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.     

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.     

Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis

Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8?g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.     

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     

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。     

Action of avermectins on lymphoid leukemia P-388 cells in vitro]

Avermectins are final products in the fermentation process with Streptomyces avermitilis. They have parasitocidic activity and are used as the main substances of insectoacaronematocides. The study of the activity of the natural avermectin complex (aversectin C) and separate avermectins A1, A2, B1 and B2 in the cell culture of lymphoid leukemia P-388 showed that within the concentrations of 0.1 to 1.0 microgram/ml aversectin C inhibited the growth of the tumor cells and induced their death. The inhibition was due to blocking the cell mitosis. The cell death was accompanied by internucleosomal degradation of the DNA nuclei i.e. the death was of the apoptosis type. The sensitivity of the cells to aversectin C was directly proportional to their initial proliferative activity. As for the separate avermectins only avermectin A1 had the cytotoxic activity within the concentrations used, avermectin A2 had the cytostatic activity and avermectins B1 showed no activity under the experimental conditions.     

Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces

A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin.     

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。     

多拉菌素产生菌aveD基因缺失突变株的构建

阿维链霉菌(Streptomyces avermitilis)bkd76-3在发酵过程中添加环己羧酸(CHC)可产生抗寄生虫药物多拉菌素(doramectin,阿维菌素衍生物CHC-B1),但同时还产生其它三种无效组分CHC-B2、CHC-A1、CHC-A2。利用基因缺失载体pXJ04(pKC1139∷△aveD1+△aveD2)对该菌株的aveD基因进行缺失,获得的aveD缺失突变株经摇瓶发酵和HPLC检测,发现只存在2种产物,经LC/MS分析验证,这两种产物分别为CHC-B1和CHC-B2,表明该突变株完全丧失了合成CHC-A1和CHC-A2的能力。缺失突变株的CHC-B1产量较出发菌株提高了78.19%,CHC-B2的产量提高了602.3%,发酵产物中有效组分多拉菌素的比例增加了93.16%。该缺失突变是在染色体上通过同源双交换完成的,不会发生进一步的重组,因此突变株具有良好的遗传稳定性,在工业生产上具有应用价值。     

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.     

阿维链霉菌的诱变育种

以阿维链霉菌(Streptomyces avermitilis)76-12为出发菌株,采用亚硝基胍、吖啶橙、紫外线和氯化锂分别对其孢子和原生质体进行诱变,经抗代谢物理性筛选,获得一系列高产突变株,其中N-1-2高产突变株的发酵单位是出发菌株的2.47倍。实验中同时获得了只产阿维菌素a组分的突变株G-32、Bla组分含量高的Ave8菌株和产蓝绿色孢子的突变株UA-G等。     

Alternative production of avermectin components in Streptomyces avermitilis by gene replacement

The avermectins are composed of eight compounds, which exhibit structural differences at three positions. A family of four closely-related major components, A1a, A2a, B1a and B2a, has been identified. Of these components, B1a exhibits the most potent antihelminthic activity. The coexistence of the "1" components and "2" components has been accounted for by the defective dehydratase of aveAI module 2, which appears to be responsible for C22-23 dehydration. Therefore, we have attempted to replace the dehydratase of aveAI module 2 with the functional dehydratase from the erythromycin eryAII module 4, via homologous recombination. Erythromycin polyketide synthetase should contain the sole dehydratase domain, thus generating a saturated chain at the C6-7 of erythromycin. We constructed replacement plasmids with PCR products, by using primers which had been derived from the sequences of avermectin aveAI and the erythromycin eryAII biosynthetic gene cluster. If the original dehydratase of Streptomyces avermitilis were exchanged with the corresponding erythromycin gene located on the replacement plasmid, it would be expected to result in the formation of precursors which contain alkene at C22-23, formed by the dehydratase of erythromycin module 4, and further processed by avermectin polyketide synthase. Consequently, the resulting recombinant strain JW3105, which harbors the dehydratase gene derived from erythromycin, was shown to produce only C22,23-unsaturated avermectin compounds. Our research indicates that the desired compound may be produced via polyketide gene replacement.     

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.     

Chromosomal aberrations and micronuclei induced in rat and mouse bone marrow cells by sodium nitrate

The genotoxicity of several anthraquinone compounds metabolically related to aflatoxin B1 was examined by means of the hepatocyte primary culture (HPC)/DNA repair test and the Salmonella microsome mutagenesis test, and compared to versicolorins A and B which are potent mutagenic and genotoxic intermediates of the aflatoxin biosynthetic pathway. 6,8-O-Dimethyl-versicolorins A, B and 6-deoxyversicolorin A were found to be strongly mutagenic and genotoxic. Genotoxicity of versicolorin A and 6,8-O-dimethylversicolorin A was stronger than that of versicolorin B and 6,8-O-dimethylversicolorin B, respectively, in the HPC/DNA repair test. Nidurufin and norsolorinic acid, which do not possess a bisfuran ring, exhibited questionable activities for mutagenicity and no genotoxicity. It is suspected that 6,8-O-dimethylversicolorins A, B and 6-deoxyversicolorin A as well as versicolorins A and B are genotoxic carcinogens.     

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。     

阿维菌素的生物合成及代谢工程研究进展

阿维菌素(avermectin)是由除虫链霉菌(Streptomycesavermitilis)产生的一种具有杀虫活性的大环内酯类抗生素,在农业和畜牧业中应用广泛。本文综述了有关除虫链霉菌基因组序列分析、阿维菌素的生物合成以及阿维菌素育种和代谢工程的研究进展。     

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。     

Modulation of serine/threonine protein kinase activity in chloramphenicol-resistant mutants of Streptomyces avermitilis

A mutation to chloramphenicol resistance (Cmlr) stimulates production of macrolide avermectin in Streptomyces avermitilis; production starts in early stationary growth. By labeling in vivo, the Cmlr mutation was shown to stimulate phosphorylation of Ser and Thr in several proteins in the same growth phase. Autophosphorylation of active protein kinases (PK) was analyzed in gel after one- or two-dimensional PAGE for the original S. avermitilis strain ATCC 31272, its Cmlr mutant, and a Cmls revertant. An increase in in vivo phosphorylation was associated with an increase in autophosphorylation of Ser/Thr-PK 41K, 45K, 52K, 62K, and 85K and complete suppression of autophosphorylation of PK 66K. Comparison of the PK molecular weights and pI with the parameters deduced for putative PK encoded by S. avermitilis genes identified the 41K, 45K, 52K, 62K, and 85K PK as pkn 24, pkn 32, pkn 13, pkn 12, and pkn 5, respectively. Prenylamine lactate, a modulator of calmodulin-dependent processes, substantially reduced the avermectin production, impaired the Cml resistance, and selectively inhibited Ca2+-dependent PK 85K in the Cmlr mutant. It was assumed that PK 85K is involved in regulating the avermectin production.