WO2013060073A1 - 一株产庆大霉素C1a的工程菌及其应用 - Google Patents

一株产庆大霉素C1a的工程菌及其应用 Download PDF

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WO2013060073A1
WO2013060073A1 PCT/CN2011/084415 CN2011084415W WO2013060073A1 WO 2013060073 A1 WO2013060073 A1 WO 2013060073A1 CN 2011084415 W CN2011084415 W CN 2011084415W WO 2013060073 A1 WO2013060073 A1 WO 2013060073A1
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gentamicin
strain
cla
fermentation
medium
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PCT/CN2011/084415
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French (fr)
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洪文荣
严凌斌
林玉双
封成军
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福州大学
常州方圆制药有限公司
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Publication of WO2013060073A1 publication Critical patent/WO2013060073A1/zh

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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.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
    • C12P19/48—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin the cyclohexyl radical being substituted by two or more nitrogen atoms, e.g. destomycin, neamin
    • C12P19/485—Having two saccharide radicals bound through only oxygen to non-adjacent ring carbons of the cyclohexyl radical, e.g. gentamycin, kanamycin, sisomycin, verdamycin, mutamycin, tobramycin, nebramycin, antibiotics 66-40B, 66-40D, XK-62-2, 66-40, G-418, G-52
    • 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
    • C12R2001/29—Micromonospora
    • C12R2001/31—Micromonospora purpurea ; Micromonospora echinospora; Micromonospora rhodorangea

Definitions

  • the invention belongs to the field of antibiotics and relates to the construction and application of an engineering bacteria. Specifically, the invention relates to a gentamicin-producing Cla engineering strain, which is applied to the manufacture of antibiotics.
  • Micromonospora can produce abundant secondary metabolites, especially aminoglycoside antibiotics such as gentamicin, sisomicin and formimicin. These micromonospora are sparsely distributed, slow in growth and have a special cell wall structure, so research progress is slow. In addition, due to the lack of a common genetic operating system, the genetic manipulation of Micromonospora is difficult, and its molecular biology research is also far behind Streptomyces.
  • Micromonospora is a gentamicin-producing bacterium.
  • Gentamicin is an aminoglycoside antibiotic. It has been used clinically for nearly half a century. It is an essential drug for anti-infection in China and the world.
  • Gentamicin is a multi-component metabolite, and the main component of the antibacterial action is the C group complex: Cl, C2 and Cla.
  • the gentamicin-producing bacteria metabolites can be developed with abundant components, such as gentamicin Cla, which is a precursor of the synthetic anti-resistant bacteria drug Etimicin; gentamicin B is further synthetically resistant.
  • the parent of the drug-resistant drug isipamicin; gentamicin, B, and X are good drugs for further development of antigenic worms; the latest research found that aminoglycoside antibiotics also have anti-HIV and other effects. Therefore, in-depth research on such rare medicinal microorganisms, especially in molecular genetics, is of great significance.
  • Etimicin is a new type of semi-synthetic gentamicin. It is a new type of antibiotics in China. It has been applied for patents in many countries. Its intellectual property rights have been effectively protected and the demand is large. It is the drug of choice for this class of antibiotics.
  • Gentamicin Cla needs to be isolated from the metabolites of gentamicin-producing bacteria.
  • all metabolites of gentamicin-producing bacteria are complexes, mainly including gentamicin Cl, C2 and Cla. These compounds have similar chemical structures and similar physical and chemical properties. It is very difficult to separate single-component gentamicin Cla, which leads to the shortage of gentamicin Cla, and the production cost is high, the chromatographic separation period is long, and the yield is low. High consumption, serious pollution, complex extraction and refining process, and unstable product quality.
  • the engineered strain is an inactivated function of Mr. iM/Wra purpurea GK1101 strain, which was registered and preserved on September 13, 2011 at the General Microbiology Center of China Microbial Culture Collection Management Committee. For CGMCC No. 5245.
  • the fermentation method of the engineered bacteria is as follows: Before the fermentation of the strain GK1101, the spore-rich single bacteria are separated by the dilution plate method, and then transferred to the inclined medium, cultured at 37 ° C for 10 days, and the excavated seeds are inoculated into the seeds. The medium was incubated at 37 ° C for 28 h at a speed of 280 rpm, then transferred to the fermentation medium at a 10% inoculum, and incubated at 37 ° C for 124 h at a speed of 280 rpm.
  • the seed medium glucose 0.1 -1.0%, corn starch 1.0-2.0%, corn flour 0.5-2.5%, peptone 0.1-0.8%, soybean cake powder 0.5-1.5%, KN0 3 0.05-0.10%, CaC0 3 0.1-0.5%, pH 6.5- 7.5
  • Fermentation medium corn starch 3.0-6.0%, corn flour 1.0-2.0%, peptone 0.1-0.8%, soybean cake powder 1.0-4.0%, KN0 3 0.01-0.10%, (NH 4 ) 2 S0 4 0.1- 0.5%, CaC0 3 0.1-0.5%, amylase 0.001-0.025%, ⁇ 6 ⁇ 5-7 ⁇ 5.
  • the screening of strain GK1101 mainly includes the following steps:
  • the replacement plasmid is transformed into Micromonospora erythraea
  • the method of gene replacement is to PCR amplify fragments of about 2000 bp in the upstream and downstream as homologous exchange arms, and insert the erythromycin resistance gene as a screening marker between the two exchange arms, and simultaneously connect the three fragments to the shuttle vector.
  • the apramycin resistance gene on the vector pKC1139 can also be used in subsequent screening.
  • the transformation is a binding transfer method mediated by E ET12657 (pUZ8002).
  • the process is to first screen strains containing both apramycin resistance (Am R ) and erythromycin resistance (ermE R ), and then screening for erythromycin-containing resistance (ermE R ) but relaxing against A. Susceptible (Am s ) strain.
  • the components of the fermentation broth were detected by thin layer chromatography (TLC), and the components were accurately determined by HPLC-MS.
  • the invention obtains an engineering strain for producing gentamicin Cla, which is a strain of Micromonospora purpurea GK1101, which was commonly used on the Chinese Microbial Culture Collection Management Committee on September 13, 2011. Microbiology Center Registration and Deposit, referred to as CGMCC, is located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 5245.
  • the engineered bacteria of the invention are used for producing gentamicin Cla, which greatly simplifies the production process, reduces production costs, reduces environmental pollution, and improves product quality. Achieved the goal of mass production of gentamicin Cla.
  • Figure 1 shows the chemical structure of gentamicin.
  • Figure 2 is a map of the recombinant plasmid pFD306.
  • Figure 3 is a schematic diagram of gene functional homologous exchange inactivation (knockout).
  • I chromosomal gene position of parent strain
  • II single exchange with chromosome KB 1 side
  • III single exchange with chromosome KB2 side
  • V and: chromosome double exchange.
  • Figure 4 shows the results of TLC analysis of the metabolites of Pseudomonas aeruginosa S-1212 and the engineered microorganism Pseudomonas aeruginosa GK1101; wherein A is the metabolite of the engineering strain M. minimonas GK1101; B is the pro Metabolite of Micromonospora S. 12-12.
  • Figure 5 is a HPLC-MS spectrum of the metabolite of S. cerevisiae S-1212.
  • the peak 1 of the total ion current map corresponds to gentamicin Cla, molecular weight 450.2; peak 2, corresponding to gentamicin C2, molecular weight 464.3; peak 3, corresponding to gentamicin C2b, molecular weight 464.3; Peak 4, corresponding to gentamicin C2a, molecular weight 464.3; peak 5, corresponding to gentamicin Cl, molecular weight 478.3.
  • Figure 6 is a HPLC-MS spectrum of the metabolite of the engineering strain M. sphaeroides GK1101.
  • the peak 15 of the total ion current map corresponds to gentamicin Cla, molecular weight 450.2; peak 20, corresponding to gentamicin C2b, molecular weight 464.3; no gentamicin Cl, C2 and C2a components detected trace.
  • the invention includes the following main steps:
  • gentamicin biosynthesis gene cluster (refer to GenBank Accession Number AY524043), two pairs of primers LB1 and LB2 and LB3 and LB4 were designed upstream and downstream of the gntK gene (SEQ.NO.l), respectively, to the starting strain S-1212 ( Zhou Xiaolan, Huang Jianzhong, Shi Bihong, Shi Qiaoqin. Studies on several major factors affecting the spore germination of M. melil O3 ⁇ 4wra purpure a S-1212.
  • chromosomal DNA (CTAB method) is used as a template, PCR is performed separately, and the DNA sequence at both ends of gntK is amplified as a homology exchange arm; the upstream exchange arm is called KB1 (LB1/LB2), and the length is 2076 bp; the downstream exchange arm is called KB2 (LB3/LB4) and has a length of 2044 bp.
  • CTAB method chromosomal DNA
  • E1 and E2 are primers
  • the amplification resistance screening marker gene ermE was 1711 bp in length (Fig. 2).
  • KBl, KB2 and ermE were cloned by pMD19-T (TA), respectively, to obtain positive plasmids, which were named pFD301, pFD302 and pFD303.
  • pFD303 was digested with Spe I/Xba I, and the 1711 bp fragment was recovered and ligated with pFD301 (4758 bp) digested with Xba I.
  • the positive plasmid was designated as pFD304.
  • the pFD302 was digested with Xba I.
  • the 2048 bp fragment was recovered and ligated with pFD304 (6463 bp) digested with Xba I, and the positive plasmid was selected and designated as pFD305.
  • the pFD305 was digested with Bgl II, and the 5801 bp fragment was recovered and cut with BamH I.
  • the pKC1139 (6500 bp) was ligated and screened to obtain a positive plasmid, which was named pFD306 (Fig. 2).
  • Replacement plasmid pFD306 transforms Micromonospora erythraea S-1212
  • the replacement plasmid pFD306 was transferred into E. coli ET12567 (pUZ8002) to obtain a donor strain E. coli ET12567 (pUZ8002, pFD306). Then, the spores of Micromonospora erythraea S-1212 were transformed by the method of conjugative transfer, cultured at 33 °C for 20 h, and covered with an aqueous solution containing erythromycin and nalidixic acid (final concentration of 25 g / mL), 37 ° C continued to culture for 5 days to grow transformants, and the obtained ermE R transformants were ligated to contain erythromycin (25-50 g / mL), apramycin (25-50 ⁇ g I mL) and nalidixic acid.
  • the single exchange strain GK1008 (pFD306) was continuously transferred for 3 generations on the anti-antibiotic-free slope, and then isolated and purified, and single colonies were picked and erythromycin-resistant (erythromycin 50 ⁇ g/mL). Plates and strains containing apramycin resistance (ampimycin 50 ⁇ g/mL) were screened for strains sensitive to apramycin and having erythromycin resistance. One of the strains was selected, and the chromosomal DNA was extracted as a template.
  • Three pairs of primers (P1/P2, P3/P4 and P5/P6; see Table 1 and Figure 3) were designed for PCR verification and sequencing, and after being identified as a double-exchange strain, Named as Micromonospora erythraea GK1101.
  • the strain was fermented (see step 1 of Example 2), and the fermentation broth was collected by filtration.
  • the filtrate was detected by silica gel GF254 thin layer chromatography.
  • the main component was Cla, which contained a small amount of C2b (Fig. 4A), and no Cl, C2 and C2a components were observed (Fig. 4B); the filtrate was qualitatively detected by HPLC-MS (Fig. 4B) Figure 6), compared with the metabolite of the parent strain C.
  • the inactivated engineering strain M. minimonas GK1101 provided by the present invention can be directly used for the manufacture of gentamicin Cla.
  • the preparation of gentamicin Cla is as follows (Hong Wenrong. Optimization of gentamicin fermentation process. Chinese Journal of Pharmaceutical Industry. 1994, 25(l).pl-3, ):
  • Seed medium glucose 0.1%, corn starch 1.0%, corn flour 1.5%, peptone 0.2%, soy cake powder 1.0% KNO3 0.05 , CaC03 0.5%, ⁇ 7 ⁇ 0.
  • Fermentation medium corn starch 6.0%, corn flour 1.0%, peptone 0.4%, soybean cake powder 2.0%, ⁇ 3 0.01 , (NH 4 ) 2 SO 4 0.1 , CaCO 3 0.5 , amylase 0.025%, ⁇ 7 ⁇ 5.
  • Shake flask fermentation The Micromonospora erythraea GK1101 obtained in Step 3 of Example 1 was fermented. Before fermentation, the spore-rich single colonies were separated by dilute plate method and then transferred to the slant medium. The cells were cultured at 37 ° C for 10 days, and inoculated into the seed medium (loading 50 mL I 250 mL flask), 37 ° C The shaker was incubated for 28 h (speed 280 rpm).
  • the fermentation medium 50 mL/250 mL flask
  • a fermentation medium loading of 50 mL/250 mL
  • Expanded fermentation 20000 liter fermenter production, stirring speed 180 rpm, ventilation 1: 0.5-1.0 (M 3 /M 3 -min), medium, culture temperature, inoculum ratio, fermentation time is equivalent to shake flask fermentation .
  • the eluent is concentrated by film to about 25-300,000 u / mL (Cla content is more than 90%), and adjusted to pH 5.5-6.0 with concentrated sulfuric acid.
  • the activated carbon is decolored to a transmittance of 92% or more, under stirring.
  • the 85% ethanol solution is rinsed to obtain a wet product.
  • the wet product is vacuum dried (vacuum degree above 500mmHg, temperature 600C, drying for 6 hours), that is, the finished product of gentamicin sulfate Cla, the yield is over 80%.

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Abstract

提供了一种灭活gntK功能的产庆大霉素C1a的工程菌及其应用，其中，工程菌为绛红色小单孢菌GK1101，已于2011年9月13日在中国微生物菌种保藏管理委员会普通微生物中心登记保藏，保藏编号为CGMCC No.5245。该工程菌能够用于制备抗菌药物。

Description

一株产庆大霉素 Cla的工程菌及其应用 技术领域
本发明属于抗生素制药领域， 涉及一种工程菌的构建及其应用， 具体地说， 本发明涉及 一株产庆大霉素 Cla工程菌， 应用于抗生素制造。
背景技术
小单孢菌可产生丰富的次级代谢产物， 特别是氨基糖苷类抗生素， 如庆大霉素、 西索米 星和福提米星等。 这些小单孢菌分布奇特， 生长缓慢且细胞壁结构特殊， 故研究进展缓慢。 此外， 由于缺乏通用的基因操作系统， 小单孢菌遗传操作困难， 使得对其分子生物学的研究 也大大落后于链霉菌。
小单孢菌是庆大霉素产生菌， 庆大霉素是氨基糖苷类抗生素， 已在临床上应用了近半个 世纪， 是我国、 乃至全世界抗感染必备的基本药物。 庆大霉素属多组分代谢产物， 起抗菌作 用的主要组分为 C族复合物： Cl、 C2和 Cla。庆大霉素产生菌代谢产物可开发的组分十分丰 富， 如庆大霉素 Cla是合成抗耐药菌药物依替米星 （Etimicin) 的前体； 庆大霉素 B是进一 步合成抗耐药菌药物异帕米星的母体； 庆大霉素 、 B、 X是进一步开发抗原虫的良好药物； 最新研究发现氨基糖苷类抗生素还具有抗 HIV等功效。 因此对该类稀有药用微生物进行深入 研究， 特别是分子遗传学方面的研究， 具有极大意义。
尽管人们对小单孢菌和庆大霉素的研究已近五十年， 也取得了一些可喜的成果， 但一直 未有重大的突破。 青霉素在五十余年的研究开发过程中， 效价提高了近万倍， 而庆大霉素仅 提高了几十倍， 差别非常悬殊。 二十世纪八十年代兴起的链霉菌基因工程， 已在抗生素生物 合成基因的克隆、 产量提高、 组分改善及杂合抗生素生产等方面取得了一定的进展， 但在小 单孢菌中的应用进展缓慢。
依替米星是新型半合成庆大霉素， 是中国独创的抗生素一类新药， 已在多国申请专利， 其知识产权得到了有效保护， 需求量大， 是本类抗生素临床首选药物。
但由于合成依替米星的母体是庆大霉素 Cla (图 1 )。 而庆大霉素 Cla需要从庆大霉素产 生菌的代谢产物中分离才能得到。 不幸的是， 到目前为止， 所有庆大霉素产生菌的代谢产物 是复合物， 主要包括庆大霉素 Cl、 C2和 Cla等。 这些复合物化学结构相近， 理化性质相似， 从中分离单组份庆大霉素 Cla非常困难，导致庆大霉素 Cla供不应求，且生产成本居高不下， 层析分离周期长， 收率低， 料耗大， 污染严重， 提取精制工艺复杂， 而且产品质量不稳定， 与庆大霉素 Cla结构相似的副产物随时出现干扰， 给依替米星产品质量控制造成了极大的不 确定性， 严重影响了药品的稳定性、 安全性和有效性， 是迫切需要解决的科学难题。 若能获得专一性庆大霉素 Cla产生菌， 则以上所有问题将迎刃而解， 其所带来的经济效 益， 社会效益和环境效益等是极其巨大的， 更重要的是给清洁生产， 安全用药， 提供了可靠 保障。
发明内容
本发明的目的在于提供一株产庆大霉素 Cla的工程菌。
该工程菌为灭活 功能的絳红色小单孢菌 (M rami i«/wra purpurea) GK1101菌株， 已于 2011年 9月 13日在中国微生物菌种保藏管理委员会普通微生物中心登记保藏， 保藏编 号为 CGMCC No. 5245。
所述的工程菌的发酵方法， 具体如下： 菌株 GK1101发酵前先用稀释平板法分离出产孢 丰富的单菌落后， 再转接于斜面培养基， 37°C培养 10天， 挖块接种于种子培养基， 37°C摇床 培养 28 h， 转速为 280rpm， 然后按 10%接种量转接于发酵培养基， 37°C摇床培养 124 h， 转 速为 280rpm; 所述种子培养基： 葡萄糖 0.1-1.0%， 玉米淀粉 1.0-2.0%， 玉米粉 0.5-2.5%， 蛋 白胨 0.1-0.8%， 黄豆饼粉 0.5-1.5%， KN03 0.05-0.10% , CaC03 0.1-0.5% , pH6.5-7.5 , 发酵培 养基： 玉米淀粉 3.0-6.0%， 玉米粉 1.0-2.0%， 蛋白胨 0.1-0.8%， 黄豆饼粉 1.0-4.0 % , KN03 0.01-0.10% , (NH4)2S040.1-0.5% , CaC03 0.1-0.5% , 淀粉酶 0.001-0.025%， ρΗ6·5-7·5。
所述的工程菌在制备抗菌药物中的应用。
菌株 GK1101的筛选， 主要包括以下几个步骤：
A. g^/基因置换质粒的构建；
B. 置换质粒转化絳红色小单孢菌；
C. 转化子的筛选；
D. 工程菌组分的鉴定。
基因置换的方法为 PCR扩增 上下游各 2000 bp左右的片段作为同源交换臂，并将红 霉素抗性基因作为筛选标记插入两交换臂之间， 将此三个片段同时连接到穿梭载体如 PKC1139上， 另外载体 pKC1139上的安普霉素抗性基因也可用于之后的筛选环节。
其中转化是以 E ET12657(pUZ8002)介导的结合转移方法。 过程为先筛选同时含安普 霉素抗性（AmR)和红霉素抗性（ermER)的菌株，松弛培养后再从中筛选含红霉素抗性（ermER) 但对安普霉素敏感 （Ams ) 的菌株。 发酵液组分检测采用薄层层析 （TLC), 组分精确测定采 用 HPLC-MS法。
本发明获得了一株产生庆大霉素 Cla 的工程菌， 该工程菌为絳红色小单孢菌 (Micromonospora purpurea) GK1101菌株， 已于 2011年 9月 13日在中国微生物菌种保藏管理 委员会普通微生物中心登记保藏， 其简称 CGMCC, 地址为北京市朝阳区北辰西路 1号院 3号， 保藏编号为 CGMCC No. 5245。 本发明的工程菌用于产庆大霉素 Cla， 大大简化了生产工艺， 并降低生产成本， 减少环境污染， 同时提高了产品质量。 达到了可大规模生产庆大霉素 Cla 的目标。
附图说明
图 1为庆大霉素化学结构式。
图 2为重组质粒 pFD306图谱。
图 3为 基因功能同源交换灭活 （敲除） 示意图。
I： 亲株染色体基因位置； II： 与染色体 KB 1侧单交换； III： 与染色体 KB2侧单交换； IV 染色体回复突变； V： 与:染色体双交换。
图 4为亲株絳红色小单孢菌 S-1212与工程菌絳红色小单孢菌 GK1101代谢产物的 TLC比较 分析结果； 其中 A 为工程菌絳红色小单孢菌 GK1101 代谢产物； B 为亲株絳红色小单孢菌 S- 1212代谢产物。
图 5为亲株絳红色小单孢菌 S-1212代谢产物的 HPLC-MS图谱。 其中总离子流图谱的峰 1对 应的是庆大霉素 Cla， 分子量 450.2; 峰 2， 对应的是庆大霉素 C2， 分子量 464.3 ; 峰 3， 对 应的是庆大霉素 C2b， 分子量 464.3 ; 峰 4， 对应的是庆大霉素 C2a， 分子量 464.3 ; 峰 5， 对 应的是庆大霉素 Cl， 分子量 478.3。
图 6为工程菌絳红色小单孢菌 GK1101代谢产物的 HPLC-MS图谱。 其中总离子流图谱的峰 15对应的是庆大霉素 Cla， 分子量 450.2; 峰 20， 对应的是庆大霉素 C2b， 分子量 464.3 ; 未 检测到庆大霉素 Cl、 C2和 C2a组分的痕迹。
具体实施方式
实施例 1 :
本发明包括以下主要步骤：
1 . 构建 基因置换质粒
根据庆大霉素生物合成基因簇 （可参考 GenBank Accession Number AY524043) , 分别在 gntK 基因（SEQ.NO.l )上下游设计两对引物 LB1和 LB2以及 LB3和 LB4, 以出发菌株 S-1212 (周 晓兰， 黄建忠， ，施碧红， 施巧琴. 影响絳红色小单孢菌 (M rami O¾wra purpure a)S-1212孢子 萌发的几个主要因子的研究. 福建师范大学学报（自然科学版)， Vol.18 Νο. Ι , ρ72-75 ) 的染 色体 DNA ( CTAB法）为模板， 分别进行 PCR， 扩增得到 gntK两端的 DNA序列， 作为同源 交换臂；上游交换臂称为 KB1 ( LB1/LB2) ,长度为 2076 bp;下游交换臂称为 KB2(LB3/LB4)， 长度为 2044bp。 以质粒 pAGe为模板 (朱碧银， 洪文荣， 严绍德， 朱宝泉， 林玉双， 封成军. 黑 暗链霉菌 DNA同源重组系统的构建. 生物技术通报 2011 ( 4)， 162— 166. )， E1和 E2为引物， 扩增抗性筛选标记基因 ermE,长度为 1711bp( Fig.2)。KBl、KB2和 ermE分别经 pMD19-T(TA) 克隆， 得到阳性质粒， 依次命名为 pFD301、 pFD302和 pFD303。 pFD303经 Spe I/Xba I酶切， 回收 1711 bp片段， 与经 Xba I酶切过的pFD301 ( 4758 bp ) 进行酶连， 转化和筛选， 得到阳 性质粒，命名为 pFD304;用 Xba I酶切 pFD302，回收 2048bp片段，与经 Xba I酶切过的 pFD304 ( 6463 bp )进行酶连，筛选得到阳性质粒，命名为 pFD305 ;用 Bgl II酶切 pFD305， 回收 5801 bp片段， 与经 BamH I酶切过的 pKC1139 ( 6500 bp )进行酶连， 经筛选， 最终得到阳性质粒， 命名为 pFD306 (图 2)。
表 1 实施例所涉及的引物
Figure imgf000006_0001
Restriction enzyme site are indicated by single underlines and box
2. 置换质粒 pFD306转化絳红色小单孢菌 S-1212
将置换质粒 pFD306 转入 E.coli ET12567 (pUZ8002)中， 得到供体菌 E.coli ET12567 (pUZ8002, pFD306)。 然后通过接合转移的方法转化絳红色小单孢菌 S-1212孢子， 33 °C培养 20 h后用含有红霉素和萘啶酸 （终浓度均为 25 g / mL) 的水溶液覆盖， 37 °C继续培养 5 天长出转化子，将所获得的 ermER转化子点接至含有红霉素 (25-50 g / mL)、安普霉素 (25-50 μ g I mL)和萘啶酸（25 μ g / mL)的平板培养基上放置 37 °C进行培养。由于置换质粒 pFD306 含温敏型复制启动子， 当培养温度超过 34 °C时， 该游离质粒在链霉菌中不能自我复制， 只 有质粒 pFD306 同源交换整合到絳红色小单孢菌染色体上的接合子才能生长， 表现出对红霉 素和安普霉素抗性， 8d后孢子生长良好， 初步判断为单交换菌株， 将长好的孢子转接到斜面 培养基上。进一步提取染色体 DNA进行 PCR验证并对 PCR产物进行测序都证实了单交换插 入。 得到一株单交换菌株 GK1008 (pFD306)。
3. 双交换菌株筛选与发酵代谢产物变化的定性检测
将单交换菌株 GK1008(pFD306)在不含抗生素的斜面上连续传 3代后，进行分离纯化，挑 取单菌落分别点种到含红霉素抗性 (红霉素 50 μ g/mL)的平板和含安普霉素抗性（安普霉素 50 μ g/mL)的平板上， 从中筛选对安普霉素敏感而具有红霉素抗性的菌株。挑选其中一株， 提取 其染色体 DNA作为模板， 设计 3对引物 （P1/P2, P3/P4和 P5/P6; 见表 1和图 3 ) 进行 PCR 验证， 并测序， 确定为双交换菌株后， 命名为絳红色小单孢菌 GK1101。 对该菌株进行发酵 (见实施例 2步骤 1 )， 过滤收集发酵液。滤液通过硅胶 GF254薄层层析检测， 结果发现其主 要成分为 Cla，含少量 C2b (图 4 A)，未见 Cl、 C2和 C2a组分（图 4 B );滤液通过 HPLC-MS 定性检测 （图 6)， 与亲株絳红色小单孢菌 S-1212的代谢产物 （图 5 ) 比较， 结果是主要成分 为 Cla (峰 1 )， 少量 C2b (峰 3)， 未见 C1 (峰 5)、 C2 (峰 2)和 C2a (epi-C2峰 4) 组分的 痕迹。
组分的 HPLC-MS分析（图 5和图 6): 电喷雾离子阱质谱， C18拄； 流动相： 0.2M 三氟 乙酸水溶液： 甲醇 （95:5 ); 流速 0.6 ul/min柱温： 30°C， 进样量 luL。
实施例 2: 絳红色小单孢菌 GK1101代谢产物 Cla的制备
本发明提供的 灭活工程菌絳红色小单孢菌 GK1101可直接用于制造庆大霉素 Cla。 庆大霉素 Cla的制备如下（洪文荣. 庆大霉素发酵工艺最佳化. 中国医药工业杂志. 1994， 25(l).pl-3, ):
1. 絳红色小单孢菌 GK1101菌株的发酵培养
种子培养基： 葡萄糖 0.1%， 玉米淀粉 1.0%， 玉米粉 1.5%， 蛋白胨 0.2%， 黄豆饼粉 1.0% KNO3 0.05 , CaC03 0.5%, ρΗ7·0。
发酵培养基：玉米淀粉 6.0%，玉米粉 1.0%，蛋白胨 0.4%，黄豆饼粉 2.0 %， ΚΝΟ3 0.01 , (NH4)2SO40.1 , CaCO3 0.5 , 淀粉酶 0.025%， ρΗ7·5。
摇瓶发酵： 将实例 1中步骤 3获得的絳红色小单孢菌 GK1101进行发酵。 发酵前先用稀 释平板法分离出产孢丰富的单菌落再转接于斜面培养基， 37°C培养 10天， 挖块接种于种子培 养基（装量为 50mL I 250mL三角瓶）， 37°C摇床培养 28 h(转速为 280rpm)。 然后按 10%接种 量转接于发酵培养基 （装量为 50mL/250mL三角瓶）， 37°C摇床培养 124 h(转速为 280rpm)。
扩大发酵： 20000升发酵罐生产，搅拌转速 180转 /分钟，通气量 1 : 0.5-1.0 (M3 /M3 -min), 培养基， 培养温度， 接种量比例， 发酵时间等同于摇瓶发酵。
2代谢产物提取精制 A、 粗提
扩大发酵后的发酵液加 10%自来水稀释后， 加入盐酸， 酸化到 pH2.0-3.0， 充分搅拌 4小 时； 然后用氢氧化钠溶液回调至 pH5.0-6.5， 投入 732NH4 +树脂静态吸附 6-8小时。 树脂投放 量按 5万 u/mL左右计算。 收集吸附饱和树脂， 用自来水漂洗干净 （无漂浮菌丝体为止）。 饱 和树脂装柱，用两倍饱和树脂体积的 0.5M HC1溶液酸洗饱和树脂，再用无离子水洗涤至中性， 然后改用 0.01%氨水进行碱洗， 当流出液达 pH 9.0以上时， 串联到等体积的 711树脂柱上， 改用 4%的氨水进行洗脱， 氨水用量为饱和树脂体积的 8-10倍， 洗脱时间控制在 8-10小时， 收集洗脱液。
B、 精制与结晶
洗脱液经薄膜浓縮到约 25-30万 u /mL( Cla含量达 90%以上），用浓硫酸调至 pH5.5-6.0.活 性炭脱色到透光度达 92%以上， 在搅拌下， 缓慢向浓縮液中滴加入 95%以上乙醇， 进行结晶， 时间 3— 4小时， 之后经固液分离， 85%乙醇溶液淋洗即得湿成品。 湿成品经真空干燥（真空 度 500mmHg以上， 温度 600C, 干燥 6小时）， 即得硫酸庆大霉素 Cla成品， 收率 80%以上。

Claims

1. 一株产庆大霉素 Cla工程菌， 其特征在于， 所述工程菌为灭活 功能的絳红色小单孢 飄 Micwmonospom purpurea) GK1101 , 已于 2011年 9月 13日在中国微生物菌种保藏管理委 员会普通微生物中心登记保藏， 保藏编号为 CGMCC No. 5245 ο
2. —种如权利要求 1所述的工程菌的发酵方法， 其特征在于， 菌株 GK1101发酵前先用稀释 平板法分离出产孢丰富的单菌落后， 再转接于斜面培养基， 37°C培养 10天， 挖块接种于种子 培养基， 37°C摇床培养 28 h， 转速为 280rpm， 然后按 10%接种量转接于发酵培养基， 37°C摇 床培养 124 h， 转速为 280rpm; 所述种子培养基： 葡萄糖 0.1-1.0%， 玉米淀粉 1.0-2.0%， 玉 米粉 0.5-2.5%， 蛋白胨 0.1-0.8%， 黄豆饼粉 0.5-1.5%， KN03 0.05-0.10% , CaC03 0.1-0.5%, ρΗβ.5-7.5, 发酵培养基： 玉米淀粉 3.0-6.0% , 玉米粉 1.0-2.0% , 蛋白胨 0.1-0.8% , 黄豆饼粉 1.0-4.0 % , KN03 0.01-0.10% , (NH4)2S040.1-0.5% , CaC03 0.1-0.5%, 淀粉酶 0.001-0.025%， ρΗ6·5-7·5。
3. 如权利要求 1所述的工程菌在制备抗菌药物中的应用。
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