host cells as described herein include, but are not limited to the genus Saccharomyces (e.g., Saccharomyces cerevisiae), the genus Pichia (e.g., P. pastoris, or P. methanolica) , the genus Komagataella (K. pastoris, K. pseudopastoris or K. phaffii), Hansenula polymorpha or Kluyveromyces lactis.

The most recent bibliography divides and renames Pichia pastoris as Komagataella pastoris, Komagataella phaffii and Komagataella pseudopastoris. At present, Pichia pastoris is used as a synonym for all, Komagataella pastoris, Komagataella phaffii and Komagataella pseudopastoris.

Preferred yeast host cells are derived from methylotrophic yeasts, such as Pichia or Komagataella, for example, Pichia pastoris, or Komagataella pastoris, or K. phaffii, or K. pseudopastoris. Examples of the host include yeasts, such as P. pastoris. Examples of P. pastoris strains include CBS 704 (= NRRL Y-1603 = DSMZ 70382), CBS 2612 (= NRRL Y-7556), CBS 7435 (= NRRL Y-11430), CBS 9173-9189 (CBS strains: CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands), and DSMZ 70877 (German Collection of Micro-organisms and Cell Cultures), but also Invitrogen strains, such as X-33, ^g S115, KM71 and SMD1168 . Examples of S. cerevisiae strains include W303, CEN.PK and the BY series (EUROSCARF collection). All strains described above have been used successfully to produce transformants and express heterologous genes.

A preferred yeast host cell, such as a P. pastoris or S. cerevisiae host cell, contains sequences of recombinant or heterologous promoters, which may be derived from a strain of P. pastoris or S. cerevisiae, different from the production host. In another specific embodiment, the host cell comprises a recombinant expression construct as described herein comprising the promoter originating from the same genus, species or strain as the host cell.

The promoter may be a promoter as described herein or any other DNA sequence that shows transcriptional activity in the host cell and that may be derived from genes encoding homologous or heterologous proteins with the host. The promoter is preferably derived from a gene that encodes a homologous protein with the host cell.

For example, a promoter as described herein may be derived from a yeast, such as a S. cerevisiae strain, and used to express a ^p D ⁱ in a yeast. A specifically preferred embodiment relates to a promoter that originates from P. pastoris for use in a method of producing a recombinant PDI in a host cell line producing P. pastoris. The homologous origin of the nucleotide sequence facilitates its incorporation into a host cell of the same genus or species, thus allowing the stable production of a PDI, with the possibility of increasing performance in industrial manufacturing processes. In addition, functionally active promoter variants from other suitable yeasts or from other fungi or other organisms, such as vertebrates or plants, can be employed.

If the PDI is a homologous protein with the host cell, that is, a naturally occurring protein in the host cell, the expression of the PDI in the host cell can be modulated by exchanging its native promoter sequence for a sequence. of promoter as described herein.

This objective can be achieved, for example, by transforming a host cell with a recombinant DNA molecule comprising homologous sequences of the target gene to allow site-specific recombination, the promoter sequence and a selective marker suitable for the host cell. Site-specific recombination is performed to operably link the promoter sequence with the nucleotide sequence encoding the PDI. This causes the expression of the PDI from the promoter sequence, as described herein, instead of the native promoter sequence.

In a specifically preferred embodiment, the promoter sequence has a greater promoter activity relative to the native promoter sequence of the PDI.

As described herein, it is preferred to provide a line of P. pastoris host cells comprising a promoter sequence according to the invention operably linked to the nucleotide sequence encoding the PDI.

According to a specific embodiment, it is also possible to provide a host cell or wildcard vector comprising a promoter as described herein, and which is ready to incorporate a gene of interest encoding a p Di. The wildcard cell line, therefore, is a preformed host cell line that is characterized by its ability to express. It follows an innovative “wild card” platform strategy for the generation of producer cell lines for the production of PDI, for example, using the exchange of modules mediated by site-specific recombinase. This new host cell facilitates the cloning of a gene of interest (GDI), for example, at predetermined genomic hot spots in a matter of days to obtain reproducible and very effective production cell lines.

According to a preferred embodiment, the method provided herein employs a recombinant nucleotide sequence encoding the PDI, which is provided on a plasmid suitable for genome integration. of the host cell, in a single copy or in multiple copies per cell. The recombinant nucleotide sequence encoding the PDI can also be provided on an autonomous replication plasmid in a single copy or in multiple copies per cell.

The preferred method as described herein employs a plasmid, which is a eukaryotic expression vector, preferably a yeast expression vector. Expression vectors may include, but are not limited to cloning vectors, modified cloning vectors and specifically designed plasmids. The preferred expression vector as used in the method provided herein may be any expression vector suitable for the expression of a recombinant gene in a host cell, and is selected depending on the host organism. The recombinant expression vector can be any vector that is capable of replicating or integrating into the genome of host organisms, also called a host vector, such as a yeast vector, that carries a DNA construct. A preferred yeast expression vector is for expression in yeasts selected from the group consisting of methylotrophic yeasts represented by the genera Hansenula, Pichia, Candida and Torulopsis.

Here, it is preferred to use plasmids derived from pPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis or pPUZZLE as a vector.

According to a preferred embodiment, a recombinant construct is obtained by coupling the relevant genes into a vector. These genes can be stably integrated into the genome of the host cell by transforming the host cell using these vectors. The polypeptides encoded by the genes can be produced using the recombinant host cell line by culturing a transformant thus obtained in an appropriate medium, isolating the expressed PDI from the culture, and purifying it by an appropriate method for the expressed product. , in particular to separate the PDI from contaminating proteins.

Expression vectors may comprise one or more phenotypic selectable markers, for example, a gene that encodes a protein that confers antibiotic resistance or that provides an autotrophic requirement. Yeast vectors usually contain an origin of replication from a yeast plasmid, an autonomous replication sequence (ARS) or, alternatively, a sequence used for integration into the host genome, a promoter region, sequences for polyadenylation, sequences for termination of transcription, and a selectable marker.

The procedures used to couple the DNA sequences, for example, which encode the precursor sequence and / or the PDI, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for integration or replication in the host, are well known to those skilled in the art, for example, as described in J. Sambrook et al., "Molecular Cloning", 2nd edition, Cold Spring Harbor Laboratory Press (1989).

It will be understood that the vector, which employs the regulatory elements described herein and / or the PDI as an integration target, can be constructed by first preparing a DNA construct containing the complete DNA sequence encoding the regulatory elements and / or the PDI, and subsequently inserting this fragment into a suitable expression vector, or by sequential insertion of DNA fragments containing genetic information for the individual elements, such as the signal, conductive or heterologous protein, followed by coupling.

Multicloning vectors, which are vectors containing a multicloning site, can also be employed herein, in which a desired heterologous gene can be incorporated into a multicloning site to provide an expression vector. In expression vectors, the promoter is placed upstream of the PDI gene and regulates gene expression. In the case of multi-cloning vectors, because the PDI gene is introduced into the multi-cloning site, the promoter is placed upstream of the multi-cloning site.

The DNA construct, as provided to obtain a recombinant host cell as described herein, can be prepared synthetically by conventional methods established, for example, the phosphoramidite method. The DNA construct can also have a genomic or cDNA origin, for example, obtained by preparing a cDNA or genomic bank and selecting DNA sequences that encode all or part of the polypeptide provided herein by hybridization using synthetic oligonucleotide probes according to conventional techniques ( Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989). Finally, the DNA construct can have a synthetic and genomic mixed origin, synthetic mixed and cDNA, or mixed genomic and cDNA, being prepared by associating fragments of synthetic, genomic or cDNA origin, as appropriate, and the fragments correspond with various parts of the complete DNA construction, according to conventional techniques.

In another preferred embodiment, the yeast expression vector is capable of stably integrating into the yeast genome, for example, by homologous recombination.

A transforming host cell obtained by transforming the cell with the regulatory elements provided herein and / or the genes of the PDI can preferably be cultured firstly under conditions to grow effectively until a large number of cells is reached without the burden of expressing a heterologous protein When the cell line is prepared for PDI expression, culture techniques are chosen to produce the expression product.

The issues of the following definitions are considered embodiments described herein:

1. A method of producing a protein of interest (PDI) by culturing a line of recombinant eukaryotic cells comprising an expression construct comprising an adjustable promoter and a nucleic acid molecule encoding a PDI under the transcriptional control of said promoter, which comprises the steps of:

a) cultivate the cell line with a basal carbon source that represses the promoter,

b) culturing the cell line without a supplemental carbon source, or with a limited amount thereof, that depresses the promoter to induce the production of the PDI at a transcription rate of at least 15%, compared to the native pGAP promoter of the cell, and

c) produce and recover the POI.

2. The method according to definition 1, wherein the basal carbon source is selected from the group consisting of glucose, glycerol, ethanol, one of its mixtures and a complex nutrient material.

3. The method according to definition 1 or 2, wherein the supplemental carbon source is a hexose, such as glucose, fructose, galactose or mannose, a disaccharide, such as sucrose, an alcohol, such as glycerol or ethanol, or One of their mixtures.

4. The method according to any of definitions 1 to 3, wherein the source of basal carbon is glycerol, and the source of supplemental carbon is glucose.

5. The method according to any of definitions 1 to 4, wherein step b) employs a feeding medium that does not provide the supplemental carbon source or provides it in a limited amount, preferably at 0-1 g / l in The culture medium.

6. The method according to definition 5, wherein the feeding medium is chemically defined and does not contain methanol.

7. The method according to any of definitions 1 to 6, wherein the limited amount of the supplemental carbon source limits growth to maintain the specific growth rate within the range of 0.02 h-1 to 0.2 h -1, preferably from 0.02 h-1 to 0.15 h-1.

8. The method according to definition 7, wherein the limited amount of the supplementary source provides a residual amount in the cell culture that is below the detection limit.

9. The method according to any of definitions 1 to 8, wherein the promoter is capable of controlling the transcription of a gene in a wild type eukaryotic cell, and said gene is selected from the group consisting of G1 (SEQ ID NO : 7), G3 (SEQ ID NO: 8), G4 (SEQ ID NO: 9), G6 (SEQ ID NO: 10), G7 (SEQ ID NO: 11), or G8 (SEQ ID NO: 12), or one of its functionally active variants.

10. The method according to definition 9, wherein said functionally active variants are selected from the group consisting of homologues with at least about 60% nucleotide sequence identity, homologs that can be obtained by modifying the sequence of nucleotides of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, which preferably comprise or consist of a nucleotide sequence of at least 200 bp, and analogues derived from species other than Pichia pastoris.

11. The method according to definition 9 or 10, wherein the functionally active variant of pG1 is selected from the group consisting of pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO : 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46).

12. The method according to any of definitions 1 to 11, wherein the promoter is a Pichia pastoris promoter or one of its functionally active variants.

13. The method according to any of definitions 1 to 12, wherein the cell line is selected from the group consisting of mammalian, insect, yeast, filamentous and plant cell lines, preferably a yeast.

14. The method according to definition 13, wherein the yeast is selected from the group consisting of Pichia, Candida, Torulopsis, Arxula, Hensenula, Yarrowia, Kluyveromyces, Saccharomyces, Komagataella, preferably a methylotrophic yeast.

15. The method according to definition 14, in which the yeast is Pichia pastoris, Komagataella pastoris, K. phaffii, or K. pseudopastoris.

16. The method according to any of definitions 1 to 15, wherein the promoter is not natively associated with the nucleotide sequence encoding the PDI.

17. The method according to any of definitions 1 to 16, wherein the PDI is a heterologous protein, preferably selected from therapeutic proteins, including antibodies or their fragments, enzymes and peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and proteins or vaccine-like particles, process enzymes, growth factors, hormones and cytokines, or a metabolite of a PDI.

18. The method according to any of definitions 1 to 17, wherein the PDI is a eukaryotic protein, preferably a mammalian protein.

19. The method according to any of definitions 1 to 18, wherein the PDI is a multimeric protein, preferably a dimer or a tetramer.

20. The method according to any of definitions 1 to 19, wherein the PDI is an antigen binding molecule, such as an antibody, or one of its fragments.

21. The method according to any of definitions 1 to 20, in which a fermentation product is manufactured using the PDI, a metabolite or a derivative thereof.

22. A method for controlling the expression of a PDI in a recombinant eukaryotic cell under the transcriptional control of a promoter adjustable by a carbon source having a transcription potency of at least 15%, compared to the cell's native pGAP promoter , in which the expression is induced under conditions that limit the carbon source.

23. A method for producing a PDI in a recombinant eukaryotic cell under the transcriptional control of a promoter adjustable by a carbon source, wherein said promoter has a transcription potency of at least 15%, compared to the native pGAP promoter of the cell.

24. The method according to any of definitions 1 to 23, wherein the adjustable promoter comprises a nucleic acid sequence selected from the group consisting of:

a) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 ( SEQ ID NO: 6);

b) a sequence that has at least 60% homology with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6);

c) a sequence that hybridizes under stringent conditions with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6); and

d) a fragment or variant derived from a), b) or c),

wherein said promoter is a functionally active promoter, which is a promoter adjustable by a carbon source capable of expressing a PDI in a recombinant eukaryotic cell at a transcription rate of at least 15%, compared to the native pGAP promoter of the cell.

25. The method according to definition 24, wherein the variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3) , pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6) is a functionally active variant selected from the group consisting of homologues with at least 60% nucleotide sequence identity, homologs that can be obtained by means of the modification of the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, which preferably comprise or consist of a nucleotide sequence of at least 200 bp, and analogues derived from species other than Pichia pastoris.

26. The method according to definition 24 or 25, wherein the functionally active variant of pG1 is selected from the group consisting of pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO : 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46).

27. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of:

a) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 ( SEQ ID NO: 6);

b) a sequence that has at least 60% homology with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6);

c) a sequence that hybridizes under stringent conditions with pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6); and

d) a fragment or variant derived from a), b) or c),

wherein said nucleic acid comprises a functionally active promoter, which is a promoter adjustable by a carbon source capable of expressing a PDI in a recombinant eukaryotic cell at a transcription rate of at least 15%, compared to the native pGAP promoter of the cell.

28. A nucleic acid according to definition 27, wherein the variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG6 (SEQ ID NO: 3 ), pG7 (SEQ ID NO: 5), or pG8 (SEQ ID NO: 6) is a functionally active variant selected from the group consisting of homologues with at least 60% nucleotide sequence identity, homologs that can be obtained through of the modification of the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, preferably with a nucleotide sequence of at least 200 bp, and analogues derived from species other than Pichia pastoris.

29. A nucleic acid according to definition 27 or 28, wherein the functionally active variant of pG1 is selected from the group consisting of pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46).

30. An expression construct comprising a nucleic acid according to any of definitions 27 to 29, operably linked to a nucleotide sequence encoding a PDI under the transcriptional control of said promoter, and said nucleic acid is not natively associated with the sequence of nucleotides encoded by the PDI.

31. A vector comprising the construction according to definition 30.

32. A recombinant eukaryotic cell comprising the construction of definition 30, or the vector of definition 31.

33. A cell according to definition 31, which is selected from the group consisting of mammalian, insect, yeast, filamentous and plant cell lines, preferably a yeast.

34. A cell according to definition 32, in which the yeast is selected from the group consisting of Pichia, Candida, Torulopsis, Arxula, Hensenula, Yarrowia, Kluyveromyces, Saccharomyces, Komagataella, preferably a methylotrophic yeast.

35. A cell according to definition 34, in which the yeast is Pichia pastoris, Komagataella pastoris, K. phaffii, or K. pseudopastoris.

36. A cell according to any of the definitions 32 to 35, which has a higher specific growth rate in the presence of an excess carbon source, relative to limited carbon source conditions.

37. A method for identifying a promoter adjustable by a carbon source from eukaryotic cells, comprising the steps of:

a) culturing eukaryotic cells in the presence of a carbon source in a discontinuous culture under cell growth conditions,

b) then culturing the cells in a batch feed culture in the presence of a limited amount of a supplemental carbon source,

c) provide samples of the cell culture of stage a) and b), and

d) perform a transcription analysis on said samples to identify an adjustable promoter that shows a higher transcriptional potency in the cells of stage b) than in the cells of stage a).

38. The method according to definition 37, in which the transcription analysis is quantitative or semi-quantitative, and preferably employs DNA microarrays, RNA sequencing and transcriptome analysis.

The specific examples refer to a batch feed fermentation of a recombinant production line of P. pastoris that produces indicator proteins, which employs a discontinuous glycerol medium and a discontinuous glucose feed medium. Comparative promoter activity studies have shown that the promoter described herein can be successfully activated to induce the production of recombinant proteins.

According to another example, human serum albumin (HSA) was produced as a POI under the control of promoters induced by a limitation in glucose, and the yield of HSA and the number of copies of the gene were determined.

According to another example, a batch feed culture of P. pastoris strains expressing HSA was performed under the control of a promoter described herein. It was found that the induction of promoter activity under glucose limitation conditions was even greater than 120 times with pG1, and greater than 20 times with pG6, compared to the repressed state.

Other examples relate to expressing a porcine carboxypeptidase B as a model protein under the transcriptional control of the pG1 and pG6 promoter.

Another example refers to the expression of an antibody fragment under the transcriptional control of pG1.

Another example demonstrates the functional activity of variants of a promoter described herein, such as fragments of pG1 with a length in the range of 300 to 1000 bp. Other experiments have shown that even shorter pG1 fragments were functionally active in a similar scenario, such as fragments that vary from 200 to 1000 bp, or fragments that vary between 250 and 1000.

The above description will be understood more fully by referring to the following examples. However, such examples are simply representative of methods for practicing one or more embodiments of the present invention and should not be construed as limiting the scope of the invention.

Examples

The following examples illustrate the materials and methods used to identify new regulators and to analyze their expression properties in Pichia pastoris.

Example 1: Identification of potent genes effectively regulated in P. pastoris under glucose limitation conditions

To identify potent genes effectively regulated and their respective P. pastoris promoters under glucose limitation conditions, an analysis of gene expression patterns was performed using microarrays. P. pastoris cells in a discontinuous glycerol culture (excess carbon source) were compared with cells that were grown in conditions where glucose limited growth (chemostat), thereby mimicking the development of a production process of proteins, which are usually performed in a discontinuous feeding mode.

a) strain

A strain of P. pastoris (CBS2612, CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used, which can grow in minimal medium without supplements.

b) Cultivation of P. pastoris

Fermentations were carried out with Minifors reactors (Infors-HT, Switzerland) with a final workload of 2.5 l. The following means were used:

Mother solution of oligosales PTMi contained per liter

6.0 g of CuSO4.5H2O, 0.08 g of NaI, 3.36 g of MnSO ⁴ .H ² O, 0.2 g of Na2MoO4.2H2O, 0.02 g of H ³ BO ³ , 0.82 g of CoCl ² , 20.0 g of ZnCl ² , 65.0 g of FeSO4.7H2O, 0.2 g of biotin and 5.0 ml of H ² SO ⁴ (95% -98%).

Discontinuous medium of glycerol contained per liter

2 g of citric acid monohydrate (C ⁶ H ⁸ O ^7. H ² O), 39.2 g of glycerol, 20.8 g of NH ⁴ H ² PO ⁴ , 0.5 g of MgSO4.7H2O, 1.6 g of KCl, 0.022 g of CaCh.2H2O, 0.8 mg of biotin and 4.6 ml of stock solution of oligosales PTM1. HCl was added to adjust the pH to 5.

Glycerol discontinuous feed medium contained per liter

632 g of glycerol, 8 g of MgSO4.7H2O, 22 g of KCl, and 0.058 g of CaCl2.2H2O.

Chemostat medium contained per liter

2 g of citric acid monohydrate (C ⁶ H ⁸ O ^7. H ² O), 99.42 g of glucose monohydrate, 22 g of NH ⁴ H ² PO ⁴ , 1.3 g of MgSO4.7H2O, 3.4 g of KCl, 0.02 g of CaCl2.2H2O, 0.4 mg of biotin and 3.2 ml of stock solution of oligosales PTM1. HCl was added to adjust the pH to 5.

The dissolved oxygen was controlled at OD = 20% with the agitator speed (500-1250 rpm). The aeration rate was of 60 lh ¹ of air, the temperature was controlled at 25 ° C, and the pH set point of 5 was controlled with the addition of NH ⁴ OH (25%).

To begin the fermentation, 1.5 l of discontinuous medium was sterilized by filtration into the fermenter and P. pastoris (from a preculture overnight in YPG, 180 rpm, 28 ° C) was inoculated with an initial optical density (DO600) of 1. The discontinuous phase of approximately 25 h reached a dry biomass concentration of approximately ^{2 0} g / l, and was followed by an exponential discontinuous feeding phase of ¹⁰ h with glucose medium that led to a dry biomass concentration of approximately 50 g / l. Then the volume was reduced to 1.5 and the culture began in a chemostat with a feed / harvest rate of 0.15 l h-1, producing a constant growth rate of p = 0.1. The fermentation ended 50 h after the start of the use of the chemostat.

This fermentation was carried out three times to obtain the biological duplicates necessary for a reliable microarray analysis.

Carbon limitation conditions (no detectable residual glucose exists) during chemostat use were verified by an HPLC analysis of the culture supernatant.

c) Sampling

Samples were taken at the end of the discontinuous glycerol phase and under steady state conditions of the glucose chemostat. A usual sample was taken as a determination of the optical density or dry mass of yeasts, a qualitative microscopic inspection and an analysis of the viability of the cells during each fermentation. For the microarray analysis, samples were taken and treated as follows: for optimal extinction, 9 ml of the cell culture broth was immediately mixed with 4.5 ml of ice-cold 5% phenol solution (Sigma) (in ethanol absolute), and aliquots were formed. Every 2 ml was centrifuged (13200 rpm for 1 minute) in precooled collection tubes (GE Healthcare, NJ), the supernatant was completely removed, and the tubes were stored at -80 ° C until RNA purification.

d) RNA purification and sample preparation for microarray hybridization

RNA was isolated using the TRI reagent according to the instructions of the suppliers (Ambion, USA). The cell pellets were resuspended in TRI reagent and homogenized with glass spheres using a FastPrep 24 (MP Biomedicals, CA) at 5 ms ^-1 for 40 seconds. After the addition of chloroform, the samples were centrifuged and the total RNA was precipitated from the aqueous phase by adding isopropanol. The sediment was washed with 70% ethanol, dried and resuspended in water without RNase. RNA concentrations were determined by measuring OD260 using a Nanodrop 1000 spectrophotometer (NanoDrop Products, DE). The remaining DNA was removed from the samples using the DNA Free Kit without DNA (Ambion, CA). A sample volume equal to 10 pg of RNA was diluted to 50 pl in water without RNase, the DNase I buffer and rDNAase I was added and incubated at 37 ° C for 30 minutes. After the addition of the DNase inactivation reagent, the sample was centrifuged and the supernatant was transferred to a new tube. RNA concentrations were determined again as described above. In addition, RNA integrity was analyzed using RNA nanochips (Agilent). The Spike In Kit (Agilent, product no .: 5188-5279) was used as a positive control to control the microarray workflow from amplification and labeling to sample hybridization. It contains 10 different polyadenylated transcripts from an adenovirus, which are amplified, labeled and inhibited together with the RNA samples themselves. Samples were labeled with Cy 3 and Cy 5 using the Quick Amp marking kit (Agilent, product no .: 5190-0444). To do this, 500 ng of the purified RNA sample was diluted in 8.3 µl of water without RNase, and 2 µl of seeding A or B, and 1.2 µl of T7 promoter primer were added. The mixture was denatured for 10 minutes at 65 ° C and kept on ice for 5 minutes. Then 8.5 pl of cDNA master mix was added (per sample: 4 pl of 5 * first strand buffer, 2 pl of 0.1 M DTT, 1 pl of 10 mM dNTP mixture, 1 pl of MMLV-RT , 0.5 pl of RNase Out), were incubated at 40 ° C for 2 hours, then transferred to 65 ° C for 15 minutes and placed on ice for 5 minutes. The transcription master mix was prepared (per sample: 15.3 pl of water without nuclease, 20 pl of transcription buffer, ⁶ pl of 0.1 M DTT, 6.4 pl of 50% p Eg, 0.5 pl of RNase inhibitor, 0.6 pl of inorganic phosphatase, 0.8 pl of T7 RNA polymerase, 2.4 pl of cyanine 3 or cyanine 5) and added to each tube and incubated at 40 ° C for 2 hours. To purify the labeled cRNA obtained, the RNeasy Mini Kit (Qiagen, catalog no. 74104) was used. Samples were stored at -80 ° C. Quantification of the concentration of the cRNA and the efficacy of the marking with the Nanodrop spectrophotometer was performed.

e) Microarray analysis

To identify strong genes effectively regulated in chemostat cultures with glucose limitation, three duplicates of biological samples from these were compared with the same reference and in one exchange of dyes each. The reference sample was generated by combining the glycerol batch culture samples in equal amounts.

The gene expression hybridization kit (Agilent, catalog No. 5188-5242) was used for hybridization of the cRNAs of the labeled samples. For the preparation of hybridization samples, every 300 ng of cRNA (Cy3 and Cy 5) and 6 µl of block agent in 10 times were diluted with water without nuclease to a final volume of 24 µl. After the addition of 1 µl of fragmentation buffer in 25 times, the mixture was incubated at 60 ° C for 30 minutes. Then 25 µl of GEx Hybridization Buffer HI-RPM hybridization buffer was added to stop the reaction. After centrifugation for one minute at 13,200 rpm, the sample was cooled on ice and used for hybridization immediately. P. pastoris specific oligonucleotide matrices designed in the inventors' laboratory (AMAD-ID: 026594, 8 * 15K basic matrices, Agilent) were used. Hybridization with microarrays was performed according to the user's guide of microarray hybridization chambers (Agilent G2534A). First, the board stage was discovered and placed on the base of the chamber with the Agilent label facing up. The sample (40 ml per matrix) was loaded in the middle of each of the eight squares. Then the microarray stage was carefully placed on the board stage (Agilent label face down) and the camera cover was placed and fixed with the clamp. Hybridization was performed in the hybridization oven for 17 hours at 65 ° C. Before scanning, the microarray chip was washed. For this, the chamber was disassembled and the interleaved plates detached from each other while they were submerged in wash buffer 1. The microarray was directly transferred to another plate with wash buffer 1, washed for 1 minute, transferred to wash buffer 2 (temperature of at least 30 ° C) and washed for another minute. After drying the micromatrix stage by touching the edge of the stage with a tissue, it was placed on the plate holder (with the Agilent label facing up). The plate stand was inserted into the carousel and scanning began.

f) Data acquisition and statistical evaluation of microarray data

The images were scanned at a resolution of 50 nm with a G2565AA microarray scanner (Agilent) and imported into the Agilent Feature Extraction 9.5 software. Agilent Feature Extraction 9.5 was used to quantify stain intensities. Then the intensity data of the gross average spots were imported into the open source program R for the subsequent normalization and analysis of the data.

For the preprocessing and normalization of the data, the R limma, vsn and marray packages were used. The intensity data was not corrected for the background and normalized with ^v S ⁿ , and after normalization they were transformed to log 2 proportions of the Cy5 channel versus the Cy3 channel. The differential expression was calculated using the lmfit and eBay function of the limma package.

The microarray data were scanned for inputs with a high difference in the level of expression between the repressed and induced state (change in number of times), as well as a high signal intensity in the induced state to identify strongly expressed genes and regulated effectively. Table 1 shows a list of the selected genes, and the change in number of times means the signal strength in the induced state divided by the signal strength in the repressed state. The data of pGAP, pMLS1 and pICL1 are added as a reference.

Table 1. Microarray data of the promoters selected for subsequent characterization and of pGAP,

ICL1 and MLS1 as controls

Example 2: Comparative promoter activity studies of newly identified promoters in P. pastoris using eGFP as an indicator gene expressed intracellularly

To analyze the properties of the newly identified promoters under glucose limitation conditions, selections were made in shake flasks as follows: preculture was carried out for 24 hours with rich medium containing glycerol as a carbon source, which simulates the discontinuous phase of the process (repressed state of the promoters), followed by the main culture with minimal medium and glucose feeding spheres, which simulates the discontinuous feeding phase with glucose limitation of the process (induced state of the promoters). a) Strain and expression vector

A wild-type strain of P. pastoris (CBS2612, CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with a vector from the inventors' laboratory called pPUZZLE (Stadlmayr et al., J. Biotechnol., December 2010, 150 (4): 519-529), which comprises an origin of the replication of E. coli (pUC19), an antibiotic resistance module (Shble gene that confers resistance to zeocin) for selection in E. coli and yeast, an expression module for the gene of interest (GDI) consisting of a multiple cloning site and transcription terminator of S. cerevisiae CYC1, and a locus for integration into the genome of P. pastoris (region 3'AOX1). b) Amplification and cloning of the newly identified promoters pG1, pG3, pG4 and pG6 in the expression vector pPUZZLE containing eGFP as GDI

Table 2 shows a list of the newly identified promoter sequences and their respective genes (see example 1). 1000 bp of the 5 'non-coding region of the respective genes were amplified to the ATG start codon by PCR (Phusion Polymerase, New England Biolabs) as promoter sequences using the primers shown in Table 2. These sequences were cloned. in the expression vector pPUZZLE pPM1aZ10_eGFP, producing pPM1aZ10_pG1_eGFP, pPM1aZ10_pG3_eGFP, pPM1aZ10_pG4_eGFP and pPM1aZ10_pG6_eGFP. In addition, the vector pPM1aZ10_pGAP_eGFP was used, which contains the commonly used promoter of glyceraldehyde 3-phosphate dehydrogenase (P. pastoris pGAP, in this SEQ ID NO: 25) as reference. Promoters were inserted upstream of the start codon of the eGFP gene using the Apal and Sbfl restriction sites (see Tables 2 and 3). It was verified that the promoter sequences were correct by Sanger sequencing.

Table 2. Primers for PCR amplification of promoters

Table 3. Amplification primers, cloning enzymes and length of cloned promoters

c) Expression of eGFP in P. pastoris for the analysis of promoter activity

All plasmids were linearized with AscI within the integration region in the 3'AOX genome before electroporation (2 kV, 4 ms, GenePulser, BioRad) in electrocompetent P. pastoris.

The selection of the positive transformants in YPD plates was performed (per liter: 10 g of yeast extract, 20 g of peptone, 20 g of glucose, 20 g of agar-agar) containing 25 pg / ml of zeocin (Invitrogen, AC). A colony PCR was used to ensure the presence of the transformed plasmid. To do this, genomic DNA was acquired by cooking and freezing colonies of P. pastoris for 5 minutes each and applied directly to the PCR with the appropriate primers. For the selection of the expression, a single colony was inoculated in liquid YPG-Zeo medium (per liter: 20 g of peptone, 10 g of yeast extract, 12.6 g of glycerol and 25 mg of zeocin) as a preculture. After approximately 24 h, the preculture was used to inoculate the main culture with an OD 600 of 0.1 in 10 ml of YP medium (per liter: 20 g of peptone, 10 g of yeast extract) and 2 of feeding spheres of glucose (Kuhner, CH). Growth conditions with glucose limitation were achieved due to the slow glucose release kinetics of these feeding spheres, which is described by the following equation: (glucose) = 1.63 * t0.74 [mg / disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. Cell density was determined by measuring OD600, eGFP expression was analyzed by flow cytometry as described in Stadlmayr et al. (J. Biotechnology, December 2010, 150 (4): 519-529). 10,000 cells were analyzed for each sample. Autofluorescence of P. pastoris was measured using wild-type P. pastoris untransformed cells and subtracted from the signal. Relative eGFP expression levels (fluorescence intensity related to cell size) as a percentage of the eGFP expression level under the control of the constitutive pGAP promoter. Similar studies were performed with the pG7 and pG8 promoters. Cloning is performed as described in example 2b, except that the wild-type strain of P. pastoris X-33 (Invitrogen) was used for the transformation of pPM1aZ10_pG7_eGFP and pPM1aZ10_pG8_eGFP. The primers and cloning fragments used are listed in Tables 2 and 3. The results are shown in Table 4.

Table 4. Results of the selection of P. pastoris clones expressing eGFP under the control of the new promoters; The data shown (fluorescence / cell size) refer to pGAP.

d) Determination of the number of copies of the eGFP gene ("Gene Copy Numbers", GCN) in clones expressing selected eGFP

Expression power is often correlated with the number of expression modules integrated into the genome of P. pastoris. Therefore, the number of copies of the eGFP gene was determined. Genomic DNA was isolated using the DNeasy blood and tissue kit (Quiagen, catalog No. 69504). The number of copies of the gene was determined using a quantitative PCR. For this, the SensiMix SYBR kit (Bioline, QT605-05) was used. Sensi Mix SYBR was mixed with the primers and the sample, and applied for real-time analysis in a real-time PCR cycler (Rotor Gene, Qiagen). Table 5 shows a list of the primers. All samples were analyzed in triplicate or quadruplicate. The Rotor Gene software program was used for data analysis. Actin gene ACT1 was used as a calibrator. The results are shown in table 6.

Table 5. Primers for the determination of the number of copies of the gene by real-time PCR

Table 6. Selection results (fluorescence / cell size relative to pGAP) and the number of gene copies of selected P. pastoris clones expressing eGFP under the control of pG1 and pG6

e) Analysis of the potency of the pG1 promoter in a discontinuous feed fermentation of an eGFP clone Fermentations were performed with discontinuous feed in DASGIP reactors with a final working volume of 0.7 l.

The following means were used:

Mother solution of oligosales PTMi contained per liter

6.0 g of CuSO4.5H2O, 0.08 g of NaI, 3.36 g of MnSO ⁴ .H ² O, 0.2 g of Na2MoO4.2H2O, 0.02 g of H ³ BO ³ , 0.82 g of CoCl ² , 20.0 g of ZnCl ² , 65.0 g of FeSO4.7H2O, 0.2 g of biotin and 5.0 ml of H ² SO ⁴ (95% -98%).

Discontinuous medium of glycerol contained per liter

2 g of citric acid monohydrate (C ⁶ H ⁸ O ^7. H ² O), 39.2 g of glycerol, 12.6 g of NH ⁴ H ² PO ⁴ , 0.5 g of MgSO4.7H2O, 0.9 g of KCl, 0.022 g of CaCh.2H2O, 0.4 mg of biotin and 4.6 ml of stock solution of oligosales PTM1. HCl was added to adjust the pH to 5.

Glycerol discontinuous feed medium contained per liter

464 g of glucose monohydrate, 5.2 g of MgSO4.7H2O, 8.4 g of KCl, 0.28 g of CaCh.2H2O, 0.34 mg of biotin and 10.1 ml of stock solution of oligosales PTM1.

Dissolved oxygen was controlled at OD = 20% with the agitator speed (400-1200 rpm). The aeration rate was 24 l h-1 of air, the temperature was controlled at 25 ° C, and the pH set point of 5 was controlled with the addition of NH ⁴ OH (25%).

To begin the fermentation, 400 ml of discontinuous medium was sterilized by filtration into the fermenter and P. pastoris clone pG1_eGFP # 8 (from a preculture) with an initial optical density (DO600) of 1 was inoculated. The discontinuous phase of approximately 25 h (a dry biomass concentration of approximately 20 g / l is reached) was followed by a discontinuous feeding phase with glucose limitation that begins with an exponential feeding for 7 h and a constant feeding rate of 15 g / l for 13 h, which led to a final dry biomass concentration of approximately 100 g / l. Samples were taken during the discontinuous phase and the discontinuous feed phase, and analyzed for eGFP expression using a plate reader (Infinite 200, Tecan, CH). For this, the samples were diluted to an optical density (OD600) of 5. The results are shown in Table 7 as relative fluorescence per bioreactor (FL / r).

Table 7. Relative bioreactor fluorescence of two different P. pastoris clones expressing eGFP under the control of pGAP or pG1 in an optimized discontinuous feed fermentation

Example 3: Comparative promoter activity studies of newly identified promoters in P. pastoris using human serum albumin (HSA) as an indicator gene expressed extracellularly

To analyze the properties of the newly identified promoters under glucose limitation conditions, selections were made in shake flasks as follows: preculture was carried out for 24 hours with rich medium containing glycerol as a carbon source, which simulates the discontinuous phase of the process (repressed state of promoters), followed by the main culture with minimal medium and glucose feeding spheres, which simulates the discontinuous feeding phase with glucose limitation of the process (induced state of the promoters). a) Strain and expression vector

A wild-type strain of P. pastoris (CBS2612, CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with a vector from the inventors' laboratory called pPUZZLE (Stadlmayr et al., J. Biotechnol., December 2010, 150 (4): 519-529), and the selection of positive transformants was based on zeocin resistance. For the secretory expression of human serum albumin (HSA), its native secretion conductor was used.

b) Amplification and cloning of the newly identified promoters pG1, pG3, pG4 and pG6 into an expression vector of the inventors' laboratory

The four promoters amplified in example 2b were cloned into the expression vector pPUZZLE pPM1aZ10_HSA, producing pPM1aZ10_pG1_HSA, pPM1aZ10_pG3_HSA, pPM1aZ10_pG4_HSA and pPM1aZ10_pG6_HSA. In addition, the vector pPM1aZ10_pGAP_HSA, which contains the commonly used promoter of glyceraldehyde 3-phosphate dehydrogenase (pGAP), was used as a reference. Promoters were inserted upstream of the start codon of the HSA gene using the ApaI and SbfI restriction sites (see table 3). It was verified that the promoter sequences were correct by Sanger sequencing.

c) Expression of HSA in P. pastoris under the control of promoters induced by a newly identified glucose limitation

All plasmids were linearized using the restriction enzyme AscI before electroporation (using a conventional transformation protocol for P. pastoris) in P. pastoris. Selection of the positive transformants in YPD plates (per liter: 10 g of yeast extract, 20 g of peptone, 20 g of glucose, 20 g of agar-agar) containing 25 pg / ml of zeocin was made. Colony PCR was used to ensure the presence of the transformed plasmid as described in example 2c.

For the selection of HSA expression, a single colony was inoculated in liquid YPG-Zeo medium (per liter: 20 g of peptone, 10 g of yeast extract, 12.6 g of glycerol and 25 mg of zeocin) as preculture . After approximately 24 h, the preculture was used to inoculate the main culture with an OD600 of 1 in YP medium (per liter: 20 g of peptone, 10 g of yeast extract) and glucose feeding spheres (Kuhner, CH) . Growth conditions with glucose limitation were achieved due to the slow glucose release kinetics of these feeding spheres, which is described by the following equation: (glucose) = 1.63 * t074 [mg / disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. The biomass concentration was determined by measuring DO600 or wet cell weight. The concentration of HSA in the culture supernatant was quantified using the human albumin ELISA quantification kit (catalog No. E80-129, Bethyl Laboratories, TX, USA) according to the supplier's instruction manual. The HSA standard was used with an initial concentration of 400 ng ml-1. The samples were diluted accordingly in sample diluent (50 mM Tris-HCl, 140 mM NaCl, 1% BSA (in w / v), 0.05% Tween20 (in v / v), pH 8.0) . Table 8 shows the HSA qualifications of the selection of several clones of each construction.

Table 8. Results of the selection of P. pastoris clones expressing HSA under the control of pGAP, pG1 and pG6

d) Determination of the number of copies of the HSA gene

Isolation of genomic DNA and measurement with qPCR were performed as in example 2d, using the primers shown in table 9. The results are shown in table 10.

Table 9. Primers for the determination of the number of copies of the gene by real-time PCR

Table 10. Results of the selection and number of gene copies of selected P. pastoris clones expressing HSA under the control of pGAP, pG1 and pG ⁶

e) Discontinuous feeding culture of P. pastoris strains expressing HSA under the control of the pG1 and pG ⁶ promoter

The fermentations were carried out in DASGIP bioreactors with a final workload of 0.7 l. Strain pG1_HSA # 23 has two copies of the HSA gene, and strain pG6_HSA # 36 carries only one copy of the HSA gene. Therefore, two different P. pastoris strains expressing HSA were grown under the control of pGAP (pGAP_HSA # 3 with one copy of the HSA gene, and pGAP_HSA # 4 with two copies of the HSA gene) as a reference. All fermentations were performed in duplicate.

The following means were used:

Mother solution of oligosales PTMi contained per liter

6.0 g of CuSO ⁴ . ⁵ H ² O, 0.08 g of NaI, 3.36 g of MnSO ⁴ .H ² O, 0.2 g of Na ² MoO ⁴ . ² H ² O, 0.02 g of H ³ BO ³ , 0.82 g of CoCl ² , 20.0 g of ZnCl ² , 65.0 g of FeSO ⁴ . ⁷ H ² O, 0.2 g of biotin and 5.0 ml of H ² SO ⁴ (95% -98%).

Discontinuous medium of glycerol contained per liter

39.2 g of glycerol, 27.9 g of H ³ PO ⁴ (85%), 7.8 g of MgSO ⁴ . ⁷ H ² O, 2.6 g of KOH, 9.5 g of K ² SO ⁴ , 0.6 g of CaSO ⁴ . ² H ² O, 0.4 mg of biotin and 4.6 ml of stock solution of oligosales PTM1. The pH was adjusted to 5.85 after sterilization by filtration to the fermenter.

Glycerol discontinuous feed medium contained per liter

550 g of glucose monohydrate, 6.5 g of MgSO ⁴ . ⁷ H ² O, 10 g of KCl, 0.35 g of CaCl ² . ² H ² O, 0.4 mg of biotin and 12 ml of stock solution of oligosales PTM1.

Dissolved oxygen was controlled at OD = 20% with the agitator speed (400-1200 rpm). The aeration rate was 24 lh ^-1 of air, the temperature was controlled at 25 ° C, and the pH setpoint of 5.85 was controlled with the addition of NH ⁴ OH (25%).

To begin the fermentation, 400 ml of discontinuous medium was sterilized by filtration into the fermenter and P. pastoris (from a preculture) was inoculated with an initial optical density (OD 600) of 1. The discontinuous phase of approximately 25 h reached a concentration of Dry biomass of approximately 20 g / l and was followed by a constant feeding phase (for ¹⁰ hours) with glucose medium that led to a dry biomass concentration of approximately 100 g / l. The pH was 5.85 during the discontinuous phase and was maintained at 5.85 throughout the fermentation. Samples were taken during the discontinuous phase and the discontinuous feeding phase. The concentration of HSA was quantified using the human albumin ELISA quantification kit (Bethyl, catalog No. E80-129) as described in example 3c. Table 11 shows the biomass concentration and HSA titres, and table 12 shows the product yield (amount of HSA secreted by biomass, HSA / YDM) at the end of the discontinuous phase (repression conditions for pG1 and pG ⁶ ) and at the end of the discontinuous feed (induction conditions for pG1 and pG ⁶ ). This can verify the induction strategy. The pG1 and pG ⁶ promoters are repressed under an excess of carbon source (in the discontinuous phase of glycerol), and almost show no detectable HSA, in contrast to the clones driven by pGAP. The induction of pG1 and pG ⁶ occurred after the change to conditions with C limited at the beginning of the discontinuous feeding phase. The induction of pG1 (HSA / YDM) was more than 120 times compared to the repressed state, the induction of pG ⁶ was more than 20 compared to the repressed state, and hardly any change was observed for pGAP (3-fold increase in HSA / YDM, compared to the discontinuous phase).

Table 11. Dry mass of yeasts and HSA titers at the end of the discontinuous phase and the discontinuous feeding of 7 fermentations of P. pastoris clones expressing HSA under the control of pGAP, pG1 or pG ⁶

Table 12. Titration of HSA by dry mass of yeasts at the end of the discontinuous phase and at the end of the discontinuous feeding of 7 fermentations of P. pastoris clones expressing HSA under the control of pGAP, pG1 or pG ⁶

Example 4: Comparative promoter activity studies at various glucose concentrations of newly identified promoters in P. pastoris using eGFP as an indicator gene expressed intracellularly To analyze the properties of newly identified promoters at various glucose concentrations, selections were made in shake flasks as follows: preculture was carried out for 24 hours with rich medium containing glycerol as a carbon source (repressed state of the promoters), followed by the main culture with minimum medium and glucose as a carbon source (induced state of the promoters).

a) Strain and expression vector

A wild-type strain of P. pastoris (CBS2612, CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with a vector from the inventors' laboratory called pPUZZLE (Stadlmayr et al., J. Biotechnol., December 2010, 150 (4): 519-529), and the selection of positive transformants was based on zeocin resistance.

b) Amplification and cloning of the newly identified promoters pG1, pG3, pG4 and pG ⁶ in the expression vector pPUZZLE containing eGFP as GDI

Amplification and cloning are performed as described in example 2.

c) Expression of eGFP in P. pastoris for the analysis of promoter activity

Transformation and selection of clones are performed as described in example 2.

For the selection of the expression, a single colony was inoculated in liquid YPG-Zeo medium (per liter: 20 g of peptone, 10 g of yeast extract, 12.6 g of glycerol and 25 mg of zeocin) as preculture. After approximately 24 h, the preculture was used to inoculate the main culture with an OD 600 of 0.01 in 10 ml of YP medium (per liter: 20 g of peptone, 10 g of yeast extract) and glucose as a carbon source . Glucose is used in various concentrations of ^{2 0} to ^{0.00 1} g l-1.

Samples were taken 1-8 after inoculation of the main culture. The expression of eGFP is analyzed by flow cytometry as described in Stadlmayr et al. (J. Biotechnology, December 2010, 150 (4): 519-529), and the selection of positive transformants was based on zeocin resistance. 10,000 cells were analyzed for each sample. Autofluorescence of P. pastoris was measured using wild-type P. pastoris non-transformed cells.

Example 5: Comparative promoter activity studies of newly identified promoters in P. pastoris using porcine carboxypeptidase B (CpB) as an indicator gene expressed extracellularly

To analyze the properties of the newly identified promoters under glucose limitation conditions, selections were made in shake flasks as follows: preculture was carried out for 24 hours with rich medium containing glycerol as a carbon source, which simulates the discontinuous phase of the process (repressed state of the promoters), followed by the main culture with minimal medium and glucose feeding spheres, which simulates the discontinuous feeding phase with glucose limitation of the process (induced state of the promoters). a) Strain and expression vector

A wild-type strain of P. pastoris (CBS2612, CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with a vector from the inventors' laboratory called pPUZZLE (Stadlmayr et al., J. Biotechnol., December 2010, 150 (4): 519-529), and the selection of positive transformants was based on zeocin resistance. For the secretory expression of porcine carboxypeptidase B (CpB) the yeast mating factor alpha was used.

b) Amplification and cloning of the newly identified promoters pG1, pG3, pG4 and pG ⁶ into an expression vector of the inventors' laboratory

Two promoters amplified in Example 2b were cloned into the expression vector pPUZZLE pPM1aZ30_aMF_CpB, producing pPM1aZ30_pG1_aMF_CpB and pPM1aZ30_pG6_aMF_CpB. In addition, the vector pPM1dZ30_pGAP_CPB, which contains the commonly used promoter of glyceraldehyde 3-phosphate dehydrogenase (pGAP), was used as a reference. Promoters were inserted upstream of the start codon of the CpB gene using the ApaI and SbfI restriction sites. It was verified that the promoter sequences were correct by Sanger sequencing.

c) Expression of CpB in P. pastoris under the control of promoters induced by a newly identified glucose limitation

Plasmids were linearized using the SpeI or SapI restriction enzyme before electroporation (using a conventional transformation protocol for P. pastoris) in P. pastoris. Selection of the positive transformants in YPD plates (per liter: 10 g of yeast extract, 20 g of peptone, 20 g of glucose, 20 g of agar-agar) containing 25 pg / ml of zeocin was made. A colony PCR was used to ensure the presence of the transformed plasmid as described in example ² c.

For the selection of CpB expression, a single colony was inoculated in liquid YPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 g glycerol and 25 mg zeocin) as preculture . After approximately 24 h, the preculture was used to inoculate the main culture with an OD600 of 1 in YP medium (per liter: 20 g of peptone, 10 g of yeast extract) and glucose feeding spheres (Kuhner, CH) . Growth conditions with glucose limitation were achieved due to the slow glucose release kinetics of these feeding spheres, which is described by the following equation: (glucose) = 1.63 * t074 [mg / disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. The biomass concentration was determined by measuring DO600 or wet cell weight. The concentration of CpB in the culture supernatant was quantified by an enzymatic assay, based on the conversion of hippyl-L-arginine to hippuric acid by CpB. The reaction kinetics was measured by controlling the absorption at 254 nm at 25 ° C using a Hitachi U-2910 spectrophotometer when the reaction was initiated. Samples and standards are stamped with assay buffer (25 mM Tris, 100 mM HCl, pH 7.65) and activated using activation buffer (0.01 mgl-1 trypsin, 300 mM Tris, ² pM ZnCl ² , pH 7.65). Activation buffer without trypsin was used instead of the sample as a negative control. The reaction begins by adding the substrate solution ( ¹ mM hippyl-L-arginine in assay buffer).

d) Discontinuous feeding culture of P. pastoris strains expressing CpB under the control of the pG ⁶ promoter. The discontinuous feeding fermentation is performed as described in example 3e. Clone pPM1aZ10_pG6_CpB # 4 did not produce detectable CpB in the discontinuous phase and produced more than 210 mg / l CpB at the end of discontinuous feeding.

Example ⁶ : Comparative promoter activity studies of newly identified promoters pG1 and pG ⁶ in multiple-copy clones of P. pastoris using human serum albumin (HSA) as an indicator gene expressed extracellularly

To analyze the properties of the newly identified promoters under glucose limitation conditions, selections were made in shake flasks as follows: preculture was carried out for 24 hours with rich medium containing glycerol as a carbon source, which simulates the discontinuous phase of the process (repressed state of the promoters), followed by the main culture with minimal medium and glucose feeding spheres, which simulates the discontinuous feeding phase with glucose limitation of the process (induced state of the promoters). a) Strain and expression vector

A wild-type strain of P. pastoris (CBS2612, CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with a vector from the inventors' laboratory called pPUZZLE (Stadlmayr et al., J. Biotechnol., December 2010, 150 (4): 519-529), and the selection of positive transformants was based on zeocin resistance. For the secretory expression of human serum albumin (HSA), its native secretion conductor was used.

b) Amplification and cloning of the newly identified promoters pG1 and pG ⁶ in an expression vector of the inventors' laboratory

Two promoters amplified in Example 2b were cloned into the expression vector pPUZZLE pPM1nZ30_HSA, producing pPM1nZ30_pG1_HSA and pPM1 nZ30_pG6_HSA.

Promoters were inserted upstream of the start codon of the HSA gene using the ApaI and SbfI restriction sites. It was verified that the promoter sequences were correct by Sanger sequencing.

c) Expression of HSA in P. pastoris under the control of promoters induced by a newly identified glucose limitation

All plasmids were linearized using the restriction enzyme AscI before electroporation (using a conventional transformation protocol for P. pastoris) in P. pastoris. Selection of the positive transformants in YPD plates (per liter: 10 g of yeast extract, 20 g of peptone, 20 g of glucose, 20 g of agar-agar) containing 25 pg / ml of zeocin was made. The gene copy number amplification was performed as described in Marx et al. (FEMS Yeast Res., December 2009, 9 (8): 1260-1270). A colony PCR was used to ensure the presence of the transformed plasmid as described in example ² c.

For the selection of HSA expression, a single colony was inoculated in liquid YPG-Zeo medium (per liter: 20 g of peptone, 10 g of yeast extract, 12.6 g of glycerol and 25 mg of zeocin) as preculture . After approximately 24 h, the preculture was used to inoculate the main culture with an OD600 of 1 in YP medium (per liter: 20 g of peptone, 10 g of yeast extract) and glucose feeding spheres (Kuhner, CH) . Growth conditions with glucose limitation were achieved due to the slow glucose release kinetics of these feeding spheres, which is described by the following equation: (glucose) = 1.63 * t0.74 [mg / disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. The biomass concentration was determined by measuring DO600 or wet cell weight. The concentration of HSA in the culture supernatant was quantified using the human albumin ELISA quantification kit (catalog No. E80-129, Bethyl Laboratories, TX, USA) according to the supplier's instruction manual. The HSA standard was used with an initial concentration of 400 ng ml-1. The samples were diluted accordingly in sample diluent (50 mM Tris-HCl, 140 mM NaCl, 1% BSA (in w / v), 0.05% Tween20 (in v / v), pH 8.0) . Table 13 shows the HSA titers of the selection of several multi-copy clones and single copy clones of Example 3c.

Table 13. Results of the selection of clones of multiple copies of P. pastoris expressing HSA under the control of pGAP, pG1 and pG ⁶

d) Determination of the number of copies of the HSA gene

Isolation of genomic DNA and measurement with qPCR were performed as in example 2d, using the primers shown in table 9. The results are shown in table 14.

Table 14. Results of the selection and number of gene copies of clones of multiple copies of P. pastoris expressing HSA under the control of pGAP, pG1 and pG ⁶

e) Discontinuous feeding culture of multiple copies of P. pastoris expressing HSA under the control of the pG1 and pG ⁶ promoter

Discontinuous feed fermentations are performed as described in example 3e. Clones pPM1nZ30_pG1_HSA # 4 * 1000 and pPM1nZ30_pG6_HSA # C6 reached 1060 and 728 mg / l of HSA at the end of discontinuous feeding, respectively.

Example 7: Comparative promoter activity studies of the newly identified promoter in P. pastoris PG1 using an antibody fragment (Fab) as an indicator gene expressed extracellularly

To analyze the properties of the newly identified promoters under glucose limitation conditions, selections were made in shake flasks as follows: preculture was carried out for 24 hours with rich medium containing glycerol as a carbon source, which simulates the discontinuous phase of the process (repressed state of the promoters), followed by the main culture with minimal medium and glucose feeding spheres, which simulates the discontinuous feeding phase with glucose limitation of the process (induced state of the promoters).

a) Strain and expression vector

A wild-type strain of P. pastoris (CBS2612, CBS-KNAW Fungal Biodiversity Center, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) was used as the host strain. The strain was transformed with a vector from the inventors' laboratory called pPUZZLE (Stadlmayr et al., J. Biotechnol., December 2010, 150 (4): 519-529), and the selection of positive transformants was based on zeocin resistance. For the secretory expression of an antibody Fab fragment, the yeast mating factor alpha conductor was used.

b) Amplification and cloning of the newly identified promoter pG1 into an expression vector of the inventors' laboratory

The amplified pG1 promoter in example 2b was cloned into the pPUZZLE expression vector containing Fab as GDI, as described in example 5b. The promoter is inserted upstream of the Fab gene start codon using the ApaI and SbfI restriction sites. It was verified that the promoter sequence was correct by Sanger sequencing.

c) Fab expression in P. pastoris under the control of the promoter induced by a newly identified glucose limitation pG1

Plasmids were linearized using the SpeI or SapI restriction enzyme before electroporation (using a conventional transformation protocol for P. pastoris) in P. pastoris. The selection of the positive transformants in YPD plates was made (per liter: 10 g of yeast extract, 20 g of peptone, 20 g of glucose, 20 g agar agar) containing 25 µg / ml zeocin. A colony PCR was used to ensure the presence of the transformed plasmid as described in example ² c.

For the selection of Fab expression, a single colony was inoculated in liquid YPG-Zeo medium (per liter: 20 g of peptone, 10 g of yeast extract, 12.6 g of glycerol and 25 mg of zeocin) as preculture . After approximately 24 h, the preculture was used to inoculate the main culture with an OD600 of 1 in YP medium (per liter: 20 g of peptone, 10 g of yeast extract) and glucose feeding spheres (Kuhner, CH) . Growth conditions with glucose limitation were achieved due to the slow glucose release kinetics of these feeding spheres, which is described by the following equation: (glucose) = 1.63 * t074 [mg / disc]. Samples were taken at the end of preculture and 24 and 48 hours after inoculation of the main culture. The biomass concentration was determined by measuring DO600 or wet cell weight. Fab expression levels are quantified by ELISA using human kappa (bound and free) light-chain alkaline phosphatase antibodies produced in goats. Table 15 shows the Fab titers of the selection of several clones that express Fab under the control of pGAP and pG1.

Table 15. Results of the selection of P. pastoris clones expressing Fab under the control of pGAP and pG1

d) Discontinuous feed culture of P. pastoris strains expressing Fab under the control of the pG1 promoter Discontinuous feed fermentations are performed as described in example 3e, but discontinuous glucose feeding is employed as described in the example 2e. The clones pPM1aZ30_pG1_Fab # C4 and pPM1aZ30_pG1_Fab # C7 reached 165 and 131 mg / l of Fab at the end of the discontinuous feed, respectively. Example ⁸ : Exponential discontinuous feed fermentation to control the specific growth rate at maximum volumetric productivity of newly identified promoters

Chemostat cultures of P. pastoris clones that express an indicator gene under the control of newly identified promoters are used to determine specific and volumetric productivity at different growth rates. As described in Maurer et al. (Microb. Cell Fact., December 2006, 11, 5:37), exponential discontinuous feed fermentations can be used to grow a clone of P. pastoris at a certain growth rate to improve production during the entire feeding phase. This can optimize space-time performance. Optimized feeding was applied, and the space-time performance of the discontinuous feeding phase improved by more than 35%.

Example 9: Determination of transcription / promoter potency: study of comparative promoter activity to identify promoter regulation with different glucose concentrations using eGFP as an indicator gene expressed intracellularly

The regulatory properties of a promoter are analyzed by the selection of clones expressing eGFP under the control of said promoter. For this, a single colony was inoculated in liquid YPG-Zeo medium (per liter: 20 g of peptone, 10 g of yeast extract, 12.6 g of glycerol and 25 mg of zeocin) as a preculture. After approximately 24 h, the preculture was used to inoculate the main culture with an OD 600 of 0.01 in 10 ml of YP medium (per liter: 20 g of peptone, 10 g of yeast extract) and glucose in different concentrations ( 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.020, 0.010, 0.005 and 0.002 g / l). A sample is taken after ⁶ hours and analyzed by flow cytometry as described in Stadlmayr et al. (J. Biotechnol., December 2010, 150 (4): 519-529). Fluorescence related to cell size (direct dispersion up to 1.5 power) is calculated for each cell / data point, and its geometric average is used to compare eGFP expression levels produced at different glucose concentrations. A clone expressing eGFP under the control of pGAP is used as a reference (P. pastoris pGAP, in this SEQ ID NO: 25). Autofluorescence of P. pastoris was measured using wild-type P. pastoris untransformed cells and subtracted from the signal. Table 16 shows the complete induction of the pG1 promoter at approximately 40 mg / l glucose or less and its transcription potency, compared to the native pGAP promoter.

Table 16. Expression of relative eGFP (in relation to pGAP) of a P. pastoris clone expressing eGFP under the control of the pG1 promoter at different glucose concentrations (20-0.002 g / l)

Similar studies were conducted to compare the relative transcription potency of the derepressed promoters pG1, pG3, pG4, pG ⁶ and pG7. A clone expressing eGFP under the control of one of the promoters was cultured in YPG (glycerol 20 g / l, repressed state) and then inoculated in YP medium containing different amounts of glucose (from 20 to 0.002 g / l (D20 , D10, ... D0.002), induced state) and was cultured for 5-6 hours. The cells were analyzed by flow cytometry and the results were evaluated as follows: fluorescence was related to cell size (direct dispersion up to 1.5 power) for each cell, and its geometric average was used to compare the different concentrations of glucose The conclusive result of these selections is shown in Figure 14, a diagram showing logarithmic glucose concentrations versus relative fluorescence, which provides a good illustration of the induction behavior of promoters adjustable by glucose limitation. Figure 14 shows the complete induction of the pG1 promoter at about 40 mg / l glucose or less, and of the pG3, pG4 and pG ⁶ promoters at about 4 g / less, and their transcription potency, compared to the pGAP promoter. native. The induction behavior of pG7 is similar to pG1 (data not shown). Based on the previous results with pG ⁸ , it was assumed that their induction behavior is in the range of the other promoters.

Example 10: Comparison of the prior art promoters pICL1 and pMLS1 with pG1 in the glucose concentration selection assay

The study of comparative promoter activity is performed according to example 9, using the pICL1 and pMLS1 promoters as a reference for comparison with the pG1 promoter described herein.

It was found that the activity of both promoters pICL1 and pMLS1 was very weak, without significant differences at a high glucose concentration (D20: 20 g / l, repression) or low (D0.04: 0.04 g / l, induction = derepression). In any case, the activity is much less than the activity of the repressed pG1 promoter in the same scenario. The results are shown in table 17 as a promoter activity in percentage in relation to the pGAP promoter.

Table 17. Relative fluorescence of strains expressing eGFP under the control of pG1, pICL1 and pMLS1, respectively, grown in medium containing glucose 20 g / l (D20) or 0.04 g / l (D0.04)

Example 11: Comparison of pG1 variants

Shorter variants of the pG1 promoter were cloned as described in example 2a and were selected similarly to that described in example 2c, but in a smaller scale transformed configuration using 24-well plates (Whatman, UK, article n. 7701-5110) and quarters of feeding spheres (12 mm, Kuhner, CH) instead of complete spheres. Clones that are expressed under the control of pG1 and pGAP were used as controls. Table 18 lists the direct primers and the lengths of pG1 and their variants. There was no significant difference in the relative fluorescence of cells expressing eGFP under the control of pG1 and the variants of pG1 a-f.

Table 18. pG1 and its variants: direct primers and 5 'and 3' end positions in the sequence of pG1 (SEQ ID NO: 1). Sequences of pG1a-f, see Figure 15 (SEQ ID NO: 41-46).

Claims (19)

1. - A method for producing a protein of interest (PDI) by culturing a line of recombinant eukaryotic cells comprising an expression construct comprising an adjustable promoter and a nucleic acid molecule encoding a PDI under the transcriptional control of said promoter, comprising the steps of: a) culturing the cell line with a basal carbon source that represses the promoter, in which the basal carbon source is a suitable carbon source for cell growth, b) culturing the cell line without a supplemental carbon source, or with a limited amount of it, which depresses the promoter to induce the production of the PDI at a transcription rate of at least 20%, compared to the native pGAP promoter of the cell, and c) produce and recover the POI; in which the promoter is selected from the group consisting of: (i) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG ⁶ (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG ⁸ (SEQ ID NO: ⁶ ); and (ii) a sequence comprising a nucleotide sequence of at least ^{2 0 0} bp and having at least 80% sequence identity with any one of pG1 (SEQ ID NO: 1), pG3 (SEQ ID No: 2) , pG4 (SEQ ID NO: 4), pG ⁶ (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG ⁸ ( ^{s Eq} ID NO: ⁶ ); or with a part of any of the above with a length of at least ^{2 0 0} bp. 2. - The method according to claim 1, wherein the basal carbon source is selected from the group consisting of glucose, glycerol, ethanol, one of its mixtures and a complex nutrient material. 3. - The method according to claim 1 or 2, wherein the supplementary carbon source is a hexose, such as glucose, fructose, galactose or mannose, a disaccharide, such as sucrose, an alcohol, such as glycerol or ethanol, or one of their mixtures. 4. - The method according to any of claims 1 to 3, wherein step b) employs a feeding medium that does not provide the supplementary carbon source or provides it in a limited amount, preferably at ^0-1 g / l in the culture medium. 5. - The method according to any of claims 1 to 4, wherein the limited amount of additional carbon source limited growth to maintain the specific growth rate within the range of ^{0, 0 2} h ^{-1 to 0,} 2 h-1, preferably from 0.02 h ^-1 to 0.15 h-1. ⁶ . - The method according to claims 1 to 5, wherein the promoter is capable of controlling the transcription of a wild-type eukaryotic cell gene, and said gene is selected from the group consisting of G1 (SEQ ID NO: 7) , G3 (SEQ ID NO: ⁸ ), G4 (SEQ ID NO: 9), G ⁶ (SEQ ID NO: 10), G7 (SEQ ID NO: 11) or G ⁸ (SEQ ID NO: 12), or one of its functionally active variants comprising a nucleotide sequence of at least ^{2 0 0} bp and a sequence identity of at least 80% with any of the promoter sequences or with one of its parts with a length of at least ^{2 0 0} pb. 7. - The method according to claim ⁶ , wherein the promoter is pG1 (SEQ ID NO: 1) or one of its functionally active variants. ⁸ . - The method according to claims ⁶ or 7, wherein said functionally active variants are selected from the group consisting of homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, and analogues derived from species other than Pichia pastoris. 9. - The method according to any of claims 1 to 5, wherein the promoter is selected from the group consisting of pG1 (SEQ ID NO: 1), pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46). 10. - The method according to any of claims 1 to 9, wherein the cell line is selected from the group consisting of mammalian, insect, yeast, filamentous and plant cell lines, preferably a yeast. 11. - The method according to any of claims 1 to 10, wherein the PDI is a heterologous protein, preferably selected from therapeutic proteins, including antibodies or their fragments, enzymes and peptides, protein antibiotics, toxin fusion proteins , carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccines and proteins or vaccine-like particles, process enzymes, growth factors, hormones and cytokines, or a metabolite of a PDI. 12. - The method according to claim 1, wherein the promoter is a functionally active variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG ⁶ (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG ⁸ (SEQ ID NO: ⁶ ), which is selected from the group consisting of homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, and analogues derived from species other than Pichia pastoris. 13. - The method according to claim 12, wherein the promoter is selected from the group consisting of pG1 (SEQ ID NO: 1), pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO: 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46). 14. - An isolated promoter nucleic acid that is operably linked to a nucleotide sequence encoding a PDI, and said promoter nucleic acid is not natively associated with the nucleotide sequence encoding the PDI, in which the nucleic acid of promoter comprises a nucleic acid sequence selected from the group consisting of: a) pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG ⁶ (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG ⁸ (SEQ ID NO: ⁶ ); and b) a sequence comprising a nucleotide sequence of at least ^{2 0 0} bp and having at least 80% sequence identity with any one of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4), pG ⁶ (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG ⁸ (s Eq ID NO: ⁶ ); or with a part of any of the above with a length of at least ^{2 0 0} bp; wherein said nucleic acid comprises a functionally active promoter, which is a promoter adjustable by a carbon source capable of expressing a PDI in a recombinant eukaryotic cell at a transcription rate of at least 20%, compared to the native pGAP promoter of the cell. 15. - A promoter nucleic acid according to claim 14, wherein the promoter is a functionally active variant of pG1 (SEQ ID NO: 1), pG3 (SEQ ID NO: 2), pG4 (SEQ ID NO: 4) , pG ⁶ (SEQ ID NO: 3), pG7 (SEQ ID NO: 5), or pG ⁸ (SEQ ID NO: ⁶ ), which is selected from the group consisting of homologs that can be obtained by modifying the nucleotide sequence of origin by insertion, deletion or substitution of one or more nucleotides within the sequence or at either or both of the distal ends of the sequence, and analogues derived from species other than Pichia pastoris. 16. - A promoter nucleic acid according to claim 14 or 15, wherein the promoter is selected from the group consisting of pG1 (SEQ ID NO: 1), pG1a (SEQ ID NO: 41), pG1b (SEQ ID NO : 42), pG1c (SEQ ID NO: 43), pG1d (SEQ ID NO: 44), pG1e (SEQ ID NO: 45) and pG1f (SEQ ID NO: 46). 17. - A vector comprising the promoter nucleic acid according to claims 14 to 16, wherein the nucleotide sequence encoding a PDI is under the transcriptional control of said promoter, and said promoter nucleic acid is not natively associated with the nucleotide sequence encoding the PDI. 18. - A recombinant eukaryotic cell comprising the vector of claim 17. 19. - A method for producing the protein of interest (PDI) using the recombinant eukaryotic cell of claim 18.