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。     

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.     

Cloning of a DNA fragment involved in pigment production in Streptomyces avermitilis

Streptomyces avermitilis has the ability to synthesize a diffusible, brown, melanin-like pigment, a common property among many Streptomyces species. A region of the S. avermitilis chromosome involved in the production of this pigment was cloned in Escherichia coli. Production of the brown pigment was attained in E. coli, and is optimal when medium is supplemented with copper ions, tyrosine and IPTG. The cloned S. avermitilis pigment-producing DNA fragment is under the control of the lac promoter carried in the E. coli vector. The gene involved in pigment production could be used as a tool to analyse gene expression in S. avermitilis, and as an alternative cloning marker in Streptomyces-Escherichia coli vectors.     

Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis

Species of the genus Streptomyces are of major pharmaceutical interest because they synthesize a variety of bioactive secondary metabolites. We have determined the complete nucleotide sequence of the linear chromosome of Streptomyces avermitilis. S. avermitilis produces avermectins, a group of antiparasitic agents used in human and veterinary medicine. The genome contains 9,025,608 bases (average GC content, 70.7%) and encodes at least 7,574 potential open reading frames (ORFs). Thirty-five percent of the ORFs (2,664) constitute 721 paralogous families. Thirty gene clusters related to secondary metabolite biosynthesis were identified, corresponding to 6.6% of the genome. Comparison with Streptomyces coelicolor A3(2) revealed that an internal 6.5-Mb region in the S. avermitilis genome was highly conserved with respect to gene order and content, and contained all known essential genes but showed perfectly asymmetric structure at the oriC center. In contrast, the terminal regions were not conserved and preferentially contained nonessential genes.     

无机盐对阿维链霉菌接合转移及异源表达放线紫红素的影响

摘要：【目的】阿维链霉菌可作为异源表达抗生素生物合成基因簇的良好宿主,但是需要优化含有大片段DNA质粒的接合转移效率。【方法】我们选取MgCl2、NaCl、Ca(NO3)2 和CaCl2等4种无机盐,在0－200 mmol/L浓度范围内分别研究其对大质粒向阿维链霉菌接合转移的影响,再设计完全随机试验筛选最佳条件。【结果】CaCl2对阿维链霉菌接合转移有极明显的促进作用,MgCl2也有一定提高作用。通过完全随机试验筛选出最佳的CaCl2和MgCl2浓度组合,使大质粒的接合转移效率提高11倍。同时,本研究还发现阿维链霉菌异源表达放线紫红素的最适培养基,成功表达放线紫红素。【结论】特定无机盐对阿维链霉菌接合转移效率有明显提高作用,并且能促进放线紫红素在阿维链霉菌中的表达。     

重离子辐照诱变阿佛曼链霉菌的实验观察

目的：对阿佛曼链霉菌采用新的诱变手段,以获得稳定高产的优良菌株。方法：采用重离子束辐照阿佛曼链霉菌,研究了0.25Gy、0.5Gy、3Gy、5Gy、10Gy和15Gy剂量的12C＋粒子束辐照阿佛曼链霉菌菌株后,菌落特性的变化及对菌株产素能力的影响。结果：重离子辐照阿佛曼链霉菌后,在其各个辐照剂量区都存在变异菌株,诱变后阿佛曼链霉菌的菌落形态多样,小山状,火山口状、彗星尾状、车轮状、边缘放射状等;菌落大小不一,有的直径达4～5mm,有的小如针尖状。效价提高到7298μg/mL,获得了高产菌株。结论：重离子束辐照阿佛曼链霉菌菌株后,阿佛曼链霉菌的产素能力显著提高,可得到高产的菌株。     

Novel family of cholesterol esterases produced by actinomycetes bacteria

Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly-Xaa-Ser-Xaa-Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.     

Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis.

Streptomyces avermitilis contains a unique restriction system that restricts plasmid DNA containing N6-methyladenine or 5-methylcytosine. Shuttle vectors isolated from Escherichia coli RR1 or plasmids isolated from modification-proficient Streptomyces spp. cannot be directly introduced into S. avermitilis. This restriction barrier can be overcome by first transferring plasmids into Streptomyces lividans or a modification-deficient E. coli strain and then into S. avermitilis. The transformation frequency was reduced greater than 1,000-fold when plasmid DNA was modified by dam or TaqI methylases to contain N6-methyladenine or by AluI, HhaI, HphI methylases to contain 5-methylcytosine. Methyl-specific restriction appears to be common in Streptomyces spp., since either N6-methyladenine-specific or 5-methylcytosine-specific restriction was observed in seven of nine strains tested.     

Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis

Streptomyces avermitilis is an industrially important soil bacterium known for production of avermectins, which are antiparasitic agents useful in animal health care, agriculture, and treatment of human infections. ku genes play a key role in the non-homologous end-joining pathway for repair of DNA double strand breaks. We identified homologs of eukaryotic ku70 and ku80 genes, termed ku1 and ku2, in S. avermitilis. Mutants with deletion of ku1, ku2, and both genes were constructed and their phenotypic changes were characterized. Deletion of ku genes had no apparent adverse effects on growth, spore formation, or avermectin production. The ku mutants, in comparison to wild-type strain, were slightly more sensitive to the DNA-damaging agent ethyl methanesulfonate, but not to UV exposure or to bleomycin. Gene targeting frequencies by homologous recombination were higher in the ku mutants than in wild-type strain. We conclude that ku-deleted strains will be useful hosts for efficient gene targeting and will facilitate functional analysis of genes in S. avermitilis and other industrially important bacterial strains.     

阿维链霉菌Streptomyces avermitilis碱性脂肪酶LpsA2的表达和酶学性质分析

【目的】本研究将推测的阿维链霉菌(Streptomyces avermitilis)脂肪酶基因lpsA2在大肠杆菌(Escherichia coli)中进行异源表达及系统的酶学性质分析。【方法】提取阿维链霉菌基因组,设计特异性引物,PCR扩增脂肪酶基因lpsA2,使其在大肠杆菌中异源表达,利用6个组氨酸标签纯化脂肪酶LpsA2,并进行酶学性质分析;对LpsA2进行序列比对和进化分析。【结果】氨基酸序列比对显示LpsA2具有脂肪酶典型的由Ser、His和Asp构成的活性部位,即Ser130-Asp221-His25,其中Ser位于保守的五肽结构(Gly128-His129-Ser130-Gln131-Gly132)中;分子系统学分析显示,LpsA2是脂肪酶第一家族亚家族成员(Subfamily I.7);实验测得纯化的重组脂肪酶LpsA2的最适反应pH为8.0,最适反应温度为50℃;最适底物为对硝基苯酚豆蔻酸酯;在10℃-50℃范围内该酶的激活自由能为6.3 kcal/mol;1 mmol/L Co2+、Hg2+、Zn2+可使酶活性提高至250%以上;15%的二甲基甲酰胺和二甲基亚砜使酶活分别提高至110.7%和138%;0.1%和1%的Span-20可使酶活性分别提高至352.7%和189.7%。【结论】本研究对推测的来源于S.avermitilis的脂肪酶基因lpsA2进行了异源表达和酶学功能鉴定,不仅为脂肪酶的研究积累了更多数据,也为具有优良性能的脂肪酶生物工程菌的筛选奠定了基础,更为其在食品加工、药物合成等工业生产中的应用提供了依据。     

Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.     

Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector.

An integration vector for gene analysis in Streptomyces has been constructed. This vector replicates in Escherichia coli, and integrates into Streptomyces by homologous recombination between a cloned fragment and the genome. To overcome methylation-specific restriction barriers, an E. coli mutant triply defective in DNA methylation was constructed as a source for the integration plasmids. The frequency of integration of pVE616 derivatives into the Streptomyces avermitilis genome was proportional to the size of the cloned DNA. Derivatives of pVE616, containing fragments from pVE650, a plasmid with a 24-kb insert of S. avermitilis DNA, were used in complementation analyses of seven S. avermitilis mutants defective in glycosylation of avermectin (Av). Three complementation groups, located in a 7-kb region, were identified. Derivatives of pVE616, containing fragments from the 18-kb of DNA adjacent to the glycosylation region, were integrated into an Av producer. Av produced from the integrants was substantially reduced, indicating that the 18 kb also encodes gene products which are involved in Av biosynthesis.     

应用于二维电泳细胞蛋白质样品提取的复合蛋白酶抑制剂的优化

阿维链霉菌在复合培养基中生长时合成大量蛋白酶以满足菌体分解有机氮源进行生长代谢的需要。而大量蛋白酶的存在对二维电泳的蛋白质组分析细胞蛋白质样品的提取带来了很大的困难。根据阿维链霉菌胞内蛋白酶的组成,以EDTA、PMSF、Bestatin、Pepstatin和E-64等5种蛋白酶抑制剂为基础,通过单因子实验和正交实验优化得到了高效的蛋白酶抑制剂复合配方。验证实验表明,该复合蛋白酶抑制剂在阿维链霉菌细胞蛋白质样品提取中,具有良好的蛋白酶活性抑制效果。     

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。     

阿维链霉菌来源的磷脂酰丝氨酸合成酶基因的克隆及其在毕氏酵母中的异源表达

本文旨在构建阿维链霉菌(Streptomyces avermitilis)来源的磷脂酰丝氨酸合成酶基因(pss)的重组质粒,研究其在毕氏酵母中的异源分泌型表达。利用PCR技术克隆阿维链霉菌来源的pss基因,再通过电转化方法将重组质粒pOG-01转入毕氏酵母KM71中,构建重组工程菌KP1。实验结果表明,阿维链霉菌来源的磷酯酰丝氨酸合成酶基因在毕氏酵母KM71中成功表达,2 mL菌体上清催化50 mmol/L卵磷脂,转酯反应的转化率为58%,酶活为4.83 U/mL。     

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。     

Endospore formation by Streptomyces avermitilis in submerged culture

The capability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycles on synthetic medium CP1 with glucose under phosphate limitation, and (2) in autolysing cell suspensions of high density obtained by tenfold concentration of a stationary-phase culture grown in a synthetic medium resuspended in phosphate buffer (pH 7.2) with 0.2% CaCl2. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to pertain to the main species-defining features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in a surface culture.     

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli.

A cluster of genes encoding the E1 alpha, E1 beta, and E2 subunits of branched-chain alpha-keto acid dehydrogenase (BCDH) of Streptomyces avermitilis has been cloned and sequenced. Open reading frame 1 (ORF1) (E1 alpha), 1,146 nucleotides long, would encode a polypeptide of 40,969 Da (381 amino acids). ORF2 (E1 beta), 1,005 nucleotides long, would encode a polypeptide of 35,577 Da (334 amino acids). The intergenic distance between ORF1 and ORF2 is 73 bp. The putative ATG start codon of the incomplete ORF3 (E2) overlaps the stop codon of ORF2. Computer-aided searches showed that the deduced products of ORF1 and ORF2 resembled the corresponding E1 subunit (alpha or beta) of several prokaryotic and eukaryotic BCDH complexes. When these ORFs were overexpressed in Escherichia coli, proteins of about 41 and 34 kDa, which are the approximate masses of the predicted S. avermitilis ORF1 and ORF2 products, respectively, were detected. In addition, specific E1 [alpha beta] BCDH activity was detected in E. coli cells carrying the S. avermitilis ORF1 (E1 alpha) and ORF2 (E1 beta) coexpressed under the control of the T7 promoter.