RU2840101C1 - Nucleic acids, vectors, host cells and methods of producing beta-fructofuranosidase from aspergillus niger - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a modified polypeptide for producing fructooligosaccharides, in which the polypeptide is β-fructofuranosidase Aspergillus niger containing the amino acid sequence SEQ ID NO: 1, fused with a signal peptide selected from a group, including S. cerevisiae alpha-factor (FAK), S. cerevisiae alpha-factor (FAKS), S. cerevisiae alpha-factor_T (AT), Aspergillus niger alpha-amylase (AA), Aspergillus awamori glucoamylase (GA), Inulinase Kluyveromyces maxianus (IN), Invertase S. cerevisiae (IV), killer protein S. cerevisiae (KP), lysozyme Gallus gallus (LZ) and serum albumin Homo sapiens (SA), wherein the signal peptide comprises a portion of four LEKR amino acids. Also disclosed is a modified nucleic acid coding said polypeptide, an expression vector, host cell and use of said polypeptide in producing fructooligosaccharides.

EFFECT: invention provides high output of recombinant β-fructofuranosidase Aspergillus niger.

11 cl, 9 dwg, 13 tbl, 8 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to the field of genetic engineering. More specifically, the aim of the invention is to improve the production of a new recombinant β-fructofuranosidase encoded by the gene fopAAspergillus nigerin the form of a secreted protein.

LEVEL OF TECHNOLOGY

Fructose oligomers, also known as fructooligosaccharides (FOS), are a series of homologous oligosaccharides. Fructooligosaccharides are usually represented by the formula GFn and mainly consist of 1-kestose (GF2), nystose (GF3), and β-fructofuranosyl nystose (GF4), in which two, three, and four fructosyl units are linked at the β-2,1 position of glucose.

Fructooligosaccharides (FOS) are characterized by many beneficial properties such as low sweetness and also useful as a prebiotic. Due to the low sweetness (about one-two-thirds of sucrose) and low caloric content (about 0-3 kcal/g), fructooligosaccharides can be used in various types of food products as a sugar substitute. Furthermore, fructooligosaccharides as prebiotics have been reported to be protective against colon cancer, improving various parameters of the immune system, improving mineral absorption, beneficially affecting serum lipid and cholesterol concentrations, and exerting glycemic control to combat obesity and diabetes (Dominguez, Ana Luisa et al. “An overview of the recent developments on fructooligosaccharide production and applications” Food and bioprocess technology 7.2 (2014): 324-337).

However, fructooligosaccharides are found only in trace amounts as natural components in fruits, vegetables, and honey. Due to such low concentrations, it is virtually impossible to extract fructooligosaccharides from food.

Attempts have been made to produce fructooligosaccharides by enzymatic synthesis from sucrose by microbial enzymes possessing transfructosylating activity. However, the major limitations in previous attempts have been lower catalytic efficiency, feedback inhibition of the enzyme by glucose leading to decreased FOS yield, and the need for longer periods of time for sucrose conversion by enzymes expressed in a recombinant host system. Furthermore, industrial production of microbial enzymes exhibiting transfructosylating activity is challenging due to additional limitations associated with large-scale enzyme expression, enzyme stability, fermentation, and purification processes.

Industrial production of fructooligosaccharides requires identification and mass production of efficient enzymes. Due to the above limitations, obtaining microbial enzymes with efficient transfructosylating activity is an expensive process, which in turn increases the cost of fructooligosaccharide production.

Thus, there is a long-standing need to identify and provide efficient, low-cost, industrial-scale means for producing microbial enzymes with superior transfructosylating activity, which in turn reduces the cost of fructooligosaccharide production.

DISCLOSURE OF THE ESSENCE OF THE INVENTION

Technical task

The technical problem to be solved in this invention is to identify and increase the yield of a new β-fructofuranosidase (UniProtKB: Q96VC5_ASPNG) of Aspergillus niger .

Solution to the problem

The problem was solved by overexpressing a novel Aspergillus niger β-fructofuranosidase by engineering nucleic acid sequences, protein sequences, promoters, recombinant vectors, host cells and secretory signal peptides to achieve high yield of the novel recombinant β-fructofuranosidase.

Furthermore, the fermentation strategy was modified to obtain high yield of approximately 2-5 g/L of recombinant β-fructofuranosidase.

Overview of the invention

The present invention relates to nucleic acids, protein sequences, vectors and host cells for recombinant expression of novel β-fructofuranosidase. The present invention also relates to precursor peptides containing signal peptides fused to novel β-fructofuranosidase enzymes that provide a higher yield of the effective enzyme as a secretory protein.

The invention also relates to a method for expressing a new recombinant β-fructofuranosidase as a secreted protein. It has been established that the concentration of β-fructofuranosidase is about 2-5 g/l. The enzyme has a purity of almost 85% after filtration, which eliminates the need for expensive chromatographic procedures.

BRIEF DESCRIPTION OF DRAWINGS

The features of the present invention will become fully apparent from the following description, taken in conjunction with the accompanying figures. Since the figures depict only a few embodiments of the invention and should not be considered as limiting its scope, the invention will be described further using the corresponding figures.

Figure 1 shows the sequence alignment of the native fopA gene and the modified fopA gene encoding β-fructofuranosidase.

Figure 2 shows the scheme for constructing the pPICZαA vector.

Figure 3 shows the results of restriction digestion analysis performed on the recombinant plasmid pPICZαA-fopA.

Figure 4 shows the results of colony PCR screening performed on Pichia integrants.

Figure 5 shows the expression of β-fructofuranosidase upon induction from recombinant Pichia pastoris host cells.

Figure 6(a) shows SDS-PAGE analysis of samples collected at different time points during fermentation of Pichia pastoris strain KM71H expressing recombinant β-fructofuranosidase enzyme. Figure 6(b) shows SDS-PAGE analysis of recombinant β-fructofuranosidase enzyme after purification.

Figure 7 shows a standard curve of glucose used to assess the activity of the enzyme β-fructofuranosidase.

Figure 8 shows the production of fructooligosaccharides (FOS) from sucrose and recombinant β-fructofuranosidase enzyme.

Figure 9 shows the HPLC chromatogram of FOS samples.

SEQUENCE SUMMARY AND SEQUENCE LIST

SEQ ID NO: 1 - Amino acid sequence of novel β-fructofuranosidase (654 amino acids).

SEQ ID NO:2 - Modified nucleic acid sequence of the gene encoding a novel β-fructofuranosidase (1965 base pairs).

A four-amino acid region (LEKR) was added to all secretory signal peptide sequences to allow efficient processing of the Kex2 preprotein.

SEQ ID NO:23 - Native nucleic acid sequence of the fopA gene (1965 base pairs), encoding secreted β-fructofuranosidase.

DEFINITIONS

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods pertain. Although any vectors, host cells, methods, and compositions similar or equivalent to those described herein may also be used in the practice or testing of the vectors, host cells, methods, and compositions, representative illustrations are provided herein.

Where a range of values is provided, it is intended that each intermediate value between the upper and lower limits of that range and any other stated or intermediate value in that stated range is contemplated within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and also be contemplated within the methods and compositions, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both limits, ranges excluding one or both of those included limits are also included in the methods and compositions.

It should be understood that certain features of the methods, which for clarity are described in the context of separate embodiments of the invention, may also be provided in combination in a single embodiment of the invention. Conversely, various features of the methods and compositions, which for brevity are described in the context of a single embodiment of the invention, may also be provided separately or in any suitable subcombination. It should be noted that, as used herein and in the appended claims, the singular forms refer to the plural unless the context clearly dictates otherwise. It is further noted that the claims may be drafted so as to exclude any optional element. As such, this statement shall serve as a basis for the use of such exclusive terminology as "solely", "solely", etc. in connection with the recitation of elements of the claims or the use of a "negative" limitation.

As will be apparent to those skilled in the art after familiarization with the present invention, each of the individual embodiments of the invention described and illustrated herein has individual components and features that can be easily separated from or combined with features of any of the other embodiments of the invention without departing from the scope or spirit of the present methods. Any method described can be performed in the order of the events described or in any other order that is logically possible.

The term "host cell(s)" includes an individual cell or cell culture that can be or has been a recipient for the object of the expression constructs. Host cells include the progeny of a single host cell. Host cells for the purposes of the present invention refer to any strain of Pichia pastoris that can be suitably used for the purposes of the invention. Examples of strains that can be used for the purposes of the present invention include wild-type, mut+, mux S, mux- strains of Pichia, such as KM71H, KM71, SMD1168H, SMD1168, GS115, X33.

The term “recombinant strain” or “recombinant host cell(s)” refers to host cell(s) that have been transfected or transformed with the expression constructs or vectors of the present invention.

The term "expression vector" refers to any vector, plasmid, or vehicle designed to provide expression of an introduced nucleic acid sequence upon transformation into a host organism.

The term "promoter" refers to DNA sequences that determine where transcription of a gene begins. Promoter sequences are usually located immediately upstream or 5' of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to the promoter sequence and initiate transcription. Promoters can be either constitutive or inducible promoters. Constitutive promoters are those that allow continuous transcription of their associated genes because their expression is usually not driven by environmental and developmental factors. Constitutive promoters are very useful tools in genetic engineering because constitutive promoters stimulate gene expression under inducer-free conditions and often exhibit better performance than the commonly used inducible promoters. Inducible promoters are those that are induced by the presence or absence of biotic or abiotic and chemical or physical factors. Inducible promoters are a very powerful tool in genetic engineering because the expression of genes functionally linked to them can be turned on or off at specific stages of development or growth of an organism or in a specific tissue or cell type.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is regulated by the other. For example, a promoter is operably linked to a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

The term "transcription" refers to the process of creating an RNA copy of a gene sequence. This copy, called a messenger RNA (mRNA) molecule, leaves the cell's nucleus and enters the cytoplasm, where it directs the synthesis of the protein it encodes.

The term translation refers to the process of translating the sequence of a messenger RNA (mRNA) molecule into a sequence of amino acids during protein synthesis. The genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence it encodes. In the cytoplasm of a cell, a ribosome reads the mRNA sequence in groups of three bases to assemble a protein.

The term "expression" refers to the biological production of a product encoded by a coding sequence. In most cases, a DNA sequence, including the coding sequence, is transcribed to form messenger RNA (mRNA). The messenger RNA is then translated to form a polypeptide product that has the appropriate biological activity. In addition, the expression process may include additional processing steps of the RNA transcription product, such as splicing to remove introns and/or post-translational processing of the polypeptide product.

The term "modified nucleic acid" as used herein is used to denote a nucleic acid encoding β-fructofuranosidase fused to a signal peptide. In embodiments of the invention, the modified nucleic acid is represented by the sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or a functionally equivalent variant thereof. A functional variant includes any nucleic acid having substantial sequence identity or similarity to SEQ ID NO: 13-22 and which retains the same biological activity.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to two or more amino acid residues joined together by peptide bonds or modified peptide bonds. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical mimic of a corresponding natural amino acid, as well as to naturally occurring amino acid polymers, including those polymers containing modified residues, and to a non-natural amino acid polymer. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides, or oligomers, and long chains, commonly referred to as proteins. Polypeptides may contain amino acids in addition to the 20 amino acids encoded by a gene. Similarly, "protein" refers to at least two covalently attached amino acids, which include proteins, polypeptides, oligopeptides, and peptides. A protein may be composed of naturally occurring amino acids and peptide bonds or synthetic peptide-mimetic structures. Thus, "amino acid" or "peptide residue" as used herein refers to both naturally occurring and synthetic amino acids.

"Amino acid" includes imino acid residues such as proline and hydroxyproline. The side chains can be in either the (R) or (S) configuration.

The term "signal peptide" or "signal peptide sequence" is defined herein as a peptide sequence, typically present at the N-terminal end of newly synthesized secretory or membrane polypeptides, that directs the polypeptide across or into a cell membrane (the plasma membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes). It is typically subsequently removed. In particular, said signal peptide may be capable of directing the polypeptide into the secretory pathway of the cell.

The term "precursor peptide" as used herein refers to a peptide containing a signal peptide (also known as leader sequences) operably linked to Aspergillus niger β-fructofuranosidase. The signal peptides are cleaved during post-translational modifications within the host Pichia cells, and the mature β-fructofuranosidase (SEQ ID NO: 1) is released into the medium.

The term "variant" as used herein in relation to peptides/precursor proteins refers to peptides with amino acid substitutions, additions, deletions or changes that do not substantially reduce the activity of the signal peptide or enzyme. Variants include both structural and functional variants. The term "variant" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

Amino acid substitution tables providing functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that are considered variants of each other:

IMPLEMENTATION OF THE INVENTION

The present invention discloses nucleic acids, vectors and recombinant host cells for the efficient production of biologically active and soluble recombinant Aspergillus niger β-fructofuranosidase as a secreted protein. Furthermore, the invention provides a method for the industrial production of recombinant β-fructofuranosidase.

The invention provides a multidimensional approach for achieving a high yield of a new recombinant β-fructofuranosidase in a heterologous host. The native β-fructofuranosidase gene was modified for expression in Pichia pastoris. The modified gene was then fused to one or more signal peptides.

In one embodiment of the invention, a modified nucleic acid encoding a novel Aspergillus niger β-fructofuranosidase is represented by the sequence SEQ ID NO: 2.

In another embodiment of the invention, the modified nucleic acid is fused to one or more signal peptides.

In another embodiment of the invention, the signal peptide is selected from S. cerevisiae Alpha Factor (FAK), S. cerevisiae Complete Alpha Factor (FAKS), S. cerevisiae T-Alpha Factor (AT), Aspergillus niger Alpha Amylase (AA), Aspergillus awamori Glucoamylase (GA), Kluyveromyces maxianus Inulinase (IN), S. cerevisiae Invertase (IV), S. cerevisiae Killer Protein (KP), Gallus gallus Lysozyme (LZ), Homo sapiens Serum Albumin (SA)

In another embodiment of the invention, the signal peptide is shown in Table 5 below.

In another embodiment of the invention, the signal peptide is selected from the list of modified signal peptides as described in Table 1.

In another embodiment of the invention, the nucleic acid fused to one or more modified signal peptides is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and variants thereof.

In another embodiment, the modified nucleic acid is cloned into an expression vector.

In another embodiment of the invention, the expression vector is configured for secretory or intracellular expression of recombinant β-fructofuranosidase from Aspergillus niger .

In another embodiment of the invention, the expression vector is selected from the group consisting of pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, pGAPZαC, pPIC3, pPIC3.5, pPIC3.5K, PAOS15, pPIC9, pPIC9K, IL-D2 and pHIL-S1.

Expression of the modified β-fructofuranosidase (fopA) gene fused to a signal peptide is preferably driven by a constitutive or inducible promoter.

In another embodiment of the invention, the nucleic acid to be expressed is operably linked to a promoter.

In another embodiment, the constitutive or inducible promoter is selected from the group indicated in Table 6.

In another embodiment of the invention, the promoter is the AOX1 promoter, which is inducible by methanol and repressed by glucose.

In an embodiment of the invention, an expression vector containing a modified gene of interest (a β-fructofuranosidase gene fused to a signal peptide encoding a nucleic acid) is transformed into a suitable host.

In another embodiment of the invention, an expression vector containing a gene of interest is transformed into yeast cells.

In another embodiment, the yeast cell is Pichia pastoris.

In another embodiment of the invention, the Pichia Pastoris host cell is a mut+, mut S, or mut- strain. Mut+ is a methanol utilization plus phenotype.

In another embodiment of the invention, the Yichia Pastoris host cell strain is selected from the group consisting of KM71H, KM71, SMD1168H, SMD1168, GS115, X33.

In another embodiment, the invention discloses β-fructofuranosidase precursor peptides in which Aspergillus niger β-fructofuranosidase is fused to one or more signal peptides.

In another embodiment of the invention, the Aspergillus niger β-fructo furanosidase has an amino acid sequence as set forth in SEQ ID NO: 1 and functional variants thereof. A functional variant includes any protein sequence having substantial or significant sequence identity or similarity to SEQ ID NO: 1 and or having substantial or significant structural identity or similarity to SEQ ID NO: 1 and which retains the same biological activity.

In another embodiment of the invention, the signal peptide is selected from the group consisting of S. cerevisiae Alpha Factor Complete (FAK) as set forth in SEQ ID NO: 3, S. cerevisiae Alpha Factor Complete (FAKS) as set forth in SEQ ID NO: 4, S. cerevisiae Alpha Factor T (AT) as set forth in SEQ ID NO: 5, Aspergillus niger Alpha Amylase (AA) as set forth in SEQ ID NO: 6, Aspergillus awamori Glucoamylase (GA) as set forth in SEQ ID NO: 7, Kluyveromyces maxianus Inulinase (IN) as set forth in SEQ ID NO: 8, S. cerevisiae Invertase (IV) as set forth in SEQ ID NO: 9, S. cerevisiae Killer Protein (KP) as set forth in SEQ ID NO: 10, Gallus gallus Lysozyme (LZ) as set forth in SEQ ID NO: 11, Homo sapiens serum albumin (SA) as set forth in SEQ ID NO: 12, and variants thereof.

In an embodiment of the invention, a method for producing recombinant β-fructofuranosidase from Aspergillus niger is provided.

Aspects of the present invention relate to the fermentation of recombinant Pichia pastoris cells containing a modified recombinant β-fructofuranosidase (fopA) gene. After fermentation is complete, the fermentation broth is centrifuged and filtered using microfiltration, and the recombinant enzyme is separated. The isolated recombinant enzyme is concentrated using tangential flow ultrafiltration or evaporation, and, as a result, a concentrated enzyme is obtained.

In one embodiment of the invention, a method for expressing Aspergillus niger β-fructofuranosidase at high levels comprises the steps of:

a. culturing recombinant host cells in a suitable fermentation medium to produce recombinant β-fructofuranosidase enzyme secreted into the fermentation broth;

b. collecting the supernatant from the fermentation broth, wherein the supernatant contains recombinant β-fructofuranosidase; and

c. purification of recombinant β-fructofuranosidase.

In another embodiment of the invention, the fermentation medium is a basic salt medium as described in Table 7.

In another embodiment of the invention, the supernatant from the fermentation broth is collected by centrifugation.

In one embodiment of the invention, the percentage of inoculum or starter for initiating the culture in the fermenter is in the range of 2.0% to 15.0% (v/v).

In another embodiment of the invention, the pH of the fermentation medium is maintained in the range of 4.0 to 7.5, since the secreted enzyme undergoes proper folding and is biologically active in this pH range.

In another embodiment of the invention, the temperature of the fermentation process is in the range of 15°C to 40°C.

In another embodiment of the invention, the fermentation process time is in the range of 50-150 hours.

In another embodiment of the invention, the fermentation broth is centrifuged at a speed in the range of 2000 xg to 15000 xg using continuous online centrifugation.

The supernatant obtained after centrifugation is microfiltered and purified to isolate biologically active recombinant β-fructofuranosidase.

In one embodiment of the invention, the supernatant obtained after centrifugation is concentrated using an ultrafiltration system based on tangential flow filtration.

The cutoff size of membranes used in tangential flow filtration (TFF) systems, which can be used to remove impurities and concentrate the collected culture supernatant, can range from 5 to 100 kDa.

In another embodiment of the invention, the process does not require centrifugation due to the high yield and purity of the secreted enzyme.

It has been found that the concentration of β-fructofuranosidase obtained according to this invention is in the range of 2-5 g/l, and the purity is about 85%.

EXAMPLES

The following examples in particular describe the manner in which the invention is to be carried out. However, the embodiments of the invention disclosed herein in no way limit the scope of the invention.

Example 1: Modified nucleic acids for expression of recombinant Aspergillus niger β-fructofuranosidase in Pichia pastoris

The cDNA of native β-fructofuranosidase (fopA) of Aspergillus niger is represented by the sequence SEQ ID NO: 23, and the amino acid sequence of the new β-fructofuranosidase is represented by the sequence SEQ ID NO: 1.

Native cDNA was modified for maximal expression in Pichia pastoris. The modified nucleic acid is represented by the sequence SEQ ID NO: 2. The differences between the native and modified sequence are shown in Figure 1.

The expression cassette encoding β-fructofuranosidase was modified to maximize expression in Pichia pastoris. The modified open reading frame contains a modified nucleotide sequence (SEQ ID NO: 2) encoding β-fructofuranosidase fused to a signal peptide. The nucleic acids were designed such that the encoded signal peptides contain an additional four-amino acid region (LEKR) for efficient processing of the Kex2 precursor peptide.

Preferred codons for expression in Pichia pastoris were used instead of rare codons.

The nucleotide sequence of the modified open reading frames encoding β-fructofuranosidase fused to modified signal peptides is shown below:

- S. cerevisiae alpha factor (FAK) is represented by the sequence SEQ ID NO: 13

- Alpha-factor complete S. cerevisiae (FAKS), represented by the sequence SEQ ID NO: 14

- Alpha factor T S. cerevisiae (AT) is represented by the sequence SEQ ID NO: 15

- Aspergillus niger alpha-amylase (AA) is represented by the sequence SEQ ID NO: 16

- Aspergillus awamori glucoamylase (GA) is represented by the sequence SEQ ID NO: 17

- Kluyveromyces maxianus inulinase (IN) is represented by the sequence SEQ ID NO: 18

- S. cerevisiae invertase (IV) is represented by the sequence SEQ ID NO: 19

- Killer protein S. cerevisiae (KP), represented by the sequence SEQ ID NO: 20

- Gallus gallus lysozyme (LZ) is represented by the sequence SEQ ID NO: 21

- Homo sapiens serum albumin (SA) is represented by the sequence SEQ ID NO: 22.

The nucleic acid sequence of SEQ ID NO: 13 was chemically synthesized and cloned into the pPICZαA vector, and the remaining modified nucleic acid sequences were obtained by overlap extension PCR using the expression cassette of SEQ ID NO: 13 as a template.

Example 2: β-Fructofuranosidase polypeptide sequences fused to signal peptides

Recombinant precursor proteins were obtained by translation of the gene encoding Aspergillus niger β-fructofuranosidase in combination with signal peptides

The signal peptides used in the modified precursor peptides were S. cerevisiae alpha factor (FAK) represented by SEQ ID NO: 3, S. cerevisiae alpha factor complete (FAKS) represented by SEQ ID NO: 4, S. cerevisiae alpha α-factor T (AT) represented by SEQ ID NO: 5, Aspergillus niger alpha-amylase (AA) represented by SEQ ID NO: 6, Aspergillus awamori glucoamylase (GA) represented by SEQ ID NO: 7, Kluyveromyces maxianus inulinase (IN) represented by SEQ ID NO: 8, S. cerevisiae invertase (IV) represented by SEQ ID NO: 9, S. cerevisiae killer protein (KP) represented by SEQ ID NO: NO: 10, Gallus gallus Lysozyme (LZ) represented by SEQ ID NO: 11, and Homo sapiens Serum Albumin (SA) represented by SEQ ID NO: 12. The modified signal peptides contain an additional four amino acid region (LEKR) for efficient processing of the Kex2 precursor peptide.

The signal peptides are cleaved during post-translational modifications inside the Pichia host cells, and mature recombinant β-fructofuranosidase containing the amino acid sequence of SEQ ID NO: 1 is released into the medium.

Example 3: Production of recombinant host cells by transformation with recombinant plasmids

The vector used in this process was pPICZαA. The vectors contained modified open reading frames as described in Example 1 and an inducible AOX1 promoter. The modified sequence encoding the recombinant protein was cloned into the pPICZαA vector.

The modified nucleic acid of SEQ ID NO: 2 encoding the β-fructofuranosidase (fopA) gene was cloned between the XhoI/SacII restriction sites present in the MCS of the pPICZαA vector to introduce the S. cerevisiae alpha factor (FAK) signal sequence in frame to create the expression cassette of SEQ ID NO: 13 using a routine procedure known from molecular biology. The vector map for pPICZαA is shown in Figure 2.

Putative recombinant plasmids were selected on low-salt LB medium containing 25 μg/ml Zeocin and screened by XhoI/SacII restriction digestion analysis.

The recombinant plasmid pPICZαA-/bpL4 was confirmed by XhoI/SacII restriction digestion analysis, which resulted in the release of 1980 bp fragments. The results of the restriction digestion analysis are shown in Figure 3.

Pichia pastoris KM71H cells were then electroporated with linearized recombinant pPICZαA-fopA DNA. Pichia integrants were selected on yeast extract peptone dextrose sorbitol agar (YPDSA) containing 100 μg/ml Zeocin.

Integration was verified by colony PCR screening (cPCR). For cPCR, a template was generated from each of the Pichia integrants by alkaline lysis. The results of the colony PCR screening are shown in Figure 4.

Pichia integrants were grown for 48 h in BMD1 medium and further induced first with BMM2 medium and then sequentially with BMM10 medium, which provided a final concentration of 0.5% methanol in the culture medium. At the end of the 96-h induction period, culture supernatants from different clones were collected. Total protein from each of the collected supernatants was precipitated with 20% TCA and analyzed by SDS-PAGE.

Upon induction of β-fructofuranosidase, protein bands of approximately 110 kDa were visible, as shown in Figure 5.

The calculated molecular weight was about 70.85 kDa. The increase in molecular weight may have been due to glycosylation.

Example 4: Fermentation of recombinant Pichia pastoris expressing Aspergillus niger β-fructofuranosidase

Fermentation of recombinant Pichia pastoris cells containing a modified β-fructofuranosidase (fopA) gene as described in Example 1 was carried out in a 50-L fermenter. Fermentation was carried out in a basal salt medium as described here. The recombinant host chosen was KM71H, which is a mut S strain that slowly metabolizes methanol.

Preparation of pre-sowing and sowing material:

Pre-seeding material was prepared by inoculating glycerol stock into 25 ml of sterile YEPG medium and growing at 30°C in a temperature-controlled orbital shaker overnight. For seed production, the inoculum was grown in basal salt medium in ridged shaker flasks at 30°C in a temperature-controlled orbital shaker until an _OD600 of 15-25 was achieved.

Fermentation process

The entire fermentation process from seeding the fermenter with the seed culture to final harvest took about 130 hours. The basal salt medium was prepared and sterilized in situ in the fermenter.

The composition of the basic salt medium optimized for the fermentation process is given in Table 7.

Pichia Trace Minerals (PTM) saline was prepared as described in Table 8. PTM salts were dissolved and made up to 1 L, and the filter was sterilized. PTM saline was added at a rate of 4 mL per liter of the initial medium volume after sterilization of the basal salt medium.

Growth phase:

The growth phase begins with inoculation of the basal salt medium in a 50-liter fermenter with a 5% seed culture and continues for about 24 hours. Dissolved oxygen (DO) levels were constantly monitored and never dropped below 40%.

After 18 hours, a sharp spike in DO was observed, indicating depletion of the carbon source (glycerol). A glycerol-saturated batch was started by feeding 50% glycerol (using 12 ml of RTM salts per liter of feed) for about six hours, until an OD _{of 600} 200 was reached.

Induction phase:

Once sufficient biomass had been obtained, the induction phase was started by stopping the glycerol feed and starting the methanol feed. Methanol (with the addition of 12 ml of RTM salts per liter of feed) was fed at a rate of 0.5 g to 3 g per liter of the initial fermentation volume. DO was maintained at 40% and the methanol feed was adjusted accordingly.

Induction of the β-fructofuranosidase (fopA) gene was monitored periodically by analyzing the culture supernatant with an enzyme activity assay. The induction phase was continued for approximately 100 hours until the OD ₆₀₀ reached 600 and the wet biomass was -560 grams per liter of culture broth.

Fermentation was stopped after 130 h and the enzyme activity in the fermenter broth at the end of fermentation was determined to be 10,573 units by the DNS method (Miller, 1959). One unit represents the amount of enzyme required to liberate one micromole of reducing sugars (glucose equivalents) from a 10% sucrose solution in 100 mM citrate buffer, pH 5.5, at 55°C. The total amount of recombinant β-fructofuranosidase in the culture broth was estimated by the Bradford assay.

Fermentation conditions:

The fermentation parameters considered were as given in Table 9. These important parameters were monitored during the fermentation process.

Example 5: Collection and purification of cells

The enzyme was harvested by continuous centrifugation at 8000 rpm. The clear supernatant obtained after centrifugation was microfiltered using a 0.1 μm spiral wound TFF cut-off membrane. The filtrate was further ultrafiltered and diafiltered using a 10 kDa spiral wound TFF cut-off membrane and concentrated sufficiently to achieve the desired activity. The enzyme was developed by incorporating 35-50% glycerol and food preservatives into the final product. The final enzyme purity was observed to be 85% as determined by SDS-PAGE analysis.

Figure 6(a) shows SDS-PAGE analysis of samples collected at different time points during fermentation of Pichia pastoris strain KM71H expressing recombinant β-fructofuranosidase enzyme. Figure 6(b) shows SDS-PAGE analysis of recombinant β-fructofuranosidase enzyme after purification.

The concentration of β-fructofuranosidase was found to be about 2.4 g/L. In most batches, the concentration was 2-5 g/L. The purity of recombinant β-fructofuranosidase was observed to be about 85%.

Example 6: Evaluation of β-fructofuranosidase activity

Studies were conducted to evaluate the activity of β-fructofuranosidase. For the evaluation studies, the amount of reducing sugar formed as a result of the action of the enzyme β-fructofuranosidase was calculated using the DNS (3,5-dinitrosalicylic acid) method (G.L. Miller, "Use of dinitrosalicylic acid reagent for determination of reducing sugar", Anal. Chem., 1959, 31, 426-428).

To perform the enzyme activity assay, 10% sucrose (dissolved in 100 mM citrate buffer) was used as a substrate, β-fructofuranosidase was isolated from the fermentation broth and ultrafiltered. Then, the ultrafiltered sample was diluted 25,000 times by serial dilution in 100 mM citrate buffer and used. The reaction volume was 2.5 ml. The pH was maintained at 5.5 and the reaction was continued for 15 minutes.

After incubation, 3 ml of DNS (3,5-dinitrosalicylic acid) was added to each reaction mixture and boiled for 10 min, cooled, and the absorbance was measured spectrophotometrically at 540 nm.

The OD of glucose at different concentrations was measured as shown in Table 10 and depicted in Figure 7. Then, based on the absorbance measurement after the reaction, the enzyme activity was calculated as shown in Table 11. Figure 7 shows the standard curve of glucose used to evaluate the activity of β-fructofuranosidase enzyme.

Example 7: Production of fructooligosaccharides (FOS) from sucrose and recombinant β-fructofuranosidase enzyme

Studies were conducted to understand the ability of the enzyme to form fructooligosaccharides. 100 ml of 90% (by weight) sucrose solution was prepared in 150 mM sodium citrate buffer with pH 5.5. To this was added 96.7 μl of β-fructofuranosidase enzyme having an activity of 51,692 units/ml (equivalent to a total of 5,000 units of enzyme).

The reaction was carried out in a 250 ml conical flask and incubated at 65°C and 220 rpm. Samples were taken at regular intervals and analyzed on thin-layer chromatographic plates (TLC).

Glucose, sucrose, fructose and FOS (containing kestose, nystose and fructofuranosyl nystose) were used as standards for thin layer chromatographic analysis. The mobile phase was n-butanol:glacial acetic acid:water (4:2:2 v/v) and the developing/staining solution was urea phosphate.

Figure 8 shows the TLC analysis performed to obtain fructooligosaccharides (FOS) from sucrose and recombinant β-fructofuranosidase enzyme.

The sample was further subjected to high performance liquid chromatography (HPLC) to quantify the formation of fructooligosaccharides. HPLC analysis was performed using an amine column (Zorbax _NH2 column, Agilent Technologies) having 4.6 (ID) × 150 mm (length) and 5 μm (particle size). Standard solutions of glucose, fructose, kestose, nystose, fructofuranosyl nystose and sucrose of various concentrations were used to obtain standard curves.

Figure 9 shows the HPLC chromatogram of the FOS samples. Table 12 shows the percentage of fructooligosaccharides (FOS) and the recovered glucose, fructose, and sucrose at the end of the 60-minute reaction period.

100 ml of 90% (by weight) sucrose solution was reacted with the enzyme β-fructofuranosidase to convert sucrose to FOS. The amounts of FOS, sucrose, glucose, and fructose released from the reaction after termination of the reaction by heating at the end of the 60-min period were measured and presented as 90% and 100% sucrose base.

Studies have shown that purified enzymes are able to efficiently convert very large amounts of sugars into fructooligosaccharides.

Example 8: Characterization of recombinant β-fructofuranosidase from Aspergillus niger

The assembled β-fructofuranosidase from Aspergillus niger was investigated to identify the bioactive fragments. The following bioactive fragments of β-fructofuranosidase were found to be conserved and mediate the catalytic activity:

It was further found that the following amino acid residues in Aspergillus niger β-fructofuranosidase were involved in the formation of a hydrogen bond network around the catalytic triad. The hydrogen bond network is important for stable stereochemistry around the catalytic triad:

- Arg-190,

- Tyr-369,

- Glu-318,

- His-332,

- Asp-191,

- Thr-293,

- Asp-119,

- His-144.

The following hydrophobic residues in Aspergillus niger β-fructofuranosidase were also found to participate in the formation of a negatively charged pocket around the active site:

- Leu-78,

- Phe-118,

- Ala-370,

- Trp-398,

- Ile-143.

Furthermore, the following important residues of Aspergillus niger β-fructofuranosidase were found to participate in interactions at the entrance to the active pocket:

- Glu-405,

- His-332,

- Tyr-404.

--->

LIST OF SEQUENCES

<110> REVELATIONS BIOTECH PVT LTD

<120> NUCLEIC ACIDS, VECTORS, HOST CELLS AND METHODS OF PRODUCTION

BETA-FRUCTOFURANOSIDASES FROM ASPERGILLUS NIGER

<130> IP51142

<150> IN201941048686

<151> 2019-11-27

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 654

<212> PRT

<213> Aspergillus niger

<400> 1

Met Lys Leu Thr Thr Thr Thr Leu Ala Leu Ala Thr Gly Ala Ala Ala

1 5 10 15

Ala Glu Ala Ser Tyr His Leu Asp Thr Thr Ala Pro Pro Pro Thr Asn

20 25 30

Leu Ser Thr Leu Pro Asn Asn Thr Leu Phe His Val Trp Arg Pro Arg

35 40 45

Ala His Ile Leu Pro Ala Glu Gly Gln Ile Gly Asp Pro Cys Ala His

50 55 60

Tyr Thr Asp Pro Ser Thr Gly Leu Phe His Val Gly Phe Leu His Asp

65 70 75 80

Gly Asp Gly Ile Ala Gly Ala Thr Thr Ala Asn Leu Ala Thr Tyr Thr

85 90 95

Asp Thr Ser Asp Asn Gly Ser Phe Leu Ile Gln Pro Gly Gly Lys Asn

100 105 110

Asp Pro Val Ala Val Phe Asp Gly Ala Val Ile Pro Val Gly Val Asn

115 120 125

Asn Thr Pro Thr Leu Leu Tyr Thr Ser Val Ser Phe Leu Pro Ile His

130 135 140

Trp Ser Ile Pro Tyr Thr Arg Gly Ser Glu Thr Gln Ser Leu Ala Val

145 150 155 160

Ala Arg Asp Gly Gly Arg Arg Phe Asp Lys Leu Asp Gln Gly Pro Val

165 170 175

Ile Ala Asp His Pro Phe Ala Val Asp Val Thr Ala Phe Arg Asp Pro

180 185 190

Phe Val Phe Arg Ser Ala Lys Leu Asp Val Leu Leu Ser Leu Asp Glu

195 200 205

Glu Val Ala Arg Asn Glu Thr Ala Val Gln Gln Ala Val Asp Gly Trp

210 215 220

Thr Glu Lys Asn Ala Pro Trp Tyr Val Ala Val Ser Gly Gly Val His

225 230 235 240

Gly Val Gly Pro Ala Gln Phe Leu Tyr Arg Gln Asn Gly Gly Asn Ala

245 250 255

Ser Glu Phe Gln Tyr Trp Glu Tyr Leu Gly Glu Trp Trp Gln Glu Ala

260 265 270

Thr Asn Ser Ser Trp Gly Asp Glu Gly Thr Trp Ala Gly Arg Trp Gly

275 280 285

Phe Asn Phe Glu Thr Gly Asn Val Leu Phe Leu Thr Glu Glu Gly His

290 295 300

Asp Pro Gln Thr Gly Glu Val Phe Val Thr Leu Gly Thr Glu Gly Ser

305 310 315 320

Gly Leu Pro Ile Val Pro Gln Val Ser Ser Ile His Asp Met Leu Trp

325 330 335

Ala Ala Gly Glu Val Gly Val Gly Ser Glu Gln Glu Gly Ala Lys Val

340 345 350

Glu Phe Ser Pro Ser Met Ala Gly Phe Leu Asp Trp Gly Phe Ser Ala

355 360 365

Tyr Ala Ala Ala Gly Lys Val Leu Pro Ala Ser Ser Ala Val Ser Lys

370 375 380

Thr Ser Gly Val Glu Val Asp Arg Tyr Val Ser Phe Val Trp Leu Thr

385 390 395 400

Gly Asp Gln Tyr Glu Gln Ala Asp Gly Phe Pro Thr Ala Gln Gln Gly

405 410 415

Trp Thr Gly Ser Leu Leu Leu Pro Arg Glu Leu Lys Val Gln Thr Val

420 425 430

Glu Asn Val Val Asp Asn Glu Leu Val Arg Glu Glu Gly Val Ser Trp

435 440 445

Val Val Gly Glu Ser Asp Asn Gln Thr Ala Arg Leu Arg Thr Leu Gly

450 455 460

Ile Thr Ile Ala Arg Glu Thr Lys Ala Ala Leu Leu Ala Asn Gly Ser

465 470 475 480

Val Thr Ala Glu Glu Asp Arg Thr Leu Gln Thr Ala Ala Val Val Pro

485 490 495

Phe Ala Gln Ser Pro Ser Ser Lys Phe Phe Val Leu Thr Ala Gln Leu

500 505 510

Glu Phe Pro Ala Ser Ala Arg Ser Ser Pro Leu Gln Ser Gly Phe Glu

515 520 525

Ile Leu Ala Ser Glu Leu Glu Arg Thr Ala Ile Tyr Tyr Gln Phe Ser

530 535 540

Asn Glu Ser Leu Val Val Asp Arg Ser Gln Thr Ser Ala Ala Ala Pro

545 550 555 560

Thr Asn Pro Gly Leu Asp Ser Phe Thr Glu Ser Gly Lys Leu Arg Leu

565 570 575

Phe Asp Val Ile Glu Asn Gly Gln Glu Gln Val Glu Thr Leu Asp Leu

580 585 590

Thr Val Val Val Asp Asn Ala Val Val Glu Val Tyr Ala Asn Gly Arg

595 600 605

Phe Ala Leu Ser Thr Trp Ala Arg Ser Trp Tyr Asp Asn Ser Thr Gln

610 615 620

Ile Arg Phe Phe His Asn Gly Glu Gly Glu Val Gln Phe Arg Asn Val

625 630 635 640

Ser Val Ser Glu Gly Leu Tyr Asn Ala Trp Pro Glu Arg Asn

645 650

<210> 2

<211> 1965

<212> DNA

<213> Artificial Sequence

<220>

<223> Modified nucleic acid sequence of the gene encoding

beta-fructofuranosidase

<400> 2

atgaaattga ctactactac tttggctttg gctactggtg ctgctgctgc tgaagcttct 60

taccatttgg atactactgc tccacctcca actaatttgt ctactttgcc taacaacact 120

ttgtttcatg tttggagacc aagagcccat attttgccag ctgaaggtca aattggagat 180

ccatgtgctc actacactga tccatctact ggtttgtttc atgttggttt cttgcacgat 240

ggagatggta ttgctggtgc tactactgct aatttggcta cttatactga tacttctgat 300

aacggttctt tcttgattca accaggtggt aaaaacgatc cagttgctgt tttcgatggt 360

gctgttattc ctgttggtgt taacaatact ccaactttgt tgtacacttc tgtttctttc 420

ttgcctattc attggtctat tccatatact agaggttctg aaactcaatc tttggctgtt 480

gctagagatg gtggtagaag attcgataaa ttggatcaag gtcctgttat tgctgatcac 540

ccatttgctg ttgatgttac tgctttcaga gatccttttg tttttagatc cgctaagttg 600

gatgttttgt tgtctttgga tgaagaggtt gctagaaatg agactgctgt tcaacaagct 660

gttgatggtt ggactgaaaa gaacgctcct tggtacgttg ctgtttctgg tggtgttcat 720

ggtgttggtc cagctcaatt tttgtataga caaaacggtg gtaatgcttc tgaattccaa 780

tactgggaat atttggggtga atggtggcaa gaagctacta attcttcttg gggagatgag 840

ggtacttggg ctggtagatg gggttttaac ttcgaaactg gtaacgtttt gtttttgact 900

gaagaggtc acgatccaca aactggagag gttttcgtta ctttgggtac tgaaggttct 960

ggtttgccta ttgttccaca agtttcttct attcacgata tgttgtgggc tgctggtgaa 1020

gttggtgttg gttctgaaca agaggtgct aaggttgaat tttctccttc tatggctggt 1080

ttcttggatt ggggtttctc tgcttacgct gctgctggta aagttttgcc agcttcttct 1140

gctgtttcta aaacttctgg tgttgaggtt gatagatacg tttcttttgt ttggttgact 1200

ggagatcaat atgaacaagc tgatggtttc cctactgctc aacaaggttg gactggttct 1260

ttgttgttgc caagagaatt gaaagttcaa actngttgaga acgttgttga taatgaattg 1320

gttagagaag agggtgtttc ttgggttgtt ggagagtctg ataatcaaac tgctagattg 1380

agaactttgg gtattactat tgctagagaa actaaggctg ctttgttggc taacggttct 1440

gttactgctg aagaggatag aactttgcaa actgctgctg ttgttccttt cgctcaatct 1500

ccatcttcta agtttttcgt tttgactgct caattggagt ttcctgcttc tgctagatcc 1560

tctccatgc aatctggttt cgaaattttg gcttctgaat tggagagaac tgctatctac 1620

taccaattct ctaacgagtc tttggttgtt gatagatccc aaacttctgc tgctgctcct 1680

actaacccag gtttggattc ttttactgag tctggtaaat tgagattgtt cgatgttatc 1740

gaaaacggtc aagaacaagt tgagactttg gatttgactg ttgttgttga taacgctgtt 1800

gttgaagttt acgctaatgg tagatttgct ttgtctactt gggctagatc ctggtacgat 1860

aactctactc aaatcagatt tttccacaat ggtgaaggag aggttcaatt cagaaacgtt 1920

tctgtttctg agggtttgta taacgcttgg ccagaaagaa attga 1965

<210> 3

<211> 85

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Alpha-factor of S. cerevisiae (FAK)

<400> 3

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg

85

<210> 4

<211> 89

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Alpha-factor full of S. cerevisiae (FAKS)

<400> 4

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala

85

<210> 5

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Alpha factor_T of S. cerevisiae (AT)

<400> 5

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Leu Glu Lys Arg

20

<210> 6

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Alpha-amylase of Aspergillus niger (AA)

<400> 6

Met Val Ala Trp Trp Ser Leu Phe Leu Tyr Gly Leu Gln Val Ala Ala

1 5 10 15

Pro Ala Leu Ala Leu Glu Lys Arg

20

<210> 7

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Glucoamylase of Aspergillus awamori (GA)

<400> 7

Met Ser Phe Arg Ser Leu Leu Ala Leu Ser Gly Leu Val Cys Ser Gly

1 5 10 15

Leu Ala Leu Glu Lys Arg

20

<210> 8

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Inulinase of Kluyveromyces maxianus (IN)

<400> 8

Met Lys Leu Ala Tyr Ser Leu Leu Leu Pro Leu Ala Gly Val Ser Ala

1 5 10 15

Leu Glu Lys Arg

20

<210> 9

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Invertase of S. cerevisiae (IV)

<400> 9

Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys

1 5 10 15

Ile Ser Ala Leu Glu Lys Arg

20

<210> 10

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Killer protein of S. cerevisiae (KP)

<400> 10

Met Thr Lys Pro Thr Gln Val Leu Val Arg Ser Val Ser Ile Leu Phe

1 5 10 15

Phe Ile Thr Leu Leu His Leu Val Val Ala Leu Glu Lys Arg

20 25 30

<210> 11

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Lysozyme of Gallus gallus (LZ)

<400> 11

Met Leu Gly Lys Asn Asp Pro Met Cys Leu Val Leu Val Leu Leu Gly

1 5 10 15

Leu Thr Ala Leu Leu Gly Ile Cys Gln Gly Leu Glu Lys Arg

20 25 30

<210> 12

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> Modified Serum albumin of Homo sapiens (SA)

<400> 12

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Leu Glu Lys Arg

20

<210> 13

<211> 2220

<212> DNA

<213> Artificial Sequence

<220>

<223> Alpha-factor (FAK) of S. cerevisiae fused to modified nucleic

acid of beta-fructofuranosidase gene

<400> 13

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga agagaatgaa attgactact actactttgg ctttggctac tggtgctgct 300

gctgctgaag cttcttacca tttggatact actgctccac ctccaactaa tttgtctact 360

ttgcctaaca acactttgtt tcatgtttgg agaccaagag cccatatttt gccagctgaa 420

ggtcaaattg gagatccatg tgctcactac actgatccat ctactggttt gtttcatgtt 480

ggtttcttgc acgatggaga tggtattgct ggtgctacta ctgctaattt ggctacttat 540

actgatactt ctgataacgg ttctttcttg attcaaccag gtggtaaaaa cgatccagtt 600

gctgttttcg atggtgctgt tattcctgtt ggtgttaaca atactccaac tttgttgtac 660

acttctgttt ctttcttgcc tattcattgg tctattccat atactagagg ttctgaaact 720

caatctttgg ctgttgctag agatggtggt agaagattcg ataaattgga tcaaggtcct 780

gttattgctg atcacccatt tgctgttgat gttactgctt tcagagatcc ttttgttttt 840

agatccgcta agttggatgt tttgttgtct ttggatgaag aggttgctag aaatgagact 900

gctgttcaac aagctgttga tggttggact gaaaagaacg ctccttggta cgttgctgtt 960

tctggtggtg ttcatggtgt tggtccagct caatttttgt atagacaaaa cggtggtaat 1020

gcttctgaat tccaatactg ggaatatttg ggtgaatggt ggcaagaagc tactaattct 1080

tcttggggag atgaggtac ttgggctggt agatggggtt ttaacttcga aactggtaac 1140

gttttgtttt tgactgaaga gggtcacgat ccacaaactg gagaggtttt cgttactttg 1200

ggtactgaag gttctggttt gcctattgtt ccacaagttt cttctattca cgatatgttg 1260

tgggctgctg gtgaagttgg tgttggttct gaacaagagg gtgctaaggt tgaattttct 1320

ccttctatgg ctggtttctt ggattggggt ttctctgctt acgctgctgc tggtaaagtt 1380

ttgccagctt cttctgctgt ttctaaaact tctggtgttg aggttgatag atacgtttct 1440

tttgtttggt tgactggaga tcaatatgaa caagctgatg gtttccctac tgctcaacaa 1500

ggttggactg gttctttgtt gttgccaaga gaattgaaag ttcaaactgt tgagaacgtt 1560

gttgataatg aattggttag agaagagggt gtttcttggg ttgttggaga gtctgataat 1620

caaactgcta gattgagaac tttgggtatt actattgcta gagaaactaa ggctgctttg 1680

ttggctaacg gttctgttac tgctgaagag gatagaactt tgcaaactgc tgctgttgtt 1740

cctttcgctc aatctccatc ttctaagttt ttcgttttga ctgctcaatt ggagtttcct 1800

gcttctgcta gatcctctcc attgcaatct ggtttcgaaa ttttggcttc tgaattggag 1860

agaactgcta tctactacca attctctaac gagtctttgg ttgttgatag atcccaaact 1920

tctgctgctg ctcctactaa cccaggtttg gattctttta ctgagtctgg taaattgaga 1980

ttgttcgatg ttatcgaaaa cggtcaagaa caagttgaga ctttggattt gactgttgtt 2040

gttgataacg ctgttgttga agtttacgct aatggtagat ttgctttgtc tacttgggct 2100

agatcctggt acgataactc tactcaaatc agatttttcc acaatggtga aggagaggtt 2160

caattcagaa acgtttctgt ttctgagggt ttgtataacg cttggccaga aagaaattga 2220

<210> 14

<211> 2232

<212> DNA

<213> Artificial Sequence

<220>

<223> Alpha-factor full (FAKS) of S. cerevisiae fused to modified

nucleic acid of beta-fructofuranosidase gene

<400> 14

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga agagagaggc tgaagctatg aaattgacta ctactacttt ggctttggct 300

actggtgctg ctgctgctga agcttcttac catttggata ctactgctcc acctccaact 360

aatttgtcta ctttgcctaa caacactttg tttcatgttt ggagaccaag agcccatatt 420

ttgccagctg aaggtcaaat tggagatcca tgtgctcact acactgatcc atctactggt 480

ttgtttcatg ttggtttctt gcacgatgga gatggtattg ctggtgctac tactgctaat 540

ttggctactt atactgatac ttctgataac ggttctttct tgattcaacc aggtggtaaa 600

aacgatccag ttgctgtttt cgatggtgct gttattcctg ttggtgttaa caatactcca 660

actttgttgt acacttctgt ttctttcttg cctattcatt ggtctattcc atatactaga 720

ggttctgaaa ctcaatcttt ggctgttgct agagatggtg gtagaagatt cgataaattg 780

gatcaaggtc ctgttattgc tgatcaccca tttgctgttg atgttactgc tttcagagat 840

ccttttgttt ttagatccgc taagttggat gttttgttgt ctttggatga agaggttgct 900

agaaatgaga ctgctgttca acaagctgtt gatggttgga ctgaaaagaa cgctccttgg 960

tacgttgctg tttctggtgg tgttcatggt gttggtccag ctcaattttt gtatagacaa 1020

aacggtggta atgcttctga attccaatac tgggaatatt tgggtgaatg gtggcaagaa 1080

gctactaatt cttcttgggg agatgagggt acttgggctg gtagatgggg ttttaacttc 1140

gaaactggta acgttttgtt tttgactgaa gagggtcacg atccacaaac tggagaggtt 1200

ttcgttactt tgggtactga aggttctggt ttgcctattg ttccacaagt ttcttctatt 1260

cacgatatgt tgtgggctgc tggtgaagtt ggtgttggtt ctgaacaaga gggtgctaag 1320

gttgaatttt ctccttctat ggctggtttc ttggattggg gtttctctgc ttacgctgct 1380

gctggtaaag ttttgccagc ttcttctgct gtttctaaaa cttctggtgt tgaggttgat 1440

agatacgttt cttttgtttg gttgactgga gatcaatatg aacaagctga tggtttccct 1500

actgctcaac aaggttggac tggttctttg ttgttgccaa gagaattgaa agttcaaact 1560

gttgagaacg ttgttgataa tgaattggtt agagaagagg gtgtttcttg ggttgttgga 1620

gagtctgata atcaaactgc tagattgaga actttgggta ttactattgc tagagaaact 1680

aaggctgctt tgttggctaa cggttctgtt actgctgaag aggatagaac tttgcaaact 1740

gctgctgttg ttcctttcgc tcaatctcca tcttctaagt ttttcgtttt gactgctcaa 1800

ttggagtttc ctgcttctgc tagatcctct ccattgcaat ctggtttcga aattttggct 1860

tctgaattgg agagaactgc tatctactac caattctcta acgagtcttt ggttgttgat 1920

agatcccaaa cttctgctgc tgctcctact aacccaggtt tggattcttt tactgagtct 1980

ggtaaattga gattgttcga tgttatcgaa aacggtcaag aacaagttga gactttggat 2040

ttgactgttg ttgttgataa cgctgttgtt gaagtttacg ctaatggtag atttgctttg 2100

tctacttggg ctagatcctg gtacgataac tctactcaaa tcagattttt ccacaatggt 2160

gaaggagagg ttcaattcag aaacgtttct gtttctgagg gtttgtataa cgcttggcca 2220

gaaagaaattga 2232

<210> 15

<211> 2034

<212> DNA

<213> Artificial Sequence

<220>

<223> Alpha-factor_T (AT) of S. cerevisiae fused to modified nucleic

acid of beta-fructofuranosidase gene

<400> 15

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctctc 60

gagaagagaa tgaaattgac tactactact ttggctttgg ctactggtgc tgctgctgct 120

gaagcttctt accatttgga tactactgct ccacctccaa ctaatttgtc tactttgcct 180

aacaacactt tgtttcatgt ttggagacca agagcccata ttttgccagc tgaaggtcaa 240

attggagatc catgtgctca ctacactgat ccatctactg gtttgtttca tgttggtttc 300

ttgcacgatg gagatggtat tgctggtgct actactgcta atttggctac ttatactgat 360

acttctgata acggttcttt cttgattcaa ccaggtggta aaaacgatcc agttgctgtt 420

ttcgatggtg ctgttattcc tgttggtgtt aacaatactc caactttgtt gtacacttct 480

gtttctttct tgcctattca ttggtctatt ccatatacta gaggttctga aactcaatct 540

ttggctgttg ctagagatgg tggtagaaga ttcgataaat tggatcaagg tcctgttatt 600

gctgatcacc catttgctgt tgatgttact gctttcagag atccttttgt ttttagatcc 660

gctaagttgg atgttttgtt gtctttggat gaagaggttg ctagaaatga gactgctgtt 720

caacaagctg ttgatggttg gactgaaaag aacgctcctt ggtacgttgc tgtttctggt 780

ggtgttcatg gtgttggtcc agctcaattt ttgtatagac aaaacggtgg taatgcttct 840

gaattccaat actgggaata tttgggtgaa tggtggcaag aagctactaa ttcttcttgg 900

ggagatgagg gtacttgggc tggtagatgg ggttttaact tcgaaactgg taacgttttg 960

tttttgactg aagagggtca cgatccacaa actggagagg ttttcgttac tttgggtact 1020

gaaggttctg gtttgcctat tgttccacaa gtttcttcta ttcacgatat gttgtgggct 1080

gctggtgaag ttggtgttgg ttctgaacaa gagggtgcta aggttgaatt ttctccttct 1140

atggctggtt tcttggattg gggtttctct gcttacgctg ctgctggtaa agttttgcca 1200

gcttcttctg ctgtttctaa aacttctggt gttgaggttg atagatacgt ttcttttgtt 1260

tggttgactg gagatcaata tgaacaagct gatggtttcc ctactgctca acaaggttgg 1320

actggttctt tgttgttgcc aagagaattg aaagttcaaa ctgttgagaa cgttgttgat 1380

aatgaattgg ttagagaaga gggtgtttct tgggttgttg gagagtctga taatcaaact 1440

gctagattga gaactttggg tattactatt gctagagaaa ctaaggctgc tttgttggct 1500

aacggttctg ttactgctga agaggataga actttgcaaa ctgctgctgt tgttcctttc 1560

gctcaatctc catcttctaa gtttttcgtt ttgactgctc aattggagtt tcctgcttct 1620

gctagatcct ctccatgca atctggtttc gaaattttgg cttctgaatt ggagagaact 1680

gctatctact accaattctc taacgagtct ttggttgttg atagatccca aacttctgct 1740

gctgctccta ctaacccagg tttggattct tttactgagt ctggtaaatt gagattgttc 1800

gatgttatcg aaaacggtca agaacaagtt gagactttgg atttgactgt tgttgttgat 1860

aacgctgttg ttgaagttta cgctaatggt agatttgctt tgtctacttg ggctagatcc 1920

tggtacgata actctactca aatcagattt ttccacaatg gtgaaggaga ggttcaattc 1980

agaaacgttt ctgtttctga gggtttgtat aacgcttggc cagaaagaaa ttga 2034

<210> 16

<211> 2037

<212> DNA

<213> Artificial Sequence

<220>

<223> Alpha-amylase (AA) of Aspergillus niger fused to modified nucleic

acid of beta-fructofuranosidase gene

<400> 16

atggttgctt ggtggagtct tttcctatac ggtctacagg tggcagctcc agcccttgcc 60

ctcgagaaga gaatgaaatt gactactact actttggctt tggctactgg tgctgctgct 120

gctgaagctt cttaccattt ggatactact gctccacctc caactaattt gtctactttg 180

cctaacaaca ctttgtttca tgtttggaga ccaagagccc atattttgcc agctgaaggt 240

caaattggag atccatgtgc tcactacact gatccatcta ctggtttgtt tcatgttggt 300

ttcttgcacg atggagatgg tattgctggt gctactactg ctaatttggc tacttatact 360

gatacttctg ataacggttc tttcttgatt caaccaggtg gtaaaaacga tccagttgct 420

gttttcgatg gtgctgttat tcctgttggt gttaacaata ctccaacttt gttgtacact 480

tctgtttctt tcttgcctat tcattggtct attccatata ctagaggttc tgaaactcaa 540

tctttggctg ttgctagaga tggtggtaga agattcgata aattggatca aggtcctgtt 600

attgctgatc acccatttgc tgttgatgtt actgctttca gagatccttt tgtttttaga 660

tccgctaagt tggatgtttt gttgtctttg gatgaagagg ttgctagaaa tgagactgct 720

gttcaacaag ctgttgatgg ttggactgaa aagaacgctc cttggtacgt tgctgtttct 780

ggtggtgttc atggtgttgg tccagctcaa tttttgtata gacaaaacgg tggtaatgct 840

tctgaattcc aatactggga atatttgggt gaatggtggc aagaagctac taattcttct 900

tggggagatg agggtacttg ggctggtaga tggggtttta acttcgaaac tggtaacgtt 960

ttgtttttga ctgaagaggg tcacgatcca caaactggag aggttttcgt tactttgggt 1020

actgaaggtt ctggtttgcc tattgttcca caagtttctt ctattcacga tatgttgtgg 1080

gctgctggtg aagttggtgt tggttctgaa caagaggtg ctaaggttga attttctcct 1140

tctatggctg gtttcttgga ttggggtttc tctgcttacg ctgctgctgg taaagttttg 1200

ccagcttctt ctgctgtttc taaaacttct ggtgttgagg ttgatagata cgtttctttt 1260

gtttggttga ctggagatca atatgaacaa gctgatggtt tccctactgc tcaacaaggt 1320

tggactggtt ctttgttgtt gccaagagaa ttgaaagttc aaactgttga gaacgttgtt 1380

gataatgaat tggttagaga agagggtgtt tcttgggttg ttggagagtc tgataatcaa 1440

actgctagat tgagaacttt gggtattact attgctagag aaactaaggc tgctttgttg 1500

gctaacggtt ctgttactgc tgaagaggat agaactttgc aaactgctgc tgttgttcct 1560

ttcgctcaat ctccatcttc taagtttttc gttttgactg ctcaattgga gtttcctgct 1620

tctgctagat cctctccat gcaatctggt ttcgaaattt tggcttctga attggagaga 1680

actgctatct actaccaatt ctctaacgag tctttggttg ttgatagatc ccaaacttct 1740

gctgctgctc ctactaaccc aggtttggat tcttttactg agtctggtaa attgagattg 1800

ttcgatgtta tcgaaaacgg tcaagaacaa gttgagactt tggatttgac tgttgttgtt 1860

gataacgctg ttgttgaagt ttacgctaat ggtagatttg ctttgtctac ttgggctaga 1920

tcctggtacg ataactctac tcaaatcaga tttttccaca atggtgaagg agaggttcaa 1980

ttcagaaacg tttctgtttc tgagggtttg tataacgctt ggccagaaag aaattga 2037

<210> 17

<211> 2031

<212> DNA

<213> Artificial Sequence

<220>

<223> Glucoamylase (GA) of Aspergillus awamori fused to modified

nucleic acid of beta-fructofuranosidase gene

<400> 17

atgtctttcc gatctctttt agccctatct ggacttgttt gttcaggttt ggctctcgag 60

aagagaatga aattgactac tactactttg gctttggcta ctggtgctgc tgctgctgaa 120

gcttcttacc atttggatac tactgctcca cctccaacta atttgtctac tttgcctaac 180

aacactttgt ttcatgtttg gagaccaaga gcccatattt tgccagctga aggtcaaatt 240

ggagatccat gtgctcacta cactgatcca tctactggtt tgtttcatgt tggtttcttg 300

cacgatggag atggtattgc tggtgctact actgctaatt tggctactta tactgatact 360

tctgataacg gttctttctt gattcaacca ggtggtaaaa acgatccagt tgctgttttc 420

gatggtgctg ttattcctgt tggtgttaac aatactccaa ctttgttgta cacttctgtt 480

tctttcttgc ctattcattg gtctattcca tatactagag gttctgaaac tcaatctttg 540

gctgttgcta gagatggtgg tagaagattc gataaattgg atcaaggtcc tgttattgct 600

gatcacccat ttgctgttga tgttactgct ttcagagatc cttttgtttt tagatccgct 660

aagttggatg ttttgttgtc tttggatgaa gaggttgcta gaaatgagac tgctgttcaa 720

caagctgttg atggttggac tgaaaagaac gctccttggt acgttgctgt ttctggtggt 780

gttcatggtg ttggtccagc tcaatttttg tatagacaaa acggtggtaa tgcttctgaa 840

ttccaatact gggaatattt gggtgaatgg tggcaagaag ctactaattc ttcttgggga 900

gatgagggta cttgggctgg tagatggggt tttaacttcg aaactggtaa cgttttgttt 960

ttgactgaag agggtcacga tccacaaact ggagaggttt tcgttacttt gggtactgaa 1020

ggttctggtt tgcctattgt tccacaagtt tcttctattc acgatatgtt gtgggctgct 1080

ggtgaagttg gtgttggttc tgaacaagag ggtgctaagg ttgaattttc tccttctatg 1140

gctggtttct tggattgggg tttctctgct tacgctgctg ctggtaaagt tttgccagct 1200

tcttctgctg tttctaaaac ttctggtgtt gaggttgata gatacgtttc ttttgtttgg 1260

ttgactggag atcaatatga acaagctgat ggtttcccta ctgctcaaca aggttggact 1320

ggttctttgt tgttgccaag agaattgaaa gttcaaactg ttgagaacgt tgttgataat 1380

gaattggtta gagaagaggg tgtttcttgg gttgttggag agtctgataa tcaaactgct 1440

agattgagaa ctttgggtat tactattgct agagaaacta aggctgcttt gttggctaac 1500

ggttctgtta ctgctgaaga ggatagaact ttgcaaactg ctgctgttgt tcctttcgct 1560

caatctccat cttctaagtt tttcgttttg actgctcaat tggagtttcc tgcttctgct 1620

agatcctctc cattgcaatc tggtttcgaa attttggctt ctgaattgga gagaactgct 1680

atctactacc aattctctaa cgagtctttg gttgttgata gatcccaaac ttctgctgct 1740

gctcctacta acccaggttt ggattctttt actgagtctg gtaaattgag attgttcgat 1800

gttatcgaaa acggtcaaga acaagttgag actttggatt tgactgttgt tgttgataac 1860

gctgttgttg aagtttacgc taatggtaga tttgctttgt ctacttgggc tagatcctgg 1920

tacgataact ctactcaaat cagatttttc cacaatggtg aaggagaggt tcaattcaga 1980

aacgtttctg tttctgaggg tttgtataac gcttggccag aaagaaattg a 2031

<210> 18

<211> 2025

<212> DNA

<213> Artificial Sequence

<220>

<223> Inulinase (IN) of Kluyveromyces maxianus fused to modified

nucleic acid of beta-fructofuranosidase gene

<400> 18

atgaagttgg cttattctct tcttcttcct ctggccggag tgtctgccct cgagaagaga 60

atgaaattga ctactactac tttggctttg gctactggtg ctgctgctgc tgaagcttct 120

taccatttgg atactactgc tccacctcca actaatttgt ctactttgcc taacaacact 180

ttgtttcatg tttggagacc aagagcccat attttgccag ctgaaggtca aattggagat 240

ccatgtgctc actacactga tccatctact ggtttgtttc atgttggttt cttgcacgat 300

ggagatggta ttgctggtgc tactactgct aatttggcta cttatactga tacttctgat 360

aacggttctt tcttgattca accaggtggt aaaaacgatc cagttgctgt tttcgatggt 420

gctgttattc ctgttggtgt taacaatact ccaactttgt tgtacacttc tgtttctttc 480

ttgcctattc attggtctat tccatatact agaggttctg aaactcaatc tttggctgtt 540

gctagagatg gtggtagaag attcgataaa ttggatcaag gtcctgttat tgctgatcac 600

ccatttgctg ttgatgttac tgctttcaga gatccttttg tttttagatc cgctaagttg 660

gatgttttgt tgtctttgga tgaagaggtt gctagaaatg agactgctgt tcaacaagct 720

gttgatggtt ggactgaaaa gaacgctcct tggtacgttg ctgtttctgg tggtgttcat 780

ggtgttggtc cagctcaatt tttgtataga caaaacggtg gtaatgcttc tgaattccaa 840

tactgggaat atttgggtga atggtggcaa gaagctacta attcttcttg gggagatgag 900

ggtacttggg ctggtagatg gggttttaac ttcgaaactg gtaacgtttt gtttttgact 960

gaagagggtc acgatccaca aactggagag gttttcgtta ctttgggtac tgaaggttct 1020

ggtttgccta ttgttccaca agtttcttct attcacgata tgttgtgggc tgctggtgaa 1080

gttggtgttg gttctgaaca agaggtgct aaggttgaat tttctccttc tatggctggt 1140

ttcttggatt ggggtttctc tgcttacgct gctgctggta aagttttgcc agcttcttct 1200

gctgtttcta aaacttctgg tgttgaggtt gatagatacg tttcttttgt ttggttgact 1260

ggagatcaat atgaacaagc tgatggtttc cctactgctc aacaaggttg gactggttct 1320

ttgttgttgc caagagaatt gaaagttcaa actgttgaga acgttgttga taatgaattg 1380

gttagagaag agggtgtttc ttgggttgtt ggagagtctg ataatcaaac tgctagattg 1440

agaactttgg gtattactat tgctagagaa actaaggctg ctttgttggc taacggttct 1500

gttactgctg aagaggatag aactttgcaa actgctgctg ttgttccttt cgctcaatct 1560

ccatcttcta agtttttcgt tttgactgct caattggagt ttcctgcttc tgctagatcc 1620

tctccatgc aatctggttt cgaaattttg gcttctgaat tggagagaac tgctatctac 1680

taccaattct ctaacgagtc tttggttgtt gatagatccc aaacttctgc tgctgctcct 1740

actaacccag gtttggattc ttttactgag tctggtaaat tgagattgtt cgatgttatc 1800

gaaaacggtc aagaacaagt tgagactttg gatttgactg ttgttgttga taacgctgtt 1860

gttgaagttt acgctaatgg tagatttgct ttgtctactt gggctagatc ctggtacgat 1920

aactctactc aaatcagatt tttccacaat ggtgaaggag aggttcaatt cagaaacgtt 1980

tctgtttctg agggtttgta taacgcttgg ccagaaagaa attga 2025

<210> 19

<211> 2034

<212> DNA

<213> Artificial Sequence

<220>

<223> Invertase (IV) of S.cerevisiae fused to modified nucleic acid of

beta-fructofuranosidase gene

<400> 19

atgcttttgc aggctttcct gttcttgctg gccggattcg ctgctaaaat ttccgctctc 60

gagaagagaa tgaaattgac tactactact ttggctttgg ctactggtgc tgctgctgct 120

gaagcttctt accatttgga tactactgct ccacctccaa ctaatttgtc tactttgcct 180

aacaacactt tgtttcatgt ttggagacca agagcccata ttttgccagc tgaaggtcaa 240

attggagatc catgtgctca ctacactgat ccatctactg gtttgtttca tgttggtttc 300

ttgcacgatg gagatggtat tgctggtgct actactgcta atttggctac ttatactgat 360

acttctgata acggttcttt cttgattcaa ccaggtggta aaaacgatcc agttgctgtt 420

ttcgatggtg ctgttattcc tgttggtgtt aacaatactc caactttgtt gtacacttct 480

gtttctttct tgcctattca ttggtctatt ccatatacta gaggttctga aactcaatct 540

ttggctgttg ctagagatgg tggtagaaga ttcgataaat tggatcaagg tcctgttatt 600

gctgatcacc catttgctgt tgatgttact gctttcagag atccttttgt ttttagatcc 660

gctaagttgg atgttttgtt gtctttggat gaagaggttg ctagaaatga gactgctgtt 720

caacaagctg ttgatggttg gactgaaaag aacgctcctt ggtacgttgc tgtttctggt 780

ggtgttcatg gtgttggtcc agctcaattt ttgtatagac aaaacggtgg taatgcttct 840

gaattccaat actgggaata tttgggtgaa tggtggcaag aagctactaa ttcttcttgg 900

ggagatgagg gtacttgggc tggtagatgg ggttttaact tcgaaactgg taacgttttg 960

tttttgactg aagagggtca cgatccacaa actggagagg ttttcgttac tttgggtact 1020

gaaggttctg gtttgcctat tgttccacaa gtttcttcta ttcacgatat gttgtgggct 1080

gctggtgaag ttggtgttgg ttctgaacaa gagggtgcta aggttgaatt ttctccttct 1140

atggctggtt tcttggattg gggtttctct gcttacgctg ctgctggtaa agttttgcca 1200

gcttcttctg ctgtttctaa aacttctggt gttgaggttg atagatacgt ttcttttgtt 1260

tggttgactg gagatcaata tgaacaagct gatggtttcc ctactgctca acaaggttgg 1320

actggttctt tgttgttgcc aagagaattg aaagttcaaa ctgttgagaa cgttgttgat 1380

aatgaattgg ttagagaaga gggtgtttct tgggttgttg gagagtctga taatcaaact 1440

gctagattga gaactttggg tattactatt gctagagaaa ctaaggctgc tttgttggct 1500

aacggttctg ttactgctga agaggataga actttgcaaa ctgctgctgt tgttcctttc 1560

gctcaatctc catcttctaa gtttttcgtt ttgactgctc aattggagtt tcctgcttct 1620

gctagatcct ctccatgca atctggtttc gaaattttgg cttctgaatt ggagagaact 1680

gctatctact accaattctc taacgagtct ttggttgttg atagatccca aacttctgct 1740

gctgctccta ctaacccagg tttggattct tttactgagt ctggtaaatt gagattgttc 1800

gatgttatcg aaaacggtca agaacaagtt gagactttgg atttgactgt tgttgttgat 1860

aacgctgttg ttgaagttta cgctaatggt agatttgctt tgtctacttg ggctagatcc 1920

tggtacgata actctactca aatcagattt ttccacaatg gtgaaggaga ggttcaattc 1980

agaaacgttt ctgtttctga gggtttgtat aacgcttggc cagaaagaaa ttga 2034

<210> 20

<211> 2055

<212> DNA

<213> Artificial Sequence

<220>

<223> Killer protein (KP) of S.cerevisiae fused to modified nucleic

acid of beta-fructofuranosidase gene

<400> 20

atgaccaaac caactcaagt tttggtgagg tctgtgtcaa tcctgttctt cattacttta 60

ctgcaccttg tagtcgcact cgagaagaga atgaaattga ctactactac tttggctttg 120

gctactggtg ctgctgctgc tgaagcttct taccatttgg atactactgc tccacctcca 180

actaatttgt ctactttgcc taacaacact ttgtttcatg tttggagacc aagagcccat 240

attttgccag ctgaaggtca aattggagat ccatgtgctc actacactga tccatctact 300

ggtttgtttc atgttggttt cttgcacgat ggagatggta ttgctggtgc tactactgct 360

aatttggcta cttatactga tacttctgat aacggttctt tcttgattca accaggtggt 420

aaaaacgatc cagttgctgt tttcgatggt gctgttattc ctgttggtgt taacaatact 480

ccaactttgt tgtacacttc tgtttctttc ttgcctattc attggtctat tccatatact 540

agaggttctg aaactcaatc tttggctgtt gctagagatg gtggtagaag attcgataaa 600

ttggatcaag gtcctgttat tgctgatcac ccatttgctg ttgatgttac tgctttcaga 660

gatccttttg tttttagatc cgctaagttg gatgttttgt tgtctttgga tgaagaggtt 720

gctagaaatg agactgctgt tcaacaagct gttgatggtt ggactgaaaa gaacgctcct 780

tggtacgttg ctgtttctgg tggtgttcat ggtgttggtc cagctcaatt ttttataga 840

caaaacggtg gtaatgcttc tgaattccaa tactgggaat atttgggtga atggtggcaa 900

gaagctacta attcttcttg gggagatgag ggtacttggg ctggtagatg gggttttaac 960

ttcgaaactg gtaacgtttt gtttttgact gaagagggtc acgatccaca aactggagag 1020

gttttcgtta ctttgggtac tgaaggttct ggtttgccta ttgttccaca agtttcttct 1080

attcacgata tgttgtgggc tgctggtgaa gttggtgttg gttctgaaca agaggtgct 1140

aaggttgaat tttctccttc tatggctggt ttcttggatt ggggtttctc tgcttacgct 1200

gctgctggta aagttttgcc agcttcttct gctgtttcta aaacttctgg tgttgaggtt 1260

gatagatacg tttcttttgt ttggttgact ggagatcaat atgaacaagc tgatggtttc 1320

cctactgctc aacaaggttg gactggttct ttgttgttgc caagagaatt gaaagttcaa 1380

actgttgaga acgttgttga taatgaattg gttagagaag agggtgtttc ttgggttgtt 1440

ggagagtctg ataatcaaac tgctagattg agaactttgg gtattactat tgctagagaa 1500

actaaggctg ctttgttggc taacggttct gttactgctg aagaggatag aactttgcaa 1560

actgctgctg ttgttccttt cgctcaatct ccatcttcta agtttttcgt tttgactgct 1620

caattggagt ttcctgcttc tgctagatcc tctccattgc aatctggttt cgaaattttg 1680

gcttctgaat tggagagaac tgctatctac taccaattct ctaacgagtc tttggttgtt 1740

gatagatccc aaacttctgc tgctgctcct actaacccag gtttggattc ttttactgag 1800

tctggtaaat tgagattgtt cgatgttatc gaaaacggtc aagaacaagt tgagactttg 1860

gatttgactg ttgttgttga taacgctgtt gttgaagttt acgctaatgg tagatttgct 1920

ttgtctactt gggctagatc ctggtacgat aactctactc aaatcagatt tttccacaat 1980

ggtgaaggag aggttcaatt cagaaacgtt tctgtttctg agggtttgta taacgcttgg 2040

ccagaaagaa attga 2055

<210> 21

<211> 2055

<212> DNA

<213> Artificial Sequence

<220>

<223> Lysozyme (LZ) of Gallus gallus fused to modified nucleic acid of

beta-fructofuranosidase gene

<400> 21

atgctaggca aaaatgaccc tatgtgtttg gttctggttt tgcttggttt aaccgcttta 60

cttggtatct gtcaaggtct cgagaagaga atgaaattga ctactactac tttggctttg 120

gctactggtg ctgctgctgc tgaagcttct taccatttgg atactactgc tccacctcca 180

actaatttgt ctactttgcc taacaacact ttgtttcatg tttggagacc aagagcccat 240

attttgccag ctgaaggtca aattggagat ccatgtgctc actacactga tccatctact 300

ggtttgtttc atgttggttt cttgcacgat ggagatggta ttgctggtgc tactactgct 360

aatttggcta cttatactga tacttctgat aacggttctt tcttgattca accaggtggt 420

aaaaacgatc cagttgctgt tttcgatggt gctgttattc ctgttggtgt taacaatact 480

ccaactttgt tgtacacttc tgtttctttc ttgcctattc attggtctat tccatatact 540

agaggttctg aaactcaatc tttggctgtt gctagagatg gtggtagaag attcgataaa 600

ttggatcaag gtcctgttat tgctgatcac ccatttgctg ttgatgttac tgctttcaga 660

gatccttttg tttttagatc cgctaagttg gatgttttgt tgtctttgga tgaagaggtt 720

gctagaaatg agactgctgt tcaacaagct gttgatggtt ggactgaaaa gaacgctcct 780

tggtacgttg ctgtttctgg tggtgttcat ggtgttggtc cagctcaatt ttttataga 840

caaaacggtg gtaatgcttc tgaattccaa tactgggaat atttgggtga atggtggcaa 900

gaagctacta attcttcttg gggagatgag ggtacttggg ctggtagatg gggttttaac 960

ttcgaaactg gtaacgtttt gtttttgact gaagagggtc acgatccaca aactggagag 1020

gttttcgtta ctttgggtac tgaaggttct ggtttgccta ttgttccaca agtttcttct 1080

attcacgata tgttgtgggc tgctggtgaa gttggtgttg gttctgaaca agaggtgct 1140

aaggttgaat tttctccttc tatggctggt ttcttggatt ggggtttctc tgcttacgct 1200

gctgctggta aagttttgcc agcttcttct gctgtttcta aaacttctgg tgttgaggtt 1260

gatagatacg tttcttttgt ttggttgact ggagatcaat atgaacaagc tgatggtttc 1320

cctactgctc aacaaggttg gactggttct ttgttgttgc caagagaatt gaaagttcaa 1380

actgttgaga acgttgttga taatgaattg gttagagaag agggtgtttc ttgggttgtt 1440

ggagagtctg ataatcaaac tgctagattg agaactttgg gtattactat tgctagagaa 1500

actaaggctg ctttgttggc taacggttct gttactgctg aagaggatag aactttgcaa 1560

actgctgctg ttgttccttt cgctcaatct ccatcttcta agtttttcgt tttgactgct 1620

caattggagt ttcctgcttc tgctagatcc tctccattgc aatctggttt cgaaattttg 1680

gcttctgaat tggagagaac tgctatctac taccaattct ctaacgagtc tttggttgtt 1740

gatagatccc aaacttctgc tgctgctcct actaacccag gtttggattc ttttactgag 1800

tctggtaaat tgagattgtt cgatgttatc gaaaacggtc aagaacaagt tgagactttg 1860

gatttgactg ttgttgttga taacgctgtt gttgaagttt acgctaatgg tagatttgct 1920

ttgtctactt gggctagatc ctggtacgat aactctactc aaatcagatt tttccacaat 1980

ggtgaaggag aggttcaatt cagaaacgtt tctgtttctg agggtttgta taacgcttgg 2040

ccagaaagaa attga 2055

<210> 22

<211> 2031

<212> DNA

<213> Artificial Sequence

<220>

<223> Serum albumin (SA) of Homo sapiens fused to modified nucleic acid

of beta-fructofuranosidase gene

<400> 22

atgaagtggg taacatttat ttccctactg tttctttttt cttcagctta ctctctcgag 60

aagagaatga aattgactac tactactttg gctttggcta ctggtgctgc tgctgctgaa 120

gcttcttacc atttggatac tactgctcca cctccaacta atttgtctac tttgcctaac 180

aacactttgt ttcatgtttg gagaccaaga gcccatattt tgccagctga aggtcaaatt 240

ggagatccat gtgctcacta cactgatcca tctactggtt tgtttcatgt tggtttcttg 300

cacgatggag atggtattgc tggtgctact actgctaatt tggctactta tactgatact 360

tctgataacg gttctttctt gattcaacca ggtggtaaaa acgatccagt tgctgttttc 420

gatggtgctg ttattcctgt tggtgttaac aatactccaa ctttgttgta cacttctgtt 480

tctttcttgc ctattcattg gtctattcca tatactagag gttctgaaac tcaatctttg 540

gctgttgcta gagatggtgg tagaagattc gataaattgg atcaaggtcc tgttattgct 600

gatcacccat ttgctgttga tgttactgct ttcagagatc cttttgtttt tagatccgct 660

aagttggatg ttttgttgtc tttggatgaa gaggttgcta gaaatgagac tgctgttcaa 720

caagctgttg atggttggac tgaaaagaac gctccttggt acgttgctgt ttctggtggt 780

gttcatggtg ttggtccagc tcaatttttg tatagacaaa acggtggtaa tgcttctgaa 840

ttccaatact gggaatattt gggtgaatgg tggcaagaag ctactaattc ttcttgggga 900

gatgagggta cttgggctgg tagatggggt tttaacttcg aaactggtaa cgttttgttt 960

ttgactgaag agggtcacga tccacaaact ggagaggttt tcgttacttt gggtactgaa 1020

ggttctggtt tgcctattgt tccacaagtt tcttctattc acgatatgtt gtgggctgct 1080

ggtgaagttg gtgttggttc tgaacaagag ggtgctaagg ttgaattttc tccttctatg 1140

gctggtttct tggattgggg tttctctgct tacgctgctg ctggtaaagt tttgccagct 1200

tcttctgctg tttctaaaac ttctggtgtt gaggttgata gatacgtttc ttttgtttgg 1260

ttgactggag atcaatatga acaagctgat ggtttcccta ctgctcaaca aggttggact 1320

ggttctttgt tgttgccaag agaattgaaa gttcaaactg ttgagaacgt tgttgataat 1380

gaattggtta gagaagaggg tgtttcttgg gttgttggag agtctgataa tcaaactgct 1440

agattgagaa ctttgggtat tactattgct agagaaacta aggctgcttt gttggctaac 1500

ggttctgtta ctgctgaaga ggatagaact ttgcaaactg ctgctgttgt tcctttcgct 1560

caatctccat cttctaagtt tttcgttttg actgctcaat tggagtttcc tgcttctgct 1620

agatcctctc cattgcaatc tggtttcgaa attttggctt ctgaattgga gagaactgct 1680

atctactacc aattctctaa cgagtctttg gttgttgata gatcccaaac ttctgctgct 1740

gctcctacta acccaggttt ggattctttt actgagtctg gtaaattgag attgttcgat 1800

gttatcgaaa acggtcaaga acaagttgag actttggatt tgactgttgt tgttgataac 1860

gctgttgttg aagtttacgc taatggtaga tttgctttgt ctacttgggc tagatcctgg 1920

tacgataact ctactcaaat cagatttttc cacaatggtg aaggagaggt tcaattcaga 1980

aacgtttctg tttctgaggg tttgtataac gcttggccag aaagaaattg a 2031

<210> 23

<211> 1965

<212> DNA

<213> Aspergillus niger

<400> 23

atgaagctca ccactaccac cctggcgctc gccaccggcg cagcagcagc agaagcctca 60

taccacctgg acaccacggc cccgccgccg accaacctca gcaccctccc caacaacacc 120

ctcttccacg tgtggcggcc gcgcgcgcac atcctgcccg ccgagggcca gatcggcgac 180

ccctgcgcgc actacaccga cccatccacc ggcctcttcc acgtggggtt cctgcacgac 240

ggggacggca tcgcgggcgc caccacggcc aacctggcca cctacaccga tacctccgat 300

aacgggagct tcctgatcca gccgggcggg aagaacgacc ccgtcgccgt gttcgacggc 360

gccgtcatcc ccgtcggcgt caacaacacc cccaccttac tctacacctc cgtctccttc 420

ctgcccatcc actggtccat cccctacacc cgcggcagcg agacgcagtc gttggccgtc 480

gcgcgcgacg gcggccgccg cttcgacaag ctcgaccagg gccccgtcat cgccgaccac 540

cccttcgccg tcgacgtcac cgccttccgc gatccgtttg tcttccgcag tgccaagttg 600

gatgtgctgc tgtcgttgga tgaggaggtg gcgcggaatg agacggccgt gcagcaggcc 660

gtcgatggct ggaccgagaa gaacgccccc tggtatgtcg cggtctctgg cggggtgcac 720

ggcgtcgggc ccgcgcagtt cctctaccgc cagaacggcg ggaacgcttc cgagttccag 780

tactgggagt acctcgggga gtggtggcag gaggcgacca actccagctg gggcgacgag 840

ggcacctggg ccgggcgctg ggggttcaac ttcgagacgg ggaatgtgct cttcctcacc 900

gaggagggcc atgaccccca gacgggcgag gtgttcgtca ccctcggcac ggaggggtct 960

ggcctgccaa tcgtgccgca ggtctccagt atccacgata tgctgtgggc ggcgggtgag 1020

gtcggggtgg gcagtgagca ggagggtgcc aaggtcgagt tctccccctc catggccggg 1080

tttctggact gggggttcag cgcctacgct gcggcgggca aggtgctgcc ggccagctcg 1140

gcggtgtcga agaccagcgg cgtggaggtg gatcggtatg tctcgttcgt ctggttgacg 1200

ggcgaccagt acgagcaggc ggacgggttc cccacggccc agcaggggtg gacggggtcg 1260

ctgctgctgc cgcgcgagct gaaggtgcag acggtggaga acgtcgtcga caacgagctg 1320

gtgcgcgagg agggcgtgtc gtgggtggtg ggggagtcgg acaaccagac ggccaggctg 1380

cgcacgctgg ggatcacgat cgcccgggag accaaggcgg ccctgctggc caacggctcg 1440

gtgaccgcgg aggaggaccg cacgctgcag acggcggccg tcgtgccgtt cgcgcaatcg 1500

ccgagctcca agttcttcgt gctgacggcc cagctggagt tccccgcgag cgcgcgctcg 1560

tccccgctcc agtccggggtt cgaaatcctg gcgtcggagc tggagcgcac ggccatctac 1620

taccagttca gcaacgagtc gctggtcgtc gaccgcagcc agactagtgc ggcggcgccc 1680

acgaaccccg ggctggatag ctttactgag tccggcaagt tgcggttgtt cgacgtgatc 1740

gagaacggcc aggagcaggt cgagacgttg gatctcactg tcgtcgtgga taacgcggtt 1800

gtcgaggtgt atgccaacgg gcgctttgcg ttgagcacct gggcgagatc gtggtacgac 1860

aactccaccc agatccgctt cttccacaac ggcgagggcg aggtgcagtt caggaatgtc 1920

tccgtgtcgg aggggctcta taacgcctgg ccggagagaa attga 1965

<210> 24

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Conserved bioactive fragment of beta-fructofuranosidase of

Aspergillus niger (Position 57-62)

<400> 24

Gln Ile Gly Asp Pro Cys

1 5

<210> 25

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Conserved bioactive fragment of beta-fructofuranosidase of

Aspergillus niger (Position 119-132)

<400> 25

Asp Gly Ala Val Ile Pro Val Gly Val Asn Asn Thr Pro Thr

1 5 10

<210> 26

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Conserved bioactive fragment of beta-fructofuranosidase of

Aspergillus niger (Position 320-330)

<400> 26

Ser Gly Leu Pro Ile Val Pro Gln Val Ser

1 5 10

<210> 27

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Conserved bioactive fragment of beta-fructofuranosidase of

Aspergillus niger (Position 401-416)

<400> 27

Gly Asp Gln Tyr Glu Gln Ala Asp Gly Phe Pro Thr Ala Gln Gln Gly

1 5 10 15

<---

Claims (22)

1. A modified polypeptide for producing fructooligosaccharides, wherein the polypeptide is Aspergillus niger β-fructofuranosidase comprising the amino acid sequence of SEQ ID NO: 1 fused to a signal peptide selected from the group consisting of S. cerevisiae Alpha Factor (FAK), S. cerevisiae Complete Alpha Factor (FAKS), S. cerevisiae Alpha Factor_T (AT), Aspergillus niger Alpha Amylase (AA), Aspergillus awamori Glucoamylase (GA), Kluyveromyces maxianus Inulinase (IN), S. cerevisiae Invertase (IV), S. cerevisiae Killer Protein (KP), Gallus gallus lysozyme (LZ) and Homo sapiens Serum Albumin (SA), wherein the signal peptide comprises a region of four amino acids LEKR. 2. A modified polypeptide according to claim 1, wherein: a. FAK comprises the amino acid sequence SEQ ID NO: 3; b. FAKS contains the amino acid sequence SEQ ID NO: 4; c. AT comprises the amino acid sequence SEQ ID NO: 5; d. AA comprises the amino acid sequence SEQ ID NO: 6; e. GA comprises the amino acid sequence SEQ ID NO: 7; f. IN contains the amino acid sequence SEQ ID NO: 8; g. IV comprises the amino acid sequence of SEQ ID NO: 9; h. KP contains the amino acid sequence SEQ ID NO: 10; i. LZ comprises the amino acid sequence of SEQ ID NO: 11; and j. SA comprises the amino acid sequence SEQ ID NO: 12; and wherein the signal peptides provide for extracellular secretion of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. 3. A modified nucleic acid containing the nucleotide sequence of SEQ ID NO: 2, encoding the polypeptide according to claim 1. 4. A modified nucleic acid encoding the polypeptide according to claim 1. 5. The modified nucleic acid of claim 4, wherein the nucleic acid is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22. 6. An expression vector comprising a nucleic acid according to claim 3 or 4, operably linked to a promoter. 7. The expression vector according to claim 6, wherein the β-fructofuranosidase gene promoter is selected from the group consisting of AOX1, ADH3, DAS, FLD1, LRA3, THI11, GAP, YPT1, TEF1, GCw14 and PGK1. 8. The expression vector of claim 6, wherein the vector is selected from the group consisting of pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, pGAPZαC, pPIC3, pPIC3.5, pPIC3.5K, PAO815, pPIC9, PPIC9K, IL-D2, pHIL-S1, and the expression vectors are configured for secretory or intracellular expression of β-fructofuranosidase from Aspergillus niger as set forth in SEQ ID NO: 1. 9. A recombinant Pichia pastoris host cell for producing a modified polypeptide according to any one of claims 1 or 2, containing an expression vector according to claim 6. 10. A recombinant Pichia pastoris host cell according to claim 9, wherein the host cell is selected from the group consisting of Pichia pastoris Mut+, Mut S, Mut-, Pichia pastoris KM71H, Pichia pastoris KM71, Pichia pastoris SMD1168H, Pichia pastoris SMD1168, Pichia pastoris X33, Pichia pastoris GS115, or any other Pichia pastoris host strain. 11. Use of a modified polypeptide according to claim 1 for obtaining fructooligosaccharides.