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Streptomyces avermitilis Bjbm0006为出发菌株,用PCR的方法构建bkdAB基因簇(Ｂranched_chain α-keto acid dehydrogenase gene AB) 的基因置换质粒pHJ5821 (pHZ1358∷bkdAB&erm),并对其进行基因中断,得到重组菌株Bjbm5821。Bjbm5821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Bjbm0006的25%。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-ketoisovaleric acid dehydrogenase) 角色。     

阿维链霉菌bkdF的基因中断对阿维菌素合成的影响

以阿维菌素 B组分菌株Streptomyces avermitilis Bjbm0006为出发菌株,用PCR方法构建支链α酮酸脱氢酶基因bkdF（Branchedchain αketo acid dehydrogenase gene）的重组质粒pHJ5816 (pHZ1358/bkdF&Ermr)对其进行基因中断,得到重组菌株Bjbm5816。经HPLC检测和核磁共振分析发现,Bjbm5816发酵产物产生的单一组分新化合物为OligomycinA。     

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。     

阿维链霉菌中aveD基因阻断对阿维菌素合成的影响

利用用于基因破坏的重组质粒pCZ2 (pKC1 1 39∷ 475bpaveD)对阿维菌素 (Avermectin)产生菌阿维链霉菌 (Streptomycesavermitilis) 76- 9的aveD基因进行插入失活 ,将获得的aveD破坏子进行摇瓶发酵和阿维菌素初步提取和HPLC检测 ,发现破坏子仅产生四个主要组分 ,但它们的保留时间分别比Bla、Blb、B2a和B2b的略长。进而将粗提液纯化并获得晶体 ,以UV、IR、NMR ( 1H NMR和13C NMR)和MS进行结构分析 ,并结合HPLC检验 ,证明它们属于C5 氧 阿维菌素B。说明阿维链霉菌的aveD基因破坏 ,不仅丧失了合成阿维菌素A组分的能力 ,也造成了其下游的aveF基因不能表达 ,因此只产生了C5 氧 阿维菌素B。     

柔红霉素产生菌SIPI-1482中dnmV基因功能的阻断及恢复

dnmV基因产物为柔红霉素生物合成途径中TDP-6-脱氧己糖C4酮基还原酶,破坏该基因能阻断柔红糖胺的合成,进而阻断柔红霉素的产生。从天蓝淡红链霉菌(S. coeruleorubidus)SIPI-1482基因组DNA中经PCR扩增出dnmV及其上游dnmU基因片段,并由此构建了用于阻断dnmV基因的同源重组质粒pYG817,转化SIPI-1482菌株后成功地破坏了dnmV基因,发酵结果显示阻断突变株不再代谢产生柔红霉素,为引入新的基因来改变代谢产物的糖基结构打下了基础。通过导入dnmV基因表达质粒可重建该突变株的生物合成途径,恢复产生柔红霉素,但产量比出发菌株要低。     

阿维莲霉菌中aveD基因缺失对阿维菌素合成的影响

利用aveD基因的缺失载体pCZ8(pKC1139::△aveD)对阿维菌素（Avermectin）的产生菌阿维链霉菌（Streptomyces avermitilis)76-9的aveD基因进行缺失获得aveD缺失突变株。经摇瓶发酵和HPLC检测,发现该突变株只产生阿链菌素B组分。说明将阿维链霉菌的aveD基因缺失,并不影响下游aveF的表达。缺失突变株的阿维菌素的总产量与出发菌株的总产量基本相同,突变株中B1的产量略有提高,阿维菌素B2的含量显著提高。     

阿维链霉菌中aveD基因缺失对阿维菌素合成的影响

利用aveD基因的缺失载体pCZ8(pKC1139∷△aveD)对阿维菌素(Avermectin)产生菌阿维链霉菌(Streptomyces avermitilis)76\|9的aveD基因进行缺失获得aveD缺失突变株。经摇瓶发酵和HPLC检测,发现该突变株只产生阿维菌素B组分。说明将阿维链霉菌的aveD基因缺失,并不影响下游aveF的表达。缺失突变株的阿维菌素的总产量与出发菌株的总产量基本相同,突变株中B1的产量略有提高,阿维菌素B2的含量显著提高。     

阿维菌素的生物合成与途径工程

阿维菌素是一种高效安全的大环内酯杀虫杀螨剂。本文介绍了阿维菌素生物合成的步骤及参与合成步骤的有关酶系统和基因簇。对阿维菌素 8个组分合成的遗传控制基因 ,特别是对其中B1a组分合成的遗传控制位点进行讨论分析 ,并介绍了利用途径工程改造阿维链霉菌生产合成单一高效组分B1a和提高活性组分产量的研究进展。     

酿酒酵母adh2和ald6双基因缺失突变株的构建

酿酒酵母乙醇合成代谢过程中, 阻断或削弱乙醛至乙酸代谢流不但能增强乙醇合成流, 同时还能降低发酵乙酸含量。本研究以乙醇脱氢酶Ⅱ(adh2)基因缺陷型酿酒酵母YS2-Dadh2为出发菌株, 应用长侧翼同源两步PCR(LFH-PCR)策略构建乙醛脱氢酶Ⅵ(ald6)基因敲除组件, 转化酿酒酵母YS2-Dadh2敲除ald6基因, 之后转入表达质粒pSH65到阳性克隆中, 半乳糖诱导表达Cre重组酶切除Kanr基因筛选标记, 最后, 传代丢失质粒pSH65获得单倍体ald6基因缺失突变株。利用同样的敲除组件和技术再次敲除其等位基因, 最终获得双基因缺失突变株YS2-△adh2-Dald6。发酵实验表明与出发菌株YS2相比, 突变株乙酸合成量降低18%, 乙醇最高产量提高12.5%。     

阿维菌素B1a组分高产菌株的诱变育种

目的了解阿维菌素产生菌S.avermitilis的生长特性,提高产生菌B1a组份的产量。方法采用紫外线(UV)、诱变剂氯化锂(LiCl),亚硝基胍(NTG)并结合甲硫氨酸(Met)诱导等手段对出发菌株S.avermitilisH0065进行诱变处理,初筛、复筛。结果筛选获得总效价达到4524.3μg/ml的高产阿维菌突变株N-3-133,其中B组分含量显著提高且高达85.3%,B1a也显著提高达到2533.6μg/ml。结论采用紫外线及亚硝基胍诱变方法,结合甲硫氨酸诱导筛选,与出发菌株相比可以获得阿维菌素总效价及B1a组分显著提高的菌株。     

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.     

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.     

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.     

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.     

Cloning and analysis of a DNA fragment stimulating avermectin production in various Streptomyces avermitilis strains

To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis.     

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。     

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。