Studies on the biosynthesis of avermectins

To elucidate the pathway of avermectin biosynthesis, the biosynthetic relationships of avermectins A1a, A2a, B1a, B2a, and their respective monosaccharides and aglycones were studied. 14C-labeled avermectin compounds prepared from [1-14C]acetate were fed to Streptomyces avermitilis strain MA5502 and their metabolites were determined. Two furan ring-free aglycones, 6,8a-seco-6,8a-deoxy-5-keto avermectin B1a and B2a, have been isolated from the fermentation broth of a blocked mutant of S. avermitilis. Addition of the compounds and a semisynthetic compound, 5-keto avermectin B2a aglycone, to the fermentation medium of a second blocked mutant established that the two compounds are intermediates in the avermectin biosynthetic pathway immediately preceding avermectin aglycones.     

Engineering the aveC gene to enhance the ratio of doramectin to its CHC-B2 analogue produced in Streptomyces avermitilis

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC-B1), which is sold commercially as Dectomax is co-produced during fermentation with the undesired analogue CHC-B2 at a CHC-B2:CHC-B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The aveC gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site-specific mutagenesis and error-prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC-B1, produced relative to the undesired CHC-B2 fermentation component. High-throughput (HTP) screening of cultures grown on solid-phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An aveC gene with mutations that result in a 4-fold improvement in the ratio of doramectin to CHC-B2 was identified. Subsequent integration of the enhanced aveC gene into the chromosome of the S. avermitilis production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product.     

氮源对阿维菌素合成的影响及基于二氧化碳释放速率的阿维菌素发酵调控

中国是世界上最大的也是唯一的阿维菌素原料生产国,但在工业规模生产中与同类型大环内酯类抗生素相比其产量相对偏低。文中通过研究不同氮源对阿维链霉菌生长、代谢的影响,发现氮源在发酵中后期对菌丝活性、菌丝浓度以及阿维菌素B1a的合成都有较为显著的影响。在100 L生物反应器中,于发酵中后期基于二氧化碳释放速率(CER)控制补入酵母粉,效价达到8697mg/L,与原工艺相比,提高了26.9%。这一结论若在实际工业生产中应用,有望带来实际的经济效益。     

力达霉素产生菌球孢链霉菌C-1027中atrA同源基因的克隆及分析

目的：在天蓝色链霉菌Streptomyces coelicolor A3(2)中多效性调节因子AtrA（AtrA-c）可通过激活放线紫红素途径特异性的调节因子ActII-ORF4的转录来控制放线紫红素的产生。在灰色链霉菌Streptomyces griseus NBRC13350和阿维链霉菌Streptomyces avermitilis MA-4680中也发现了AtrA-c 编码基因（atrA-c）的同源基因,分别影响链霉素和阿维菌素的生物合成。本文目的在于探索球孢链霉菌C-1027（Streptomyces globisporus C-1027）中是否存在AtrA,克隆球孢链霉菌C-1027中atrA基因并进行生物信息学分析,为进一步确定其对力达霉素产生的调控作用及调控机制奠定基础。[方法] 采用在球孢链霉菌C-1027中异源表达AtrA-c,来确定AtrA-c对力达霉素产量的影响;通过Southern blot 分析来判断在球孢链霉菌C-1027 基因组中是否有atrA-c同源基因;PCR扩增方法获得球孢链霉菌C-1027 atrA基因（atrA-gl）并测序;通过多种生物信息学软件来分析atrA-gl及其与旁侧基因的组织结构、对已发现的AtrA蛋白进行同源性比对及亲缘关系分析。[结果]在球孢链霉菌C-1027中异源表达天蓝色链霉菌AtrA-c蛋白,发现其对力达霉素的产量有影响。以atrA-c为探针,通过Southern blot分析显示球孢链霉菌C-1027基因组中存在atrA-c的同源基因。PCR扩增得到球孢链霉菌C-1027 的atrA基因的全序列以及该基因上下游的旁侧序列（GenBank/EMBL/DDBJ 登录号GU723707）。通过对球孢链霉菌C-1027、天蓝色链霉菌A3(2)、灰色链霉菌NBRC13350以及阿维链霉菌MA-4680 AtrA蛋白序列进行同源性分析发现,四种AtrA蛋白编码氨基酸序列一致性达到65%至 87%,相似性高达70% 至89%。并且,球孢链霉菌C-1027 atrA基因与相邻基因形成的组织结构与天蓝色链霉菌和灰色链霉菌完全一致。根据蛋白质同源性绘制进化树,发现球孢链霉菌AtrA蛋白与灰色链霉菌AtrA蛋白亲缘关系最近。[结论]确定在球孢链霉菌C-1027中存在atrA同源基因并影响力达霉素的产量,克隆了首个力达霉素生物合成基因簇外的调节基因--atrA基因,通过生物信息学分析初步推测了该基因的功能,为进一步研究AtrA-gl对力达霉素途径特异性级联调控网络的调控关系奠定了基础。     

Avermectins: biotechnological features of the producing strain of Streptomyces avermitilis VKM As 1301]

The monosporic plating of the avermectin-producing strain Streptomyces avermitilis VKM Ac-1301 with low activity showed the heterogeneity of the population. By selection of natural mutants the authors obtained a strain synthesizing up to 60 micrograms avermectin B1 per ml of culture liquid. The maximum avermectin yield was observed in the medium containing 7% glucose after 100-120 h of culture growth.     

阿维链霉菌bkdAB的基因中断对阿维菌素合成的影响

以阿维菌B组分菌株StreptomycesavermitilisBjbm0 0 0 6为出发菌株 ,用PCR的方法构建bkdAB基因簇(Branched_chainα_ketoaciddehydrogenasegeneAB)的基因置换质粒pHJ582 1(pHZ13 58∷bkdAB&erm) ,并对其进行基因中断 ,得到重组菌株Bjbm582 1。Bjbm582 1的发酵产物经HPLC检测发现 ,除了产生B1a和B2a外 ,还产生一种原菌株没有的新组分 ,3个组分的总含量只有出发菌株Bjbm0 0 0 6的 2 5%。结果表明bkdAB的中断不仅部分阻断了阿维菌素的合成 ,还阻断了阿维菌素b组分的合成 ,可以推测bkdAB的产物在阿维菌素合成途径中主要承担了α酮基异戊酸脱氢酶 (α_ketoisovalericaciddehydrogenase)角色     

除虫链霉菌10个聚酮类抗生素生物合成基因簇的敲除对阿维菌素产量的影响

【目的】考察除虫链霉菌基因组中其它聚酮合成酶类(Polyketide synthase,PKS)抗生素生物合成基因簇的敲除突变对于阿维菌素产量的影响。【方法】构建了11个PKS基因簇的打靶Cosmid和质粒载体,导入除虫链霉菌中筛选突变株。【结果】在工业菌株MMR630中成功敲除了10个PKS基因簇。发酵结果显示7个PKS基因簇敲除突变株中阿维菌素的产量均有不同程度的提高,而2个突变株不能产生阿维菌素。然而,在3个连续敲除2个PKS基因簇的突变株中阿维菌素产量没有能够超过单个PKS敲除突变株的提升幅度。【结论】除虫链霉菌基因组的一些PKS基因簇的敲除可以提高阿维菌素的产量,同时暗示同一类次生代谢产物的代谢流之间存在复杂的相互作用关系。     

阿维菌素生产菌的常压室温等离子体诱变育种及培养基优化

【目的】通过诱变筛选技术选育阿维菌素高产突变株,对其发酵培养基进行响应面优化,提高阿维菌素产量。【方法】采用常压室温等离子体(ARTP)诱变技术,结合链霉抗性和卡那霉素抗性筛选法及96深孔板高通量筛选法,筛选阿维菌素高产株。在单因素实验的基础上,应用响应面分析法对其发酵培养基进行优化,最后确定最佳培养基配方。【结果】获得一株遗传性状稳定的阿维菌素高产株K-1A6,其阿维菌素产量达到4.22 g/L,比出发菌株9-39提高了23.4%,在最佳培养基中阿维菌素产量达到5.36 g/L,较优化前提高了27.01%。【结论】通过对阿维链霉菌9-39菌株进行ARTP诱变筛选及发酵培养基优化研究能显著提高阿维菌素的产量。     

Dependence of the composition of the avermectin complex of Streptomyces avermitilis on glucose content of the growth medium

The effects of glucose concentration in the medium and O-methyl-L-threonine resistance on the ratio of components of the avermectin complex produced by Streptomyces avermitilis have been studied. Glucose deficiency increases the ratio of components A and a, while decreasing that of components 1 in the complex. A mutation that renders the microorganisms resistant to O-methyl-L-threonine (an analog of isoleucine) increases the ratio of components a, while decreasing that of components 1 in the complex. The distribution of a and b in fractions 1 and 2 remains constant: the values of the ratio a/b in the fractions amount, respectively, to 1:1 and 2:1. The relation of the variations in the composition of the avermectin complex to changes in the carbohydrate metabolism of the producer stain, underlain by availability of the source of carbon, is discussed.     

Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis.

The Tn3-like Streptomyces transposon Tn4560 was used to mutagenize Streptomyces avermitilis, the producer of anthelmintic avermectins and the cell growth inhibitor oligomycin. Tn4560 transposed in this strain from a temperature-sensitive plasmid to the chromosome and from the chromosome to a plasmid with an apparent frequency of about 10(-4) to 10(-3) at both 30 and 39 degrees C. Auxotrophic and antibiotic nonproducing mutations were, however, obtained only with cultures that were kept at 37 or 39 degrees C. About 0.1% of the transposon inserts obtained at 39 degrees C caused auxotrophy or abolished antibiotic production. The sites of insertion into the S. avermitilis chromosome were mapped. Chromosomal DNA fragments containing Tn4560 insertions in antibiotic production genes were cloned onto a Streptomyces plasmid with temperature-sensitive replication and used to transport transposon mutations to other strains, using homologous recombination. This technique was used to construct an avermectin production strain that no longer makes the toxic oligomycin.     

阿维菌素衍生物CHC-B1的突变生物合成

利用成功构建的基因缺失载体pLJ04(pKC1139∷△bkdF +△bkdH)对阿维菌素(avermectin)高产菌阿维链霉菌(Streptomyces avermitilis)76-02-e的bkdFGH基因进行缺失,获得的bkdFGH缺失突变株经过摇瓶发酵和HPLC检测,发现该突变株完全丧失了产生阿维菌素的能力。2-甲基丁酸及异丁酸的前体添加试验表明,当有外源前体存在时,突变株又能恢复阿维菌素合成的能力。将该bkdFGH基因缺失突变株命名为S.avermitilis bkd76-3。环己羧酸(CHC)前体添加试验及HPLC检测发现存在4种产物,经LC/MS分析验证,其中两种产物分别为CHC-B1和CHC-A2。     

Purification and identification of dTDP-oleandrose, the precursor of the oleandrose units of the avermectins

The nucleotide sugar precursor of the oleandrose units of the avermectins has been purified from a mutant of Streptomyces avermitilis, which does not synthesize any avermectins but which converts avermectin aglycones to their respective disaccharides. This precursor has been identified as dTDP-oleandrose. The purification was achieved by anion exchange and reverse phase high performance liquid chromatography. The purified nucleotide sugar had an absorption spectra characteristic of thymidine, released dTMP when treated with phosphodiesterase, and possessed an NMR spectrum in which three resonances characteristic of oleandrose were seen in addition to the thymidine signals. The enzyme, avermectin aglycone dTDP-oleandrose glycosyltransferase, which catalyzes the stepwise addition of oleandrose to the avermectin aglycones, has been demonstrated in cell-free extracts and (NH4)2SO4 fractions of cell-free extracts of S. avermitilis. The enzyme is specific for dTDP-oleandrose as the glycosyl donor but utilizes all avermectin aglycones as glycosyl acceptors. The stoichiometry between dTDP-oleandrose consumed in the reaction and oleandrose units transferred to the avermectin mono- and disaccharide was found to be 1:1.     

Two threonine residues required for role of AfsKav in controlling morphogenesis and avermectin production in Streptomyces avermitilis

AfsKav is a eukaryotic-type serine/threonine protein kinase, required for sporulation and avermectin production in Streptomyces avermitilis. In terms of their ability to complement SJW4001 (DeltaafsK-av), afsK-av mutants T165A and T168A were not functional, whereas mutants T165D and T168D retained their ability, indicating that Thr-165 and Thr-168 are the phosphorylation sites required for the role of AfsKav. Expression of the S-adenosylmethione synthetase gene promoted avermectin production in the wild-type S. avermitilis, yet not in the mutant harboring T168D or T165D, demonstrating that tandem phosphorylation on Thr-165 and Thr-168 in AfsKav is the mechanism modulating avermectin production in response to S-adenosylmethione accumulation in S. avermitilis.     

阿维菌素B1a组分高产菌株的定向选育

以阿维链霉菌(Streptomyces avermitilis)1-17为出发菌株,分别使用紫外线及亚硝基胍并结合L-异亮氨酸诱导手段进行诱变处理,得到AVMB1a组分摇瓶发酵水平较出发菌株提高12.86%的突变株3-6.传代实验表明该菌株的高产性能稳定.结果表明,采用UV、NTG诱变结合L-Ile诱导的手段可以获得B1a组分显著提高的菌株.     

Semi-synthetic DNA shuffling of aveC leads to improved industrial scale production of doramectin by Streptomyces avermitilis

The avermectin analog doramectin (CHC-B1), sold commercially as Dectomax, is biosynthesized by Streptomyces avermitilis. aveC, a gene encoding an unknown mechanistic function, plays an essential role in the production of doramectin (avermectin CHC-B1), modulating the production ratio of CHC-B1 to other avermectins, most notably the undesirable analog CHC-B2. To improve the production ratio for doramectin, the aveC gene was subjected to iterative rounds of semi-synthetic DNA shuffling. Libraries of shuffled aveC gene variants were transformed into S. avermitilis, screened using a miniaturized 96-well growth and production format, and analyzed by high throughput mass spectrometry to determine CHC-B2:CHC-B1 ratios. Several improved aveC variants were identified; the best shuffled gene encoded 10 amino acid mutations, and conferred a final CHC-B2:CHC-B1 ratio of 0.07:1, a 23-fold improvement over the starting gene (aveC wild type). Chromosomal insertion of an improved aveC shuffled gene into a high titer S. avermitilis strain yielded an improved doramectin production strain. This strain is under development to be used commercially, and is expected to provide considerable cost savings in large-scale manufacture, as well as significantly reducing by-product levels of CHC-B2 requiring disposal.     

A second branched-chain alpha-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis: its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins.

A second cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1 alpha), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1 beta) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1 alpha subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5' end of bkdF is also described. The mutant exhibited a typical Bkd- phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an alpha-branched-chain fatty acid starter unit, either S(+)-alpha-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(+)-alpha-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.     

Targeted gene disruption of the avermectin B O-methyltransferase gene in Streptomyces avermitilis

The biological activity of avermectin B components is superior to that of avermectin A components, which are derived from avermectin B by avermectin B 5-O-methyltransferase. Gene disruption, targeting avermectin B 5-O-methyltransferase gene in Streptomyces avermitilis, was carried out to obtain a strain of avermectin B producer. Phenotype analysis of the mutant with the disrupted O-methyltransferase gene showed that only avermectin B components were produced with a significant increase in production