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WO2011160573A1 - Procédé de production de rifamycine sv présentant une activité élevée et une pureté élevée - Google Patents

Procédé de production de rifamycine sv présentant une activité élevée et une pureté élevée Download PDF

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WO2011160573A1
WO2011160573A1 PCT/CN2011/076009 CN2011076009W WO2011160573A1 WO 2011160573 A1 WO2011160573 A1 WO 2011160573A1 CN 2011076009 W CN2011076009 W CN 2011076009W WO 2011160573 A1 WO2011160573 A1 WO 2011160573A1
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rifamycin
gene
strain
cytochrome
producing
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赵国屏
赵维
袁华
钟怡
覃重军
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C12P17/189—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms containing the rifamycin nucleus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
    • C12Y202/01—Transketolases and transaldolases (2.2.1)
    • C12Y202/01001—Transketolase (2.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to the fields of bioengineering and pharmaceuticals. More specifically, the present invention relates to the use of a cytochrome P450 gene or a transketolase gene or a protein encoded thereby for regulating the conversion of rifamycin SV to rifamycin B, and the production of high activity by bioengineering methods
  • Mediterranean amyloid Meditefranei is a strain isolated from a soil sample near St. Raphael in France in 1957. It is an actinomycete that produces the important antibiotic rifamycin.
  • Rifamycin belongs to the class of ansa antibiotics (ansamycins;), mainly used for the treatment of Mycobacterium tuberculosis
  • the initially isolated Mediterranean Amycolatopsis genus was able to synthesize a structurally related mixture of rifamycin. Later, after a series of physical and chemical mutagenesis, the main component of the industrial synthesis of Mycobacterium sinensis was rifamycin B (Floss and Yu 2005). The yield of rifamycin B was relatively high. However, its antibacterial activity is relatively low. After fermentation to obtain Rf-B, it is chemically or enzymatically converted to obtain highly active rifamycin SV, and finally further clinically obtained rifampicin or other pharmaceutically acceptable derivatives (Floss and Yu 2005)
  • the rifamycin biosynthesis pathway can be used to transform the target production strains by molecular biology and genetic engineering methods. It is the goal of scientists.
  • Floss et al. used the 3-amino-5-hydroxybenzoic acid (AHBA) synthase gene rz ⁇ as a probe to clone a segment of the rifamycin-producing gene cluster of the Mediterranean amygdaloid strain about 100 kb. DNA fragment. Subsequently, the function of most of these genes was experimentally identified (Floss and Yu 2005; Xu, Wan et al., 2005).
  • AHBA 3-amino-5-hydroxybenzoic acid
  • rifamycin The synthesis of rifamycin is based on AHBA, malonyl-CoA (2 molecules;) and methylmalonyl-CoA (8 molecules) as extension units. Proxamycin X, then, pre-ansamycin X is subjected to various modifications such as redox, acetylation, methylation, etc. via Rf-SV and finally Rf-B.
  • the rifamycin synthesis pathway is represented by the following formula, wherein rifamycin S is an oxidized form of rifamycin SV, which can be transformed into each other.
  • cytochrome P450 or a transketolase gene or a protein encoded thereby for the modulation of the conversion of rifamycin SV to rifamycin B.
  • the cytochrome P450 or transketolase gene or the protein they encode is derived from a strain that produces rifamycin B. In another embodiment, the cytochrome P450 or transketolase gene or the protein they encode is derived from Amycolatopsis mediterranei. In a preferred embodiment, the strain is selected from the group consisting of: ATCC 13685 Hehe ATCC 21789 or S699.
  • cytochrome P450 or transketolase genes or proteins encoded thereby are used alone or in combination.
  • cytochrome P450 or transketolase gene is selected from the group consisting of:
  • iii hybridizes under stringent conditions to a sequence defined by (; i) or (; ii) and encodes a sequence of cytochrome P450 or transketolase; and/or
  • nucleotide sequence is (i) or (ii) has more than 85% (preferably 90% or more) sequence identity and encodes a cytochrome P450 or a transketolase.
  • sequence homologous to the sequence of (3 ⁇ 4) encodes a cytochrome P450 or a transketolase.
  • the genes are ⁇ /75 (encoding transketolase) and ⁇ /7 encoding cytochrome ⁇ 450, respectively, and the encoded products of these two genes are involved in Rf-SV to Rf-B. Conversion. Inactivation or simultaneous inactivation of any of these two genes can accumulate Rf-SV (and its oxidized Rf-S) in the Mycobacterium sinensis, but not Rf-B. . After replenishing these two genes, the phenotype recovery of rifamycin SV to B can be achieved.
  • the homologous sequence is derived from a strain producing rifamycin B, preferably Mycobacterium sinensis (_yco/ato; w ec /terra «e/).
  • the strain is selected From: ATCC 13685 and ATCC 21789 or S699.
  • the rifamycin SV is blocked by converting the cytochrome P450 or the transketolase gene or a homologous gene thereof, respectively, or inhibiting the protein they encode. Rifamycin B.
  • inactivation of Rf-B in the Mediterranean amyrobacteria inactivation of the rifl5 ⁇ rifl6 gene causes blockade of the rifamycin SV to the rifamycin B synthesis pathway.
  • the inhibition is achieved by gene knockout, gene replacement, gene silencing, RNA interference or point mutation.
  • the inhibition is such that the 84th R in the protein encoded by the gene of the Phytophthora infestans producing rifamycin B is mutated to W, for example, such that ATCC 13685 and ATCC 21789 or The 84th R mutation in the protein encoded by the gene in S699 is W.
  • the expression of the cytochrome P450 or the transketolase gene or a homologous gene thereof is enhanced separately or simultaneously, or the function of the protein they encode is enhanced, and the rifamycin SV is converted to rifamycin B.
  • the enhancement is achieved by a method selected from the group consisting of: insertion or overexpression.
  • genomic recovery of rifamycin SV to B can be achieved by gene replenishment of and/or rifl6 in R. aureus-producing Amycolatopsis.
  • a strain for producing rifamycin SV or rifamycin S characterized in that the cytochrome P450 gene and/or the transketolase gene are inactivated in the strain Or the enzyme encoded thereby is inactivated, provided that the strain is not a Mycobacterium sinensis strain having the accession number CGMCC 4.5720.
  • the amount of rifamycin B produced by the strain is at most 50%, 40%, 30%, 20%, 10%, or even 0% of the rifamycin SV production.
  • the rifamycin SV produced by the strain can be used to synthesize an antibiotic having a rifamycin SV as a precursor, and the antibiotic can be one or more selected from the group consisting of: Rifabutin, rifapentine, rifampicin or rifamycin B.
  • the cytochrome P450 gene and/or the transketolase gene are selected from the group consisting of: (i) having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 23, and / or the nucleotide sequence of the sequence shown in SEQ ID NO: 24; (ii) a sequence homologous to the sequence in (3); (iii) hybridizing to a sequence defined by (3 ⁇ 4 or (ii) under stringent conditions, and encoding a sequence of a cytochrome P450 or a transketolase; and/or a nucleotide sequence of (i) or (ii) having more than 85% (preferably more than 90%) sequence identity and encoding a cytochrome P450 or transketol The sequence of the enzyme.
  • the strain is a natural strain or a genetically engineered strain.
  • the genetic engineering is carried out by a method selected from the group consisting of gene knockout, gene replacement, gene silencing, RNA interference or point mutation.
  • the strain is rifamycin-producing Amycolatopsis mediterranei or Amycolatopsis c/Amycolatopsis c/ Terra "e/) was transformed from genetic engineering.
  • the rifamycin-producing Mycobacterium sphaeroides is selected from the group consisting of: ATCC 21789, ATCC 13685 or S699, more preferably ATCC 21789.
  • a method of obtaining a strain for producing rifamycin SV or rifamycin S comprising the steps of: (a) providing a strain for producing rifamycin; The cytochrome P450 gene and/or the transketolase gene in the strain is inactivated or the enzyme encoded thereby is inactivated.
  • the inactivation is achieved by genetic engineering selected from the group consisting of: gene knockout, gene replacement, gene silencing, RNA interference, point mutation.
  • the rifamycin-producing strain is a strain producing rifamycin B, preferably a rifamycin-producing Mediterranean Phytophthora ( ⁇ co/ato; ⁇ mediterranei) More preferably, ATCC 21789, ATCC 13685 or S699.
  • a method of screening a strain for producing rifamycin SV or rifamycin S comprising: (a') providing a strain for producing rifamycin; b') detecting the cytochrome P450 gene and/or the transketolase gene of the strain, or measuring the activity of the enzyme encoded thereby, if there is no cytochrome P450 gene and/or transketolase in the strain The activity of the gene or the enzyme encoded thereby, or the activity is significantly lower than the positive control strain producing rifamycin B, indicating that the strain can be used to produce rifamycin sv.
  • the detection of the gene is determined by a method selected from the group consisting of:
  • the positive control strain producing rifamycin B is selected from the group consisting of: ATCC 21789, ATCC 13685 or S699.
  • a method of producing rifamycin SV, rifamycin S or a derivative thereof comprising: A) providing a strain of the invention, using the invention a strain produced by the method, or a strain obtained by the method of the present invention; B) producing rifamycin SV or rifamycin by the strain And S) optionally further producing a rifamycin derivative selected from the group consisting of rifamycin SV or rifamycin S obtained in step B).
  • the derivative of rifamycin SV or rifamycin S is selected from the group consisting of: rifabutin, rifapentine, rifampicin or rifamycin B.
  • the production comprises the steps of inoculation, fermentation, extraction, concentration, and the like.
  • the method further comprises performing further selection of the strain based on the stability and yield of the strain.
  • a further aspect of the invention relates to a method of preparing a highly active rifamycin SV, the method comprising: a rifl5 and/or rifl6 gene in a strain of Mycobacterium sinensis producing rifamycin B, or Homologous genes that are highly homologous and have the same or similar function (; at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical) are manipulated The gene and the homologous gene are inactivated to obtain a mutated strain which is a strain producing rifamycin Rf-SV.
  • the strain is selected from the group consisting of Mycobacterium sinensis producing rifamycin B.
  • the and/or gene or a gene highly homologous thereto is inactivated to obtain a mutated strain.
  • the method further comprises the steps of: cultivating the mutant strain, identifying its ability to produce rifamycin SV, and screening for a further high yield, high purity rifamycin SV strain.
  • a further aspect of the invention relates to a strain prepared by the method of the invention, which is selected from the group consisting of Mycobacterium amyloliquefaciens, wherein the strain comprises and/or a gene or a gene highly homologous thereto, and/ Or genes or genes that are highly homologous to them have been inactivated.
  • Another aspect of the invention relates to a method of preparing rifamycin B, the method comprising: replenishing the Mediterranean Sea producing rifamycin B in a strain of Mycobacterium sinensis producing rifamycin SV
  • the rifl5 and/or rtfl6 gene in the strain of Mycobacterium sclerotium, or a gene highly homologous thereto (the identity is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%), the gene and the gene highly homologous thereto are functionalized to obtain a mutant strain which is a strain producing rifamycin Rf-B.
  • the strain is selected from the group consisting of Mycobacterium sphaeroides producing rifamycin SV.
  • the gene and the gene or a gene highly homologous thereto are replenished to obtain a mutant strain.
  • the method further comprises the steps of: cultivating the mutant strain, identifying the same The ability to produce rifamycin B was screened for strains with further high yield, high purity rifamycin B.
  • a further aspect of the invention relates to a strain prepared by the method of the invention selected from the group consisting of Mycobacterium sinensis, wherein the strain comprises and/or a gene or a gene highly homologous thereto.
  • Figure 1 Identification of the rifl5 mutant on the chromosome of A. oxysporum ATCC 21789.
  • Lane 1 is the lkb molecular weight marker
  • lanes 2-8 are the picked rifl5 mutant clones 1-7
  • lane 9 is the negative control (no DNA template lane 10 is the positive control.
  • Figure rifl5 gene is inactivated and inactivated back Effect of supplementation on the production of rifamycin Rf-B and Rf-SV
  • Figure 2A is a BPC spectrum analysis of ATCC 21789 fermentation broth
  • 2B is a BPC spectrum analysis of the fermentation broth of ATCC 21789 rifl5 mutant (clone 1 in Figure 1)
  • 2C is a BPC map analysis of the fermentation broth of the rifl5 mutant clone 1 transferred into the empty plasmid pDXM4, and 2D is the BPC pattern analysis of the fermentation liquid of the rifl5 mutant clone 1 replenishing strain.
  • Figure 3 PCR identification of the rifl6 mutant on the chromosome of A. oxysporum ATCC 21789.
  • Lane 1 is the lkb molecular weight marker
  • lanes 2-16 are the picked rifl6 mutant clones 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, lanes 17 is a negative control (unsuccessful DNA template) and lane 18 is a positive control (; genomic DNA;).
  • Figure 4 Effect of gene inactivation and replenishment on the production of rifamycin Rf-B and Rf-SV.
  • Fig. 4A is a BPC spectrum analysis of the fermentation broth of ATCC 21789
  • Fig. 4B is a BPC spectrum analysis of the ethyl acetate extract of the fermentation broth of Mycobacterium sinensis ATCC 21789 after gene inactivation (Cloning 2 in Fig. 3).
  • Fig. 4C shows the BPC map analysis of the fermentation broth of the strain in which the rifl6 mutant was transferred into the empty plasmid pDXM4
  • Fig. 4D shows the BPC map analysis of the fermentation broth of the strain rifl6 supplemented by the mutant clone 2.
  • Figure 5 is a thumbnail of the rz/gene cluster
  • 5b is ATCC 13685, ATCC 21789, S699 and
  • Figure 6 shows the rifamycin-producing phenotype in the medium plus apramycin antibiotic (dashed line in 6a) or no apramycin antibiotic (solid line in 6a).
  • Figure 6b shows the loss of the pDXM4-P450 plasmid in different media. Straight bars represent no apramycin antibiotics added to the U32 (pDXM4-P450) medium, and wavy columns represent the addition of apramycin to the U32 (pDXM4-P450) medium.
  • Figure 7 The three-dimensional structure of the P450 in the U32 simulated with the Swiss-Pdb Viewer.
  • the bat model indicates that the amino acid is Rifl6 R84 amino acid (Arg or Trp), and the SNP site is just at the end of a ⁇ -sheet, presumably the pocket of the binding substrate.
  • FIG. 8 Amino acid sequence alignment of S699, U32, ATCC13685 and ATCC 21789.
  • the amino acid sequences of the cytochrome P450 proteins of Mycobacterium sphaeroides strains S699, U32, ATCC 13685, and ATCC 21789 were compared with each other using CLUST software (; Alignment;). It can be seen that U32 differs from the other three in amino acid 84 (R84W); but at position 295, U32 is only different from S699 (G295E), and is consistent with the other two ATCC 13685, ATCC 21789. Therefore, we can speculate that the change of R84W affects the conversion of rifamycin SV to rifamycin B in U32 strain. Note: The initial codon prediction of U32 is 31 amino acids ahead of S699, which does not affect the true expression of its protein. Summary of the invention
  • Cytochrome P450 genes such as and / or transketolase genes (such as the activity of ⁇ / 7 is decisive for the conversion of rifamycin SV to rifamycin B)
  • strains for efficient preparation of high-purity rifamycin SV can be obtained, or assays for the activity of these key genes can be used to screen strains that can be used to efficiently prepare high-purity rifamycin SV.
  • the rifamycin-producing SV-producing strain can be transformed into a rifamycin-producing strain using the supplementation of these key genes.
  • cytochrome P450 genes such as and/or transketolase. Genes (such as the use of their encoded proteins, including the inactivation of these two genes separately or simultaneously, blocking the conversion of rifamycin SV to rifamycin B, and the replenishment of these two genes to make rifamycin SV Further converted to rifamycin 6.
  • the inventors also provide a strain for producing high activity and high purity rifamycin SV, a preparation and screening method thereof, and a high activity and high purity rifamp using the strain. The method of SV, thus completing the present invention.
  • the inventors participated in the transformation process of rifamycin SV to rifamycin B by inactivation.
  • the genes of two key enzymes (especially the genes rifl5 and/or rifl6) block the synthesis of Rf-SV to Rf-B, thereby increasing the yield of highly active rifamycin Rf-SV, and
  • the components of the fermentation product are purified.
  • the rifl6 gene is a sequence ratio of the three strains of wild-type rifamycin-producing strains after the whole genome sequencing of a strain of Mycobacterium sinensis strain U32 producing rifamycin Rf-SV. Found during the analysis.
  • the amino acid sequences of the cytochrome P450 proteins of Mycobacterium sphaeroides strains S699, U32, ATCC 13685, and ATCC 21789 were aligned with each other using CLUST software. Studies have shown that: 1 in the gene encoding cytochrome P450 is W in U32 (84 amino acids, the coding sequence is tgg;), and R is encoded in ATCC 13685 and ATCC 21789 and S699. For the egg table 1).
  • U32 is only different from S699 (G295E), and it is consistent with the other two ATCC 13685, ATCC 21789. (Note: The initial codon prediction of U32 is 31 amino acids ahead of S699, which does not affect the true expression of the protein.)
  • the inventors believe that the mutation in the U32 is most likely to cause Rf-SV to The key reason for Rf-B blockage. Therefore, the inventor replenished the plasmid containing wild-type ATCC 21789 gene (pDXM4-P450) in U32, and used an empty plasmid as a control.
  • rifamycin B can be produced in a large amount in the replenished strain, and the fermentation of the empty plasmid strain is carried out. There is still only rifamycin SV and its oxidized Rf-S in the liquid, and no Rf-B is produced (Fig. 5). This proves to play a key role in the conversion of Rf-SV to Rf-B.
  • the inventors further investigated the effect of the stability of the plasmid pDXM4-P450 on the yield of different rifamycin derivatives. As a result, it was found that Rf-SV and Rf-S in the fermentation broth rapidly accumulated with the loss of the replenishing plasmid pDXM4-P450 (Fig. 6). Thus, it is verified from another angle that it plays a role in the conversion of Rf_SV to Rf-B.
  • the inventors also interrupted the gene in the wild-type ATCC 21789 producing rifamycin B, and found that rifamycin B was no longer synthesized. Further replenishment experiments revealed that the supplemented strain recovered rifamycin Rf— The production of B, without the detection of any Rf-SV production (Fig. 4), indicates that the gene is indeed not only very important in the transformation of rifamycin SV to B, but also plays a necessary role.
  • the inventors further knocked out the rifl5 gene encoding the transketolase in ATCC 21789. Result Now, the inactivation and replenishment of this transketolase produces the same phenotype as P450 inactivation and replenishment (Fig. 2). It is proved that the function of rifl5 is also necessary for the conversion from Rf-SV to Rf-B.
  • the present inventors identified a key gene in the transformation of rifamycin SV to rifamycin B, namely a cytochrome P450 gene (such as and/or a transketolase gene (such as rifl5, and The regulation and screening of the activity of these genes successfully obtained high-yield and high-purity rifamycin SV strains, which can be used in industrial production. Cytochrome P450 gene and transketolase gene
  • cytochrome P450 gene or gene is used interchangeably and refers to a gene encoding cytochrome P450 in a rifamycin-producing bacterium, preferably having SEQ ID NO: 2 (eg, ATCC 21789 or ATCC)
  • SEQ ID NO: 2 eg, ATCC 21789 or ATCC
  • the ORF sequence of the same gene as shown in 13685 or SEQ ID NO: 24 (for example, the rifl6 gene in S699), or may be highly homologous to the sequence (for example, homology is at least 50%, 60) %, 70%, 80%, 90%, 95%), or may be a molecule that hybridizes to the gene sequence under stringent conditions or a family gene molecule that is highly homologous to the above molecule, and inhibition of expression of the gene may hinder
  • the conversion of rifamycin SV to rifamycin B can be used to efficiently produce high-purity rifamycin SV, and the replenishment of this gene can promote the
  • transketolase gene or “gene” are used interchangeably and refer to a gene encoding a transketolase in a rifamycin-producing bacterium, preferably having the SEQ ID NO: 1;
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) Hybridization is carried out with a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42. C et al; or (3) identity between only two sequences Hybridization occurs at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more.
  • a sequence that hybridizes to a particular sequence under stringent conditions can be the complement of the particular sequence.
  • the full length sequence of the gene nucleotide of the present invention or a fragment thereof can be usually obtained by a conventional method such as PCR amplification, recombinant method or artificial synthesis.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the cytochrome P450 gene and the transketolase gene of the present invention are preferably the genes of the Phytomycin-producing Phytophthora infestans ( ⁇ co/ato; ⁇ ecftterrawe/), which are obtained from other producing countries.
  • the fumycin B fungus is highly homologous to the Mediterranean amyrobacteria; if it has 50% or more, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more Other genes, more preferably 85% or more, such as 85%, 90%, 95%, or even 98% sequence identity, are also within the equivalent scope of the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
  • the homologous sequence of the gene and the gene ie, the gene encoding the cytochrome P450
  • the homologous sequence in the Mediterranean Phytophthora oxysporum producing rifamycin B has the same function, that is, has the regulation of rifampy
  • the conversion of the SV into rifamycin B is such that these homologous sequences are responsible for encoding the transketolase or cytochrome P450 in the Mycobacterium sinensis.
  • the term “inactivation” means that the cytochrome P450 gene and/or the transketolase gene are not normally expressed in a manner that the corresponding enzyme or the amount of enzyme or enzyme activity expressed is significantly reduced.
  • the term “inactivated strain” refers to a strain which does not normally express a cytochrome P450 gene and/or a transketolase or a correspondingly reduced amount of enzyme or enzyme activity expressed.
  • the amount of cytochrome P450 and/or transketolase gene expression or the activity of the expressed enzyme in genetically engineered strains is reduced by 70 compared to unmodified strains. %, 80%, 90%, 95%, 98%, 99%, or even 100%.
  • Related genes can be inactivated by methods known in the art, such as related gene knockout, gene replacement, gene silencing, RNA interference, or mutation (preferably gene knockout, point mutation; etc.) method.
  • DNA homologous recombination can be used to construct a related gene disruption vector, and the target gene can be knocked out (Ding Xiaoming et al., using homologous recombination to establish a gene replacement/interruption system of the U32 chromosome of Mycobacterium sinensis, Journal of Bioengineering , 2002, 18(4): 43 1-437).
  • the method known in the art can be used to determine whether the gene has been inactivated, for example, by Southern hybridization or by PCR amplification to detect whether the target gene is mutated; the phenotypic change of the mutant strain is detected by HPLC-MS (eg, The method of changing the composition of rifamycin or the related physiological and biochemical experiments to determine whether the mutated gene is inactivated.
  • the present invention provides a strain which can produce a highly active rifamycin SV in which a gene and/or a gene or a gene highly homologous thereto has been inactivated.
  • This strain is a Mediterranean formula that can produce Rf-B. Ecftterrawe/) or obtained by genetic engineering.
  • the present invention also provides a strain capable of producing rifamycin B, which is a gene inserted into and/or highly homologous to a strain producing rifamycin SV, and the gene and its encoding
  • the cytochrome P450 gene and/or transketolase have normal functions.
  • the strain is genetically engineered from the Rf-SV-producing Mediterranean Phytophthora (_yco/ato M/s ecftterrawe/).
  • inactivated strains can be screen for inactivated strains using the knowledge disclosed in the prior art. For example, when a vector is constructed, a resistance gene is introduced, and the related gene is interrupted by an interrupt vector. The gene-inactivated strain was screened using a culture medium containing resistance, and the screened strain was verified by the above method for verifying gene inactivation. A method for determining the ability of a strain to produce an antibiotic can be analyzed by HPLC-MS. Cultivation and utilization of inactivated strains
  • the resulting strain can be cultured and expanded under conditions conventionally used in the art, for example, amyrobacteria can be at 26 Incubate in a solid or liquid medium at °C -30 °C (Wang W et al., 2002).
  • the present invention also provides a method for efficiently preparing a high-purity rifamycin SV or a non-rifamycin B antibiotic which is a precursor thereof using an inactivated strain.
  • non-rifamycin B antibiotics with rifamycin SV as a precursor "" or “derivative of rifamycin SV” refers to an antibiotic that is first produced in industrial production by rifamycin sv, and then further synthesized with rifamycin SV as a precursor, such as rifabutin. , rifapentine or rifampicin.
  • rifamycin SV can be carried out under industrial conditions using inactivated strains obtained or screened, for example by fermentation or the like.
  • the prepared rifamycin SV can be further utilized to produce a non-rifamycin B antibiotic having rifamycin SV as a precursor, such as rifabutin, rifapentine or rifampicin.
  • the term “enhanced activity” means that the expression activity or enzyme activity of an increased or expressed expression of a cytochrome P450 gene and/or a transketolase gene is significantly enhanced.
  • activity-enhancing strain refers to a strain having an increased expression activity of a cytochrome P450 gene and/or a transketolase gene or a significant increase in the amount of the corresponding enzyme or enzyme expressed.
  • the amount of cytochrome P450 gene and/or transketolase gene expression or the activity of the expressed enzyme in genetically engineered strains is increased by 50% compared to unmodified strains, 60%, 70%, 80%, 85 %, 90% or more.
  • the activity of the relevant gene can be enhanced by methods known in the art, for example, by insertion or overexpression. Methods known in the art can be used to determine whether the gene or enzyme activity is enhanced, for example, by HPLC-MS to detect phenotypic changes in the strain.
  • the obtained strain can be cultured and amplified under the conditions conventionally used in the art, for example, Mycobacterium sinensis can be Incubate in a solid or liquid medium at 26 °C -30 °C (Wang W et al., 2002).
  • the present invention also provides a method of preparing rifamycin B or converting rifamycin B into rifamycin SV and a non-rifamycin B antibiotic thereof using the supplemental strain or activity-enhancing strain.
  • non-rifamycin B antibiotic with rifamycin SV as a precursor means that rifamycin SV is first prepared in industrial production, and then further synthesized with rifamycin SV as a precursor. antibiotic.
  • rifamycin B The production of rifamycin B can be carried out under industrial conditions using a prepared supplement strain or an activity-enhancing strain, for example, by fermentation or the like.
  • the rifamycin B produced can be further converted into rifamycin SV and a non-rifamycin B antibiotic with rifamycin SV as a precursor, such as rifabutin, rifapentine, and Rifampin.
  • rifamycin SV a non-rifamycin B antibiotic with rifamycin SV as a precursor, such as rifabutin, rifapentine, and Rifampin.
  • the culture of Mycobacterium amyloliquefaciens was carried out using Ben's medium (Bennet Medium, Wang W et al., 2002); LB medium for E. coli culture.
  • the final concentrations of antibiotics added to the medium were: ampicillin 100 ⁇ , apramycin apramycin 30 ⁇ , erythromycin 200 g/ml.
  • Example 1 Whole genome sequencing of rifamycin-producing Rf_SV strain U32 and its sequence alignment with rifamycin-producing B strain
  • the rifl6 gene is a sequence ratio of the three wild-type rifamycin-producing strains of the strain of U. sinensis strain U32 which produces rifamycin Rf-SV. Found during the analysis. in particular:
  • NA indicates that the mutation of the gene does not affect the translation codon, the product of the gene is involved in the step before the formation of rifamycin SV in the rifamycin B biosynthesis pathway, or it is reported that the mutation does not affect the rifampicin Production of vegetarian B.
  • ORF15B is Rif 15
  • ORF16 is Rif l6.
  • the encoded P450 protein is a cytoplasmic protein with no transmembrane region, and the SNP site in U32 is just at the end of a ⁇ -sheet.
  • the Swiss-Pdb Viewer to simulate the three-dimensional structure, it can be seen that the SNP is around the active pocket of the P450, suggesting his important steric hindrance (Figure 7).
  • the PCR product of the gene containing its own promoter region in ATCC 21789 (the primers used are shown in SEQ ID Nos: 19 and 20) was cloned into the Mediterranean-type Mycobacterium serovar free-type multicopy plasmid using conventional molecular biology methods.
  • a plasmid pDXM4-P450 containing the wild type ATCC 21789 rifl6 gene was obtained at the EcoRV site of pDXM4 (see Ding Xiaoming 2001).
  • pDXM4-P450 containing rifl6 was transferred to U32 by electroporation (refer to Ding Xiaoming 2001). The result is shown in Figure 5.
  • Example 3 Effect of stability of U32 replenishing plasmid pDXM4-P450 on conversion of Rf_SV to Rf_B If an apramycin antibiotic is added to the medium, the plasmid pDXM4-P450 can be stabilized in the U32 strain, whereas the plasmid will The bacteria are lost during the proliferation process. Adding apramycin antibiotics to the culture medium as a control, we sampled in the U32 fermentation broth without the addition of apramycin antibiotics for 2 days, 3 days, 5 days, and together with the fermentation broth supplemented with antibiotics. HPLC-MS detection.
  • the upstream and downstream homologous fragments of the inactivated rifl5 or rifl6 PCR product were sequentially cloned into pBluescript KS (-X was purchased from Stratagene) by molecular biological methods, and the upstream fragment was Hi III and EcoR V, and the downstream fragment was EcoR V and EcoR I; rz/7 ⁇ upstream fragment is Hi III and coR V, downstream fragment is coR V and coR I), and finally the apramycin resistance gene is inserted into the middle of the upstream and downstream homologous fragments.
  • the homologous recombination knockout plasmid was transferred to ATCC 21789 by electroporation at the restriction site 3 ⁇ 4oR V (see Ding Xiaoming 2001).
  • the homologous recombination knockout plasmid only has one homologous fragment recombined with the chromosome, The obtained apramycin-resistant transformants are single-exchange; and if both homologous fragments on the plasmid are recombined, a mutant in which the corresponding gene is inactivated is obtained.
  • the sequences of the inactivating primers Rifl5KO ll, Rifl5KO12, Rifl5KO21 and Rif 15KO22 of rifl5 are shown in SEQ ID NOs: 3-6 and rz/7 ⁇ 5 inactivation primers Rif 16KO 11, Rifl6KO 12, Rifl6KO21 and Rifl6KO22, respectively. They are shown in SEQ ID NOs: 7-10, respectively.
  • the primers shown in SEQ ID NOs: 11-12 and the primers shown in SEQ ID NOs: 13-14 were respectively used for the rif 15 mutation.
  • the body or rif 16 mutant was subjected to PCR verification.
  • the erythromycin promoter and the ORF (the sequence of which is shown in SEQ ID NO: 1) or the rif 16 ORF (the sequence of which is shown in SEQ ID NO: 2) are first linked to pBluescript KS by molecular biological methods. ) Then, the vector was transferred to the coRV site of pDXM4, and the empty plasmid was used as a control, and pDXM4 containing rtfl5 and rifl6 was transferred to the corresponding ATCC 21789 rifl5 or mutant by electroporation (Ding Xiaoming 2001).
  • the supernatant of the fermentation broth cultured for a suitable period of time was adjusted to pH 2-3, extracted with an equal volume of ethyl acetate, and filtered through a 0.22 ⁇ m filter, and the rifamycin component was directly analyzed by HPLC-MS.
  • MS parameters are: mass spectrum range: 550-1 100 m/z (MS scan rate 1.03 and resolution ⁇ 0.5 amu), nebulizer 40 psi, Gas (N 2 ) temperature 350 ° C, gas flow rate 91 / min, VCap 3500V, fragmentor 160V, separator (Skimmer) 65V, Octopole RF 750 V, Ext Dyn standard 2GHz (3200).
  • the inventors sequenced the U32 genome and aligned and analyzed with the three rifamycin-producing strain sequences (as described in Example 1).
  • One of the genes encoding the cytochrome P450 was found in U32. W (84 amino acids), and R in ATCC 13685 and ATCC 21789 and S699.
  • the rifl6-encoded P450 protein is a cytoplasmic protein with no transmembrane region, and the SNP site in U32 is just at the end of a ⁇ -sheet.
  • the inventor replenished the plasmid containing the wild type ATCC 21789 rifl6 gene (PDXM4-P450) in U32 (as described in Example 2), and simultaneously used the empty plasmid as a control, and the rifos in the fermentation broth of the obtained strain.
  • the assay was performed (as described in Example 6;). The results showed that a large amount of rifamycin B was detected in the fermentation broth of the replenished strain, while only the rifamycin was found in the fermentation broth of the empty plasmid strain.
  • the SV and its oxidized Rf-S have no Rf-B production (Fig. 5). This proves to play a key role in the conversion of Rf-SV to Rf-B.
  • the inventors also conducted an experiment to compensate for the effect of the stability of the plasmid pDXM4-P450 on the yield of different rifamycin derivatives (as described in Example 3). If apramycin antibiotic is added to the medium, the plasmid pDXM4-P450 can be stabilized in the U32 strain, whereas the plasmid is lost during bacterial proliferation. As a result, it was found that Rf-SV and Rf-S in the fermentation broth rapidly accumulated as the replenishing plasmid pDXM4-P450 was gradually lost without the addition of apramycin antibiotic. This proves from another perspective that it plays a role in the conversion of Rf-SV to Rf-B (Fig. 6).
  • P450 is a monooxygenase and its main function is to hydroxylate the substrate (Lamb, Skaug et al., 2002).
  • the inventors believe that other enzymes and related genes, such as 3 ⁇ 4 rifl5, may be involved in the biochemical conversion process from Rf-SV to Rf-B.
  • the inventors knocked out ⁇ /75 (encoding a transketolase) which may also function in ATCC 21789 (as described in Example 4;). It was found that the inactivation and replenishment of this transketolase produced the same phenotype as the inactivation of ⁇ 4500 /76) (Fig. 2).
  • ATCC 21789 produces a large amount of Rf-B (residence time is about 9 min, as shown in Fig. 2A;). It can be seen from Fig. 2B that after being inactivated, a mutant of R. sinensis-producing A. oxysporum ATCC 21789 (a candidate clone with a successful mutation in Figure 1;) accumulates rifamycin Rf-SV in the fermentation broth. (; retention time is about 10.5 min) and its oxidized Rf-S (residence time is about 15 min), and no longer synthesized into rifamycin Rf-B. The introduction of a normal gene restored the phenotype of the mutant (Fig.

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Abstract

L'invention concerne un procédé de production de rifamycine SV (Rf_SV) présentant une activité élevée et une pureté élevée, et des utilisations du gène du cytochrome P450 ou du gène de transcétolase ou des protéines codées par les deux gènes dans la régulation de la transformation de la rifamycine SV en rifamycine B (Rf_B). En inactivant l'une ou les deux enzymes clés Rif15 et Rif16 nécessaires pour la transformation de Rf_SV en Rf_B dans la voie de biosynthèse de la rifamycine dans des souches produisant la rifamycine B, le procédé de la présente invention permet d'obtenir de la rifamycine SV présentant une activité élevée et une pureté élevée.
PCT/CN2011/076009 2010-06-21 2011-06-21 Procédé de production de rifamycine sv présentant une activité élevée et une pureté élevée Ceased WO2011160573A1 (fr)

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