WO2022017523A1 - 生产脱环氧埃坡霉素b的重组菌及其用途 - Google Patents

生产脱环氧埃坡霉素b的重组菌及其用途 Download PDF

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WO2022017523A1
WO2022017523A1 PCT/CN2021/108266 CN2021108266W WO2022017523A1 WO 2022017523 A1 WO2022017523 A1 WO 2022017523A1 CN 2021108266 W CN2021108266 W CN 2021108266W WO 2022017523 A1 WO2022017523 A1 WO 2022017523A1
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epothilone
gene
medium
recombinant bacteria
desepoxidized
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French (fr)
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唐莉
邱荣国
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北京华昊中天生物医药股份有限公司
成都华昊中天药业有限公司
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Priority to CN202180006642.0A priority Critical patent/CN114761564B/zh
Priority to EP21845599.6A priority patent/EP4063509A4/en
Priority to JP2022537248A priority patent/JP7528222B2/ja
Priority to US17/758,042 priority patent/US20230340550A1/en
Publication of WO2022017523A1 publication Critical patent/WO2022017523A1/zh
Priority to JP2024018206A priority patent/JP2024079669A/ja

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Definitions

  • the present application relates to recombinant bacteria and uses thereof, and more particularly to recombinant cellulosic bacteria for producing desepoxidized epothilone B and uses thereof.
  • Epothilone is a class of macrolide antibiotics with antitumor activity produced by soil microorganisms and acting on tubulin. It was first isolated from the myxobacter Sorangium cellulosum in the early 1990s. ), there are mainly two products A and B, and their structures are as follows.
  • US Patents US5843718 and US6033883 describe the production of novel polyketides by recombination of the modular PKS gene in recombinant host cells, as well as in host cells such as Streptomyces, E. coli and Myxobacterial ) in the recombinant method for the production of polyketides by introducing heterologous PKS genes into host cells that cannot produce epothilones, such as Streptomyces coelicolor and Myxococcus xanthus, by introducing heterologous epothilone genes
  • the production of epothilone A and B has been reported (Tang, et al. Science 287:640-642; Julien & Shah, 2002, Amtimicrobial. Agent Chemother. 46(9):2772-2778, incorporated herein by reference).
  • a recombinant bacterium Sorangium cellulosum for fermentative production of desepoxidized epothilone B is provided, wherein the epoK gene in the epothilone biosynthesis gene cluster in the recombinant bacterium is inactivated .
  • the epoK gene is inactivated by point mutation, insertion, deletion, substitution.
  • the recombinant bacterium is a recombinant bacterium from So ce90 (Sorangium cellulosum So ce90).
  • the epoK gene comprises the nucleotide sequence of SEQ ID NO.1, and the insertion sequence is the 291st in the nucleotide base of SEQ ID NO.1 to 1960th. More specifically, the recombinant bacteria is C. cellulosum BG03-09K.
  • the recombinant genetic engineering strain provided by the invention comprises the modification of the epothilone biosynthesis gene, and the modified gene is the DNA of the epoK gene in the epothilone biosynthesis gene cluster in the host cell of the producing bacteria through DNA recombination technology
  • the sequence is inactivated by inserting a DNA fragment, thereby allowing the epothilone biosynthesis gene in the host cell to be modified.
  • the recombinant bacteria provided by the present invention can be obtained by recombination technology, which uses the method of homologous recombination to clone the adjacent two end regions of the gene or functional group to be changed into a suicide vector to obtain a conjugation transfer plasmid vector, Through two-hybrid recombination between the genes in the suicide vector and the homologous genes in the genome contained in the host cell nucleus, one or more genes or functional groups involved in the biosynthesis of epothilone are inserted into inactivation or replaced to obtain recombination. genetically engineered production strains.
  • one or two antibiotic resistance genes are inserted in the middle of the two regions of the gene to be changed, and DNA recombination is performed in the host cell by the homologous sequences of the epoK gene on both sides of the inserted antibiotic resistance gene to replace the host cell.
  • Wild-type gene a recombinant strain obtained by inserting the antibiotic resistance gene into the epoK group and causing its loss of function. This inactivation can also be accomplished by random or point gene mutation, deletion or substitution.
  • the resulting recombinant biosynthetic gene is different from the gene in the natural producing bacteria, so that the epothilone derivative or intermediate product can be produced as the main product in the recombinant host cell, for example, the host containing the epothilone synthesis gene cells, which can produce epothilone B as the main product, when the DNA sequence of the epoK gene in the epothilone biosynthesis gene is inserted into other DNA sequences, resulting in inactivation, resulting in the production of desepoxyepothilone B by the recombinant strain as the main product.
  • the recombinant bacteria of the present invention can be stored for a long time and have good stability.
  • BG03-09K culture medium after culturing in BCF medium for 3 ⁇ 1 days with 10-20ml of 90%-100% sterile glycerol in a sterile bottle, take 1-1.5ml of the mixed solution Packed in a labeled sterile cell cryopreservation tube to prepare a cell bank of production bacteria, and immediately placed in an ultra-low temperature below -70 °C for cryopreservation, the bacteria maintains good long-term stability.
  • PCR analysis and gene sequencing analysis (16S rRNA gene, EpoD-MT functional region, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene) of the recombinant C. fibrocystis strain BG03-09K housekeeping gene are used as strain genetic identification The goal.
  • Three pairs of oligo primers were designed for PCR amplification, and the obtained PCR products were sequenced and analyzed.
  • the partial sequence of the housekeeping gene of BG03-09K in the database it is found that the DNA sequence of BG03-09K and the sequence of S. cellulosum have little difference in the evolutionary distance at the nucleotide level, which proves that BG03 -09K is the same species as S. cellulosum.
  • a method for producing desepoxidized epothilone B comprising culturing the recombinant Helicobacter cellulosus described in the first aspect in a culture medium.
  • the medium used in the production method is a water-soluble medium, such as seed medium BCF comprising: 0.8-1.0% soy peptone, 0.5-0.7% fructose, 0.05%-0.1% MgSO 4 .7H 2 O and 25-50 mM HEPES, pH 7.2-7.6; production fermentation medium FBCF contains: 0.6-0.7% soy peptone, 0.5-0.7% fructose, 0.05-0.1% MgSO 4 .7H 2 O and a solution of trace elements 5-10 mg/L, and the pH is 7.1 ⁇ 0.3.
  • seed medium BCF comprising: 0.8-1.0% soy peptone, 0.5-0.7% fructose, 0.05%-0.1% MgSO 4 .7H 2 O and 25-50 mM HEPES, pH 7.2-7.6
  • production fermentation medium FBCF contains: 0.6-0.7% soy peptone, 0.5-0.7% fructose, 0.05-0.1% MgSO 4 .7H 2 O and a solution of trace
  • the production method comprises adding XAD-16 resin, eg, 2%-5% XAD-16 resin, to the fermentation medium for fermentative production.
  • the daily split feeding is performed during the fermentation process of the production method.
  • the production method further comprises purifying desepoxidized epothilone B using a PC chromatographic column and a C chromatographic column, the purification eluting with a 62-80% methanol solution, such as 62%, 67 %, 72%, 77%, 80% methanol.
  • Fermentation culture process parameters culture temperature is set at 34°C, acceptable range is 34 ⁇ 1°C; pH is set at 7.1, acceptable range is 7.1 ⁇ 0.3, adjusted with acid-base solution; dissolved oxygen control set point is 30%-50%, set by The air intake volume and the stirring speed are controlled together, and the defoamer is fed back to defoaming.
  • the feed was fed in batches, starting from the afternoon of the 3rd ⁇ 1st day, 2 ⁇ 1 times a day, respectively, and soy peptone solution and fructose solution were added each time.
  • Fermentation yield should be ⁇ 240mg/L, and can be as high as 500mg/L or more.
  • the seed culture and fermentation culture conditions are preferably 34°C, and the acceptable range is 34 ⁇ 1°C.
  • the fermentation culture conditions are preferably set to pH 7.1, and the acceptable range is 7.1 ⁇ 0.3.
  • the fermentation culture conditions are preferably started at the fermentation time of 3 ⁇ 1 days, and the feeding is carried out 2 ⁇ 1 times a day, respectively, and the soy peptone solution and the fructose solution are supplemented.
  • the fermentation culture conditions preferably extend the fermentation culture time to 14 days ⁇ 2 days by batch feeding, so that a higher yield can be obtained.
  • the feed liquid is separated from the XAD resin and the bacteria/bacteria liquid with a 100-200 mesh screen, the intercepted XAD is rinsed with an equal amount of water and then loaded into the XAD resin column, and the XAD resin is eluted with >90% methanol , and collect the methanol eluate containing the product as the XAD eluate.
  • the collected solution that meets the standard after the above PC column treatment is added with purified water to prepare a sample solution with a methanol concentration of about 50% ⁇ 5 (v/v), which is loaded on the C chromatographic column (C18 filler), and 62-80% methanol solution is used. Elute and collect product fractions.
  • the HPLC purity of the deepoxidized epothilone B in the collected solution that meets the standard after C column treatment can reach 98%, and the peak area of other single impurities should not be greater than 1.0%.
  • the total recovery yield after PC column and C column treatment greater than 70%.
  • the HPLC purity of the desepoxidized epothilone B in the collection solution that meets the standard after C column treatment reaches 98%, and the peak area of other single impurities is not more than 1.0%
  • Figure 1 HPLC profile of the product obtained from the fermentation of BG03-09K. Among them, desepoxidized epothilone A is at a position around 4.4 minutes, and desepoxidized epothilone B is at a position around 5.1 minutes. (Analytical method: 65% acetonitrile: 0.1% glacial acetic acid isocratic).
  • Figure 2 A: Deepoxidized epothilone B yield (between 410mg/L-480mg/L) and analysis of impurity components before and after passage of BG03-09K strain; The position near 32 minutes, and the position near 34 minutes for desepoxidized epothilone B.
  • Figure 3 SEQ ID NO.1, the DNA sequence of the epoK gene insertion inactivation region, the italic font is the epoK gene sequence, and the normal font is the inserted DNA sequence.
  • Figure 4 A: HPLC profile of the XAD eluate after fermentation, B: HPLC analysis profile after C column treatment.
  • the HPLC purity of desepoxidized epothilone B in the fermentation collection liquid of BG03-09K strain reached 98%, and the peak area of other single impurities was not more than 1.0%.
  • FIG. 5 Map of conjugation transfer plasmid (pBGS2-4).
  • Example 1 Construction of genetically engineered strains with EpoK gene inactivation
  • This example describes a genetically engineered strain with EpoK gene insertion inactivation and its construction.
  • the DNA sequences of the adjacent regions on both sides of the EpoK gene in the epothilone biosynthesis gene cluster were cloned by PCR, with primers: QK-F, 5'-GCAAGCTTCAGGATCTACAATCTCGC-3' (SEQ ID NO.2); QK-R , 5'-CGTCTAGATTAAACGACGGGTTGACGA-3'(SEQ ID NO.3); HK-F,5'-CGTCTAGAGTATACGTCACGCGCCATCGACC-3'(SEQ ID NO.4),HK-R,5'-CGGGATCCGGAGAAAGACCATCTCCCC-3'(SEQ ID NO. .5).
  • the obtained two PCR products were ligated on both sides of the AphII and Blmt antibiotic resistance genes (1.67kb) to obtain the epoK gene fragment with the insert sequence, and the fragment was ligated into the conjugation transfer vector (pUC18-oriT, Beijing North Jin).
  • the pBGS2-4 conjugation transfer plasmid was obtained from Edge Technology Co., Ltd. (the gray fragments on both sides of Blmt and AphII in Figure 5 are the homologous DNA sequences of epoK).
  • conjugation transfer method the constructed pBGS2-4 conjugation transfer plasmid was introduced into So ce90 bacteria of Hemobacterium cellulosum through E.coli S17-1. Then, phleomycin-resistant bacteria were picked on the S42 solid medium containing 30 ug/ml phleomycin.
  • BG-K1 5-ATCATATGACACAGGAGCAAGCGAATCAGAGT-3' (SEQ ID NO. 6) and BK-R: 5-GAGTTCTACCGGCAGTGCAAATC-3' (SEQ ID NO. 7) were used PCR was performed, and the strain that obtained a PCR product of about 0.59 kb was the epoK inactivated strain, while the non-inactivated strain did not have the PCR product.
  • the above epoK-inactivated C. cellulosum recombinant strain was inoculated into 5 ml of BCF seed medium (0.8-1.0% soy peptone, 0.5-0.7% fructose, 0.05%-0.1% MgSO 4 .7H 2 O and 25-50 mM HEPES , pH 7.2-7.6) in a glass test tube.
  • BCF seed medium 0.8-1.0% soy peptone, 0.5-0.7% fructose, 0.05%-0.1% MgSO 4 .7H 2 O and 25-50 mM HEPES , pH 7.2-7.6
  • the strains were grown for 4 days in a shaker at 34°C, 200 rpm, and then inoculated into 50 ml of FBCF medium (0.6-0.7% soy peptone, 0.5-0.7% fructose, 0.05-0.1% MgSO 4 .7H 2 O and a solution of trace elements 5-10 mg/L, and the pH was 7.1 ⁇ 0.3, and added XAD-16 (Rohm & Haas) resin 2-5%), and cultured for 7 days.
  • XAD-16 was collected from the culture broth and epothilone metabolites were eluted from XAD with 10 ml of methanol.
  • the fermentation product was analyzed by HPLC, and a recombinant strain named BG03-09K was obtained, which could produce desepoxidized epothilone B.
  • the bacteria used desepoxidized epothilone A/B as the main metabolite and did not produce Epothilone Primer A/B (results are shown in Figure 1).
  • Example 2 Preservation performance of strain BG03-09K
  • BG03-09K culture solution was spread on S42 agar plate and cultured at 32°C for 8 days. Then pick a single colony from this plate, inoculate it in a test tube containing 3ml of sterile BCF seed medium, and cultivate for 4 days at 34°C with a shaker at 200rpm.
  • One of the test tubes with lush growth was selected to detect the presence of impurity bacteria under the microscope, and then transferred to 50ml of BCF seed medium (250ml conical flask), and cultured for three days. Check the bacteria under the microscope to see if there are any bacteria.
  • the BG03-09K strain stored at ultra-low temperature below -70°C for a long time and its passage stability were investigated.
  • the reproduction cycle of Helicobacter cellulosum is about 16 hours. Therefore, it is fermented and cultured in batch fed-feed fermentation culture process, from the cell cryopreservation tube to the 100L fermentation production after 14 days of sampling, the strain passage can reach 35 generations; from the cell cryopreservation tube in 30ml of BCF medium for continuous passage Cultivated for 30 days (transplanted once a day), and the strains can be passaged for up to 45 generations.
  • Embodiment 3 Molecular genetic identification of BG03-09K strain
  • the cell pellet was quickly suspended in 3 mL STE (25% sucrose, 10 mM Tris pH 8, 1 mM EDTA), and then 0.6 mL RLM (5% SDS, 0.5 mM EDTA) was added. M Tris pH 7.4, 125mM EDTA), DNA was extracted as a template, PCR amplification was performed with Oligo primers, and the PCR products were sequenced and analyzed, as follows.
  • PCR was performed on the genomic DNA of BG03-09K, and a PCR fragment of about 2.8kb was obtained. The sequence analysis was performed, which proved that the epoK gene was inactivated by DNA insertion.
  • BG-K1 ATCATATGACACAGGAGGAGCAAGCGAATCAGAGT-3' (SEQ ID NO. 6)
  • BG-K2 CGCCTCGAGGCGAGCAAGGGACACCCCGGGG-3' (SEQ ID NO. 8)
  • the 16S rRNA gene, the special function gene methyltransferase (MT domain) in the biosynthetic gene cluster of epothilone and the glyceraldehyde 3-phosphate dehydrogenase gene were selected as the targets for genetic identification of the strain.
  • Three pairs of oligo primers were designed for PCR amplification, and the obtained PCR products were sequenced and analyzed.
  • the amplification primer sequences are:
  • MTW-F 5'-GCTGCTCACCACGCCGGAAT-3' (SEQ ID NO. 12)
  • MTW-R 5'-TCAGCGGAGCCATCGGCCC-3' (SEQ ID NO. 13).
  • the partial sequence of the special function gene methyltransferase (MT domain) gene in the epothilone biosynthesis gene epoD gene in BG03-09K is in the database with the MT domain DNA sequence of this segment of wild-type Helicobacter so ce90 By comparison, the results showed no difference, and the homology was 100%, which proved that BG03-09K was the same species as Soce90, and both produced epothilone.
  • MT domain special function gene methyltransferase
  • the amplification primer sequences are:
  • GAPDH-F 5'-TCGTGCTCGAGTGCAC-3' (SEQ ID NO. 15)
  • GAPDH-R 5'-AGAAGCCCCACTCGTT-3' (SEQ ID NO. 16).
  • Glyceraldehyde 3-phosphate dehydrogenase is an enzyme that catalyzes the phosphorylation and oxidation of glyceraldehyde 3-phosphate in the presence of NAD+ and phosphoric acid to form 1.3-bisphosphoglycerate in microbial glycolysis.
  • the partial sequence of the GAPDH gene of BG03-09K is compared in the database (there is no corresponding data of So ce90 in the database), and it is found that the DNA sequence of this section is the same as that of So ce56.
  • the evolutionary distances of the segment sequences differed by 1.7% or 3.0 at the nucleotide level, respectively, which proved that BG03-09K was the same species as Soce56.
  • C. cellulosum BG03-09K strain can be grown under culture conditions utilizing cellulose as the sole carbon source and potassium nitrate (KNO 3 ) as the sole nitrogen source.
  • KNO 3 potassium nitrate
  • ST2 on a filter paper of an inorganic salt agar medium medium components was 0.1% KNO 3, 0.1% MgSO 4 .7H 2 0,0.1% CaCl 2 .2H 2 O, 0.1% K 2 HPO 4, 0.01% MnSO 4. 7H 2 O, 0.02% FeCl 3 , 0.002% yeast extract, trace element solution (Beijing Beijinyuan Technology Co., Ltd.)), 1% agar), strain BG03-09K formed dark russet to dark brown fruiting bodies.
  • the vegetative mycelium of this strain in the logarithmic growth phase in liquid culture is rod-shaped, and dark cylindrical bacilli with wide rounded ends can be observed under phase contrast microscope, with an average length of 3-6 ⁇ m and a thickness of 1 ⁇ m.
  • the cells were easy to aggregate to form sunflower clusters, especially in the late stage of liquid culture or the early stage of fermentation production.
  • Example 4 Production of desepoxidized epothilone B from genetic recombinant strain Bacillus cellulans BG03-09K
  • Step 2 First-class seed tank cultivation
  • the bacterial seed liquid in the shake flask was aseptically inoculated into 5L sterile BCF seed medium (0.8-1.0% soy peptone, 0.5-0.7 % fructose, 0.05%-0.1% MgSO 4 .7H 2 O) in the first-class seed tank, cultured according to the following process conditions for 3 days ⁇ 1 day.
  • 5L sterile BCF seed medium 0.8-1.0% soy peptone, 0.5-0.7 % fructose, 0.05%-0.1% MgSO 4 .7H 2 O
  • the first-class seed culture process parameters the culture temperature is 34 ⁇ 1°C. pH7.1 ⁇ 0.3, adjusted by acid-base, dissolved oxygen control point is 30%, controlled by air intake and stirring speed. Foam is controlled by antifoam feedback.
  • the first-class seed tank seed liquid was aseptically transferred to a fermenter containing 90L of aseptic fermentation FBCF medium according to the inoculation ratio of 5 ⁇ 1%, and the tank was placed in the tank after culturing for 14 ⁇ 2 days according to the following process parameters.
  • feeding was carried out in batches, starting from day 3 ⁇ 1, feeding 2 ⁇ 1 times a day, respectively, adding soy peptone solution and fructose solution each time. From the 6th day of fermentation to the end day of fermentation (12-16 days), daily sampling was performed to detect the content of deepoxidized epothilone B and the presence or absence of bacterial contamination, and the fermentation yield was >300 mg/L.
  • Fermentation and culture process parameters culture temperature 34 ⁇ 1°C, pH 7.1 ⁇ 0.3, adjusted by acid-base, DO control point 30%, controlled by air intake and stirring speed, and feedback defoaming by defoamer.
  • the production fermentation medium formula FBCF contains: 0.6-0.7% soy peptone, 0.5-0.7% fructose, 0.05-0.1% MgSO 4 .7H 2 O, trace element solution (Beijing Beijinyuan Technology Co., Ltd.) 5-10mg/L , and added XAD resin 2-5% to it.
  • ELSD-LC analysis method HPLC gradient method, DAD combined with ELSD detection
  • Detector A Agilent DAD detector, 190nm-400nm scan
  • Detector B Agilent ELSD detector, evaporation tube, drift tube temperature: 50°C; carrier gas flow rate 1.8L/min
  • Step 4 Fermentation resin harvest and elution
  • the feed liquid is placed on a separating sieve to separate the XAD resin from the bacterial cells/bacterial liquid.
  • the XAD resin in the fermentation broth was intercepted and rinsed by a 100-mesh sieve, it was loaded onto the XAD resin column to elute the XAD resin with methanol, and the methanol eluent was collected as the XAD eluent. After the XAD elution was completed, sampling was performed for HPLC analysis. content.
  • the XAD eluent was passed through a absorbent cotton filter, and water was added to prepare a sample solution with a methanol concentration of about 45% (v/v), loaded onto a PC chromatographic column (C18 packing), and eluted with 62-80% methanol solution isocratic/gradient , collect the product components, and take samples to analyze the content by HPLC.
  • the collected solution that meets the standard after the above PC column treatment is added with water to prepare a sample solution with a methanol concentration of about 50% (v/v), which is loaded on the C chromatographic column (C18 packing), and the isocratic/gradient of 62-80% methanol solution is used. Elute and collect product fractions.
  • the HPLC purity of the desepoxidized epothilone B in the collected solution that meets the standard after C column treatment reaches 98%, and the peak area of other single impurities is not more than 1.0%, as shown in Figure 4B below.

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Abstract

用于发酵生产脱环氧埃坡霉素B的重组纤维堆囊菌(Sorangium cellulosum)，该重组菌中埃坡霉素生物合成基因簇中的epoK基因被插入失活，以及使用所述重组菌生产脱环氧埃坡霉素B的方法。

Description

生产脱环氧埃坡霉素B的重组菌及其用途 技术领域
本申请涉及重组细菌及其用途，更具体涉及生产脱环氧埃坡霉素B的重组纤维堆囊菌及其用途。
背景技术
埃坡霉素(Epothilone)是一类由土壤微生物产生的作用于微管蛋白的具抗肿瘤活性的大环内酯类抗生素，于20世纪90年代初首先从粘细菌纤维堆囊菌(Sorangium cellulosum)中分离出来，主要有A和B两种产物，其结构如下。
Figure PCTCN2021108266-appb-000001
对纤维堆囊菌菌埃坡霉素生物合成基因簇中的基因的克隆分析表明，埃坡霉素聚酮合酶(polyketide synthases)利用酰基辅酶A为单位聚合形成微生物的次级代谢产物脱环氧埃坡霉素A和B，比例为2:1，其作为中间代谢产物再通过epoK基因产物细胞色素P450氧化酶的作用，最终分别形成环氧型的埃坡霉素A和B。在天然产生菌纤维堆囊菌中，脱环氧埃坡霉素A和B只以微量存在，远远低于终产物埃坡霉素A和B的产量，并且纤维堆囊菌发酵液中至少还有35种其它埃坡霉素类似物存在(Hardt，et al.,J Nat Prod 64:847-856，2001)。美国的Danishefsky研究小组对脱环氧埃坡霉素B成功地进行了化学全合成试验，但该全合成过程复杂、难于控制、产量低、成本高，从而大大降低了其药用开发价值。
由于非环氧化形式的脱环氧埃坡霉素B在毒性上远低于埃坡霉素的环 氧化配对物，因此需要开发高效生产脱环氧埃坡霉素B的生产菌株，提高脱环氧埃坡霉素B的产量，以使其用于临床治疗。
美国专利US5843718和US6033883中描述了在重组宿主细胞内通过重组modular PKS基因而生产新型聚酮化合物，以及在宿主细胞体内，如链霉菌(Streptomyces)、大肠杆菌(E.coli)及粘细菌(Myxobacterial)中引入异源PKS基因而生产聚酮化合物的重组方法，在不能生产埃坡霉素的宿主细胞内，如链霉菌Streptomyces coelicolor和粘细菌Myxococcus xanthus中，通过引入异源埃坡霉素基因而生产埃坡霉素A和B已有报道(Tang,et al.Science 287:640-642；Julien&Shah,2002,Amtimicrobial.Agent Chemother.46(9):2772-2778，列于此以作参考)。
发明内容
第一方面，提供了用于发酵生产脱环氧埃坡霉素B的重组菌纤维堆囊菌(Sorangium cellulosum)，该重组菌中埃坡霉素生物合成基因簇中的epoK基因是失活的。
在一些具体的实施方案中，所述epoK基因通过点突变、插入、缺失、取代而失活。
在一些具体的实施方案中，所述的重组菌是来自纤维堆囊菌So ce90(Sorangium cellulosum So ce90)的重组菌。
在一些具体的实施方案中，其中所述epoK基因通过插入而失活，所述epoK基因包含核苷酸序列SEQ ID NO.1，插入序列是SEQ ID NO.1的核苷酸基中第291位至第1960位。更具体的，所述重组菌为纤维堆囊菌BG03-09K。
本发明提供的重组基因工程菌株，包含了埃坡霉素生物合成基因的修饰，所修饰的基因是通过DNA重组技术使产生菌宿主细胞中的埃坡霉素生物合成基因簇中epoK基因的DNA序列通过插入DNA片段而失活，从而使宿主细胞中的埃坡霉素生物合成基因被修饰。
本发明提供的重组菌可以通过重组技术获得，该技术利用同源重组的方法将所需被改变的基因或功能基团的相邻的两端区域克隆到自杀载体中，获得接合转移质粒载体，通过自杀载体中的基因与宿主细胞核所含基因组中的 同源基因进行双杂交重组，导致参与埃坡霉素生物合成的一个或多个基因或功能基团插入失活或被取代，而得到重组的基因工程生产菌株。例如，用一个或二个抗生素抗性基因插入在需被改变的基因两端区域中间，通过插入的抗生素抗性基因两侧的epoK基因同源序列在宿主细胞中进行DNA重组从而取代宿主细胞中野生型基因，获得抗生素抗性基因插入到epoK基团中而导致其功能丧失的重组株。这种失活作用也可通过随机或点基因突变，缺失或取代来完成。由此得到的重组生物合成基因不同于天然的产生菌中的基因，从而可以在重组宿主细胞中产生埃坡霉素衍生物或中间产物为主要产物，例如包含有埃坡霉素合成基因的宿主细胞，可产生埃坡霉素B为主要产物，当其中埃坡霉素生物合成基因中epoK基因DNA序列发生插入其他DNA序列而致使失活，导致该重组菌株生产脱环氧埃坡霉素B为主要产物。
本发明的重组菌，例如BG03-09K可长期保存且稳定性能良好。例如，通过30-50ml在BCF培养基中培养3±1天后的BG03-09K培养液与10-20ml的90％-100％无菌甘油在无菌瓶中混合，取1-1.5ml混合液分装于标记好的无菌细胞冻存管中制备为生产菌细胞库，并立即放入-70℃以下超低温中冷冻保存，该菌长期保持稳定性能良好。
本发明通过重组纤维堆囊菌菌株BG03-09K持家基因的PCR分析和基因测序分析(16S rRNA基因，EpoD-MT功能区域、甘油醛3-磷酸脱氢酶(GAPDH)基因)作为菌种遗传鉴定的目标。设计三对oligo引物进行PCR扩增，并对获得的PCR产物进行了DNA测序和分析。通过BG03-09K的持家基因部分序列在数据库中进行比较，发现BG03-09K的DNA序列与纤维堆囊菌(S.cellulosum)该段序列其进化距离在核苷酸水平差异不大，证明了BG03-09K与纤维堆囊菌(S.cellulosum)是相同种属。
第二方面，提供了生产脱环氧埃坡霉素B的方法，包括在培养基中培养第一方面所述的重组纤维堆囊菌。
在一些具体的实施方案中，所述生产方法中使用的培养基为水溶性培养基，例如种子培养基BCF包含：0.8-1.0％大豆蛋白胨、0.5-0.7％果糖、0.05％-0.1％MgSO 4.7H 2O和25-50mM HEPES，pH为7.2-7.6；生产发酵培养基FBCF包含：0.6-0.7％大豆蛋白胨、0.5-0.7％果糖、0.05-0.1％MgSO 4.7H 2O和微量元素溶液5-10mg/L，且pH为7.1±0.3。
在一些具体的实施方案中，所述生产方法包括在发酵培养基中添加XAD-16树脂，例如2％-5％的XAD-16树脂，进行发酵生产。
在一些具体的实施方案中，在所述生产方法的发酵过程中进行每天分次补料。
在一些具体的实施方案中，所述生产方法还包括使用PC色谱柱和C色谱柱纯化脱环氧埃坡霉素B，所述纯化使用62-80％甲醇溶液洗脱，例如62％、67％、72％、77％、80％甲醇。
更具体地，脱环氧埃坡霉素B高产菌株BG03-09K发酵生产脱环氧埃坡霉素B的具体操作如下：
取保存-70℃以下超低温冰箱的菌种细胞冻存管培养至规定时间后转种在种子培养基中培育，将培养特征符合要求的种子液按照5±1％的接种比例，无菌转种至含有发酵培养基的发酵罐中，按照下述工艺参数进行分批补料培养14±2天。
发酵培养工艺参数：培养温度设定34℃，可接受范围34±1℃；pH设定7.1，可接受范围7.1±0.3，以酸碱溶液调节；溶氧控制设置点30％-50％，由进气量和搅拌转速协同控制，由消泡剂反馈消泡。发酵过程中分批补料，从第3±1天下午开始，每天分别补料2±1次，每次补加大豆蛋白胨溶液及果糖溶液。
在发酵第6天～发酵结束，逐日取样检测含量及镜检菌体生长状态及纯菌情况。以XAD树脂甲醇萃取液进行HPLC检测后分析计算发酵产量(见图1)。发酵产量应≥240mg/L，可高达500mg/L以上。
具体地，所述方法中，种子培养和发酵培养条件优选34℃，可接受范围34±1℃。
具体的，所述方法中，发酵培养条件优选pH设定7.1，可接受范围7.1±0.3。
具体的，所述方法中发酵培养条件优选在发酵时间3±1天开始，每天分别进行补料2±1次，补加大豆蛋白胨溶液及果糖溶液。
具体的，所述方法中，发酵培养条件优选通过分批流加补料延长发酵培养时间至14天±2天，可以获得更高的产量。
发酵培养结束后，将料液用100-200目筛网分离XAD树脂与菌体/菌液， 拦截的XAD用等量水淋洗后装填至XAD树脂柱，用>90％甲醇洗脱XAD树脂，收集含有产品的甲醇洗脱液为XAD洗脱液。
将XAD洗脱液经脱脂棉过滤器，加水制备成约50％±5％(v/v)甲醇浓度的样品溶液加载至PC色谱柱(C18填料)上，用62-80％甲醇溶液洗脱，收集产品组分。
将上述PC柱处理后符合标准的收集液加纯化水制备成约50％±5(v/v)甲醇浓度的样品溶液，加载至C色谱柱(C18填料)上，用62-80％甲醇溶液洗脱，收集产品组分。C柱处理后符合标准的该收集液中脱环氧埃坡霉素B的HPLC纯度可达98％，其他单一杂质峰面积不得大于1.0％，PC柱和C色谱柱处理后总的回收产率大于70％。
在所述方法中，C柱处理后符合标准的收集液中脱环氧埃坡霉素B的HPLC纯度达98％，其他单一杂质峰面积不大于1.0％
附图说明
图1：BG03-09K发酵获得的产品的HPLC图。其中，脱环氧埃坡霉素A在4.4分钟附近位置，脱环氧埃坡霉素B在5.1分钟附近位置。(分析方法：65％乙腈：0.1％冰醋酸等度)。
图2：A：BG03-09K菌株传代前后的脱环氧埃坡霉素B产率(410mg/L-480mg/L间)和杂质组分分析比较；其中，脱环氧埃坡霉素A在32分钟附近位置，脱环氧埃坡霉素B在34分钟附近位置。
B：BG03-09K菌株传代前后菌种培养3-6天的显微镜菌体形态比较。左边两个小图为3天培养新鲜样品；右边两个小图为6天培养液放置后样品。
图3:SEQ ID NO.1，epoK基因插入失活区域的DNA序列，斜体字体为epoK基因序列，正体字体为插入DNA序列。
图4：A：发酵后XAD洗脱液HPLC图谱，B：C色谱柱处理后HPLC分析图谱。BG03-09K菌株发酵收集液中脱环氧埃坡霉素B的HPLC纯度达98％，其他单一杂质峰面积不大于1.0％。
图5：接合转移质粒(pBGS2-4)图谱。
具体实施方式
实施例一：构建EpoK基因失活的基因工程菌株
本实施例描述了一株EpoK基因插入失活的基因工程菌株及其构建。
利用DNA提取试剂盒(江苏康为世纪生物科技有限公司)，从野生型埃坡霉素天然生产菌株纤维堆囊菌So ce90中制备得到总DNA，方法参见文献Jaona et al.,1992,Plasmid 28:157-165。通过PCR方法来克隆埃坡霉素生物合成基因簇中EpoK基因两侧相邻区域的DNA序列，引物为：QK-F，5‘-GCAAGCTTCAGGATCTACAATCTCGC-3′(SEQ ID NO.2)；QK-R，5’-CGTCTAGATTAAACGACGGGTTGACGA-3′(SEQ ID NO.3)；HK-F,5’-CGTCTAGAGTATACGTCACGCGCCATCGACC-3′(SEQ ID NO.4),HK-R,5’-CGGGATCCGGAGAAAGACCATCTCCCC-3′(SEQ ID NO.5)。将所得到的两段PCR产物连接在AphII和Blmt抗生素抗性基因(1.67kb)两侧而获得带有插入序列的epoK基因片段，将该片段连接在接合转移载体(pUC18-oriT，北京北进缘科技有限公司)上获得pBGS2-4接合转移质粒(图5中Blmt和AphII两侧的灰色片段即为epoK同源DNA序列)。
通过接合转移方法，将构建的pBGS2-4接合转移质粒通过E.coli S17-1引入到纤维堆囊菌So ce90菌中。然后，在含有30ug/ml腐草霉素的S42固体培养基上挑取腐草霉素抗性菌。S42培养基包含0.05％胰化蛋白胨,0.15％MgSO 4.7H 2O,1.2％HEPES(PH＝7.4)和1.2％琼脂，高压灭菌后，在每升S42培养基中加入如下物质：10ml的过滤灭菌了的10％CaCl 2.2H 2O,1ml的6％K 2HPO 4,1ml的EDTA-Fe/亚硫酸氢钠溶液,8ml的40％过滤灭菌了的葡萄糖,10ml的5％硫酸胺和35ml的高压灭菌过的培养液。分析挑取约500个抗性菌落，制备DNA，采用引物BG-K1：5-ATCATATGACACAGGAGCAAGCGAATCAGAGT-3′(SEQ ID NO.6)和BK-R:5-GAGTTCTACCGGCAGTGCAAATC-3′(SEQ ID NO.7)进行PCR，获得约0.59kb PCR产物的菌株为epoK失活菌株，而非失活菌株的无该PCR产物。
将上述epoK失活的纤维堆囊菌重组菌株接种到含有5ml BCF种子培养基(0.8-1.0％大豆蛋白胨、0.5-0.7％果糖、0.05％-0.1％MgSO 4.7H 2O和25-50mM HEPES，pH为7.2-7.6)的玻璃试管中。菌株在34℃、200rpm的摇 床中培养4天，然后接种到50ml的FBCF培养基(0.6-0.7％大豆蛋白胨、0.5-0.7％果糖、0.05-0.1％MgSO 4.7H 2O和微量元素溶液5-10mg/L，且pH为7.1±0.3，并添加XAD-16(Rohm&Haas)树脂2-5％)中，培养7天。从培养液收集XAD-16，并用10ml的甲醇从XAD中洗脱埃坡霉素类代谢产物。HPLC分析发酵产物，获得一株命名为BG03-09K的重组菌能产生脱环氧埃坡霉素B，该菌以脱环氧埃坡霉素A/B为主要代谢产物，不产生埃坡霉素A/B(结果见图1)。
实施例二：菌株BG03-09K的保存性能
将适量BG03-09K培养液涂布在S42琼脂平板上，32℃下培养8天。然后从这个平板上挑取单个菌落,分别接种在含有3ml的灭菌BCF种子培养基的试管中，34℃摇床200rpm培养4天。选出其中一管生长茂盛的试管在显微镜下检测菌体有无杂菌，并转接在50ml的BCF种子培养基(250ml三角瓶)中，继续培养三天。在显微镜下检测菌体有无杂菌。取适量BCF培养液与90％甘油在无菌瓶中混合，取1-1.5ml混合液分装于标记好的无菌细胞冻存管中，并立即放入-70℃以下超低温冷冻保存。
考察了在-70℃以下超低温冷冻长期保存的BG03-09K菌株及其传代稳定性。纤维堆囊菌繁殖周期约为16个小时。因此以分批流加补料发酵培养工艺发酵培养，从细胞冻存管到100L发酵生产14天结束后取样，菌株传代可达35代；从细胞冻存管在30ml的BCF培养基中连续传代培育30天(每天转种一次)，菌株传代可达45代。通过BG03-09K细胞库在-70℃以下超低温冷冻保存1年以上冻存管菌株与BG03-09K从种子到100L发酵生产结束后收集的菌株或与BG03-09K冻存管在BCF种子培养基中连续传代30天的菌株进行发酵生产比较，结果表明，BG03-09K菌株传代前后的脱环氧埃坡霉素B产率(410mg/L-480mg/L间)和杂质组分(图2A，分析方法同实施例4)和菌体形态特征都一致(图2B)，说明BG03-09K菌株传代或长期保存稳定性极好。
对BG03-09K菌种在超低温冷冻保存1年以上的原始细胞库以及上述连续传代(35、45代)后的菌种培养液，分别提取菌体DNA，用NotI切割DNA样品并在0.8％琼脂糖凝胶上电泳。根据标准方案将凝胶印迹到带电的 尼龙膜上。将印迹与埃坡霉素生物合成基因簇DNA标记的探针进行分子杂交(Southern印迹分析)，分析显示所有样品都有相同的DNA片段模式，表明该菌细胞库在-70℃以下超低温冷冻保存或在连续传代35代或以上，其微生物遗传性能相对稳定，产品产率相对稳定，说明了菌种在保存和生产中不会发生回复突变，可保证产业化生产用样品的一致性。
实施例三、BG03-09K菌株的分子遗传学鉴定
以BG03-09K的培养液(10mL)离心获得菌体后，迅速将细胞沉淀悬于3mL STE(25％蔗糖，10mM Tris pH 8,1mM EDTA)中，然后加入0.6mL RLM(5％SDS，0.5M Tris pH 7.4,125mM EDTA)，提取DNA作为模版，以Oligo引物进行PCR扩增，并对PCR产物进行测序分析，具体如下。
4.1 epoK基因失活区域的DNA序列分析
以BG-K1和BG-K2为引物，对BG03-09K的基因组DNA进行PCR，获得约2.8kb PCR片段后进行了测序分析，证明了epoK基因被DNA插入失活。
BG-K1：ATCATATGACACAGGAGCAAGCGAATCAGAGT-3′(SEQ ID NO.6)
BG-K2:CGCCTCGAGGCGAGCAAGGGACACCCCGGGG-3′(SEQ ID NO.8)
DNA序列分析结果如图3所示，确认了BG03-09K菌株的EpoK基因在第342位核苷酸上发生了DNA插入(插入序列1.67kb)而导致该基因功能失活。BG03-09K中的埃坡霉素生物合成基因epoK部分序列与数据库中野生型纤维堆囊菌so ce90的epoK该段DNA序列进行比较，表明在核苷酸水平这两株菌在epoK基因部分的DNA序列没有差异，同源性为100％，证明了BG03-09K除插入失活外对epoK基因片段没有产生其它变异。
4.2菌株BG03-09K持家基因的PCR分析和基因测序分析
选择16S rRNA基因，埃坡霉素(epothilone)的生物合成基因簇中特殊功能基因甲基转移酶(MT结构域)以及甘油醛3-磷酸脱氢酶基因作为菌种遗传鉴定的目标。设计三对oligo引物进行PCR扩增，并对获得的PCR产物进行了DNA测序和分析。
4.2.1 16S rRNA基因序列扩增选用了细菌16SrRNA基因的通用引物，即27F和1495R，具体序列如下：
27F：5′-AGAGTTTGATCCTGGCTCAG-3′(SEQ ID NO.9)
1495R：5′-CTACGGCTACCTTGTTACGA-3′(SEQ ID NO.10)
结果：对PCR所获得的DNA产物(1.45kb)进行测序，结果显示BG03-09K菌种的16SrRNA基因序列如下：
Figure PCTCN2021108266-appb-000002
Figure PCTCN2021108266-appb-000003
将BG03-09K的16SrRNA基因序列与数据库中DNA进行比较，发现BG03-09K的16S rRNA基因序列与其它纤维堆囊菌的序列其进化距离在核苷酸水平差异小于1％，证明了BG03-09K与纤维堆囊菌是相同。
4.2.2埃坡霉素生物合成基因epoD中特殊功能基因甲基转移酶(MT结构域)片段
扩增引物序列为：
MTW-F:5’-GCTGCTCACCACGCCGGAAT-3′(SEQ ID NO.12)
MTW-R:5’-TCAGCGGAGCCATCGGCCC-3′(SEQ ID NO.13)。
结果：对PCR所获得的DNA产物(1.22kb)进行测序，结果显示，BG03-09K菌种埃坡霉素生物合成基因中甲基转移酶(MT结构域)功能基因序列如下：
Figure PCTCN2021108266-appb-000004
Figure PCTCN2021108266-appb-000005
BG03-09K中的埃坡霉素生物合成基因epoD基因中特殊功能基因甲基转移酶(MT结构域)基因部分序列在数据库中与野生型纤维堆囊菌so ce90的该段MT结构域DNA序列进行比较，结果表明没有差异，同源性为100％，证明了BG03-09K与纤维堆囊菌so ce90是相同种属，且都产生埃坡霉素。
4.2.3甘油醛3-磷酸脱氢酶基因片段
扩增引物序列为：
GAPDH-F:5’-TCGTGCTCGAGTGCAC-3′(SEQ ID NO.15)
GAPDH-R:5’-AGAAGCCCCACTCGTT-3′(SEQ ID NO.16)。
结果：对PCR对所获得的DNA产物(0.7kb)进行测序，结果如下。
GAPDH部分基因序列：
Figure PCTCN2021108266-appb-000006
Figure PCTCN2021108266-appb-000007
甘油醛3-磷酸脱氢酶(GAPDH)是在微生物糖酵解中，催化甘油醛3-磷酸在有NAD+和磷酸时被磷酸化并氧化，形成1.3-二磷酸甘油酸的酶。BG03-09K的GAPDH基因部分序列在数据库中进行比较(数据库中没有纤维堆囊菌So ce90的对应数据)，发现该段DNA序列与纤维堆囊菌so0157-2或纤维堆囊菌so ce56的该段序列其进化距离在核苷酸水平差异分别在1.7％或3.0，证明了BG03-09K与纤维堆囊菌so ce56是相同种属。
4.3菌株BG03-09K的理化特性和形态学鉴定
纤维堆囊菌BG03-09K菌株可以在利用纤维素作为唯一碳源和硝酸钾(KNO 3)作为唯一氮源的培养条件下生长。在ST2无机盐琼脂培养基的滤纸上(培养基成分为0.1％KNO 3，0.1％MgSO 4.7H 20，0.1％CaCl 2.2H 2O，0.1％K 2HPO 4，0.01％MnSO 4.7H 2O，0.02％FeCl 3，0.002％酵母提取物，微量元素溶液(北京北进缘科技有限公司))，1％琼脂),菌株BG03-09K形成了深赤褐色到深褐色的子实体。该菌株在液体培养中对数生长期的营养菌丝体为杆状，在相差显微镜下可观察到暗的带有宽的圆末端的圆柱状的杆菌,平均为3-6μm长和1μm厚，但10分钟后菌体易聚集形成向日葵菌团，尤其是在液体培养后期或发酵生产前期。
实施例四、从基因重组菌株纤维堆囊菌BG03-09K生产脱环氧埃坡霉 素B
步骤1、菌种复苏与摇瓶培养
菌种冻存管1～2支，室温融化后，无菌接种至50ml BCF2复苏培养基(含葡萄糖3g/L，果糖3g/l，酵母粉5g/L，蛋白胨4g/L，MgSO 4.7H 200.5g/L)，pH7.4中，置34℃±1℃摇床培养4±1天，目视检查，菌液应呈现雾状，菌体未聚集成大量的球状颗粒。取培养特征符合该要求的菌液，无菌接种至含有约500ml BCF种子培养基的三角摇瓶中，置34℃±1℃培养3天±1天，目视检查，菌液应呈现雾状，菌体未聚集成大量的球状颗粒，无杂菌污染。
步骤2、一级种子罐培养
将摇瓶培养结束后摇瓶中的菌种液在无菌条件下，按照10±2％的接种比例无菌接种至含有5L无菌BCF种子培养基(0.8-1.0％大豆蛋白胨，0.5-0.7％果糖，0.05％-0.1％MgSO 4.7H 2O)的一级种子罐中，按照下述工艺条件培养3天±1天。
一级种子培养工艺参数：培养温度34±1℃。pH7.1±0.3，以酸碱调节，溶氧控制点30％，由进气量和搅拌转速协同控制。由消泡剂反馈控制泡沫。
步骤3、发酵培养
将一级种子罐种子液按照5±1％的接种比例，无菌转种至含有90L无菌发酵FBCF培养基的发酵罐中，按照下述工艺参数，培养14±2天后放罐。发酵过程中进行分批补料，从第3±1天开始，每天分别补料2±1次，每次补加大豆蛋白胨溶液及果糖溶液。发酵第6天至发酵终止日(12-16天)，逐日取样检测脱环氧埃坡霉素B的含量及有无杂菌污染，发酵产率>300mg/L。
发酵培养工艺参数：培养温度34±1℃，pH 7.1±0.3，以酸碱调节，DO控制点30％，由进气量和搅拌转速协同控制，由消泡剂反馈消泡。
生产发酵培养基配方FBCF包含有：0.6-0.7％大豆蛋白胨，0.5-0.7％果糖，0.05-0.1％MgSO 4.7H 2O，微量元素溶液(北京北进缘科技有限公司)5-10mg/L，并向其中添加了XAD树脂2-5％。
发酵的产率通过发酵培养液取样，以XAD甲醇洗脱液进行HPLC检测。HPLC分析图谱见图4A，根据如下分析方法：
ELSD-LC分析方法：HPLC梯度法，DAD联用ELSD检测
1.1)色谱条件
液相色谱：Agilent 1260型HPLC
流动相A：0.1％冰醋酸
流动相B：乙腈
流速：1.0ml/min
洗脱梯度
Figure PCTCN2021108266-appb-000008
色谱柱：Agilent ZORBAX Eclipse Plus C18 4.6*150mm,5μm
柱温：35℃
检测器A：Agilent DAD检测器，190nm-400nm扫描
检测器B：Agilent ELSD检测器，蒸发管、漂移管温度：50℃；载气流速1.8L/min
步骤4：发酵树脂收获与洗脱
发酵培养结束后，将料液放到分离筛上将XAD树脂和菌体/菌液分开。发酵液中的XAD树脂经100目筛网拦截并淋洗后，装填至XAD树脂柱上用甲醇洗脱XAD树脂，收集甲醇洗脱液为XAD洗脱液，XAD洗脱完成后，取样HPLC分析含量。
步骤5：PC柱预处理
将XAD洗脱液经脱脂棉过滤器，加水制备成约45％(v/v)甲醇浓度的样品溶液加载至PC色谱柱(C18填料)上，用62-80％甲醇溶液等度/梯度洗脱，收集产品组分，取样HPLC分析含量。
步骤6：C柱分离纯化
将上述PC柱处理后符合标准的收集液加水制备成约50％(v/v)甲醇浓度的样品溶液，加载至C色谱柱(C18填料)上，用62-80％甲醇溶液等度/梯度洗脱，收集产品组分。C柱处理后符合标准的该收集液中脱环氧埃坡霉素B的HPLC纯度达98％，其他单一杂质峰面积不大于1.0％，见下图4B。
通过分批流加补料延长发酵培养时间至14天±2天可以获得更高的产量。利用该发酵工艺，BG03-09K在发酵罐中，利用补料发酵工艺，脱环氧埃坡霉素的产量可高达500mg/L。通过PC柱和C柱处理后，收集的产品HPLC纯度可达98％以上。
尽管已参考某些实施方案公开本发明，但显而易见的是，可以在不偏离如本文中公开和如随附权利要求书提供的本发明的精神和范围的情况下作出修改和变化。此外，应理解，虽然公开中的所有实施例说明了本发明的实施方案，但它们仅是作为非限制性实施例而提供，因此不应视为限制由此说明的本发明的各个方面。本发明意图具有由本公开、以下权利要求书的语言及其任何等效物界定的全部范围。因此，附图和详述应被视为是说明性的而不是限制性的。

Claims (10)

  1. 用于发酵生产脱环氧埃坡霉素B的重组菌纤维堆囊菌(Sorangium cellulosum)，该重组菌中埃坡霉素生物合成基因簇中的epoK基因是失活的。
  2. 权利要求1所述的重组菌，其中所述epoK基因通过点突变、插入、缺失、取代而失活。
  3. 前述任一项权利要求所述的重组菌，其是来自纤维堆囊菌So ce90(Sorangium cellulosum So ce90)的重组菌。
  4. 前述任一项权利要求所述的重组菌，其中所述epoK基因通过插入而失活，所述epoK基因包含核苷酸序列SEQ ID NO.1，插入序列是SEQ ID NO.1的核苷酸基中第291位至第1960位。
  5. 前述任一项权利要求所述的重组菌，该菌株生产以脱环氧埃坡霉素B为主要代谢产物的高产菌株。
  6. 一种生产脱环氧埃坡霉素B的方法，包括在培养基中培养权利要求1至5任一项中所述的重组菌。
  7. 权利要求6所述的方法，其中所述培养基为水溶性培养基，例如种子培养基BCF包含：0.8-1.0％大豆蛋白胨、0.5-0.7％果糖、0.05％-0.1％MgSO 4.7H 2O和25-50mM HEPES，pH为7.2-7.6；生产发酵培养基FBCF包含：0.6-0.7％大豆蛋白胨、0.5-0.7％果糖、0.05-0.1％MgSO 47H 2O和微量元素溶液5-10mg/L，且pH为7.1±0.3。
  8. 权利要求6或7所述的方法，包括在生产发酵培养基中添加XAD-16树脂，例如2％-5％的XAD-16树脂，进行发酵生产。
  9. 权利要求6-8任一项所述的方法，其中所述发酵过程中进行每天分次补料。
  10. 权利要求6-9任一项所述的方法，还包括使用PC色谱柱和C色谱柱纯化脱环氧埃坡霉素B，所述纯化使用62-80％甲醇溶液洗脱。
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