A 2ND 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 2ND 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
复制标题
DOI：
10.1128/jb.177.12.3504-3511.1995
复制
发表时间：
1995-06-01
影响因子：
3.2
通讯作者：
WERNAU, WC
中科院分区：
文献类型：
--
作者：
DENOYA, CD;FEDECHKO, RW;WERNAU, WC
第二个基因簇编码支链α-酮酸脱氢酶（BCDH）的E1 α、E1 β和E2亚基，bkdFGH，已从除虫链霉菌（Streptomycesavermiatum）中克隆并鉴定，除虫链霉菌是一种产生驱虫除虫菌素的土壤微生物。打开阅读帧1（ORF 1）（bkdF，编码E1 α），将编码44，394 Da的多肽（406个氨基酸），不完全测序的ORF 2的推定起始密码子（bkdG，编码E1 β）位于ORF 1末端下游83 bp处，推导的氨基酸序列与几种原核和真核BCDH复合物的相应E1 α亚基相似，An S.还描述了通过缺失包含bkdF的5 ′末端的基因组区域而构建的阿维菌素bkd突变体，该突变体表现出典型的Bkd（-）表型：它缺乏E1 CDH活性，并且丧失了在含有异亮氨酸、亮氨酸和缬氨酸作为唯一碳源的固体基本培养基上生长的能力。由于BCDH提供了或支链脂肪酸起始单元，S（+）-α-甲基丁酰辅酶A或异丁酰辅酶A，其对于起始S.在缺乏S（+）-α-甲基丁酸盐和异丁酸盐的培养基中，被破坏的突变体不能产生天然阿维菌素。补充这些化合物中的任一种可恢复相应天然阿维菌素的生产，而补充替代脂肪酸的培养基可导致新阿维菌素的形成。这些结果证实了BCDH催化的支链氨基酸催化剂反应是为S.阿维菌素。
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 CDH 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 or-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.