Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin
Genetic engineering of Streptomyces bingchenggensis to produce milbemycins A3/A4 as main components and eliminate the biosynthesis of nanchangmycin
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DOI：
10.1007/s00253-013-5255-5
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发表时间：
2013-09
影响因子：
5
通讯作者：
Ji Zhang;Jing An;Ji-Jia Wang;Yi-Jun Yan;Hai-Rong He;Xiang-Jing Wang;Wen-Sheng Xiang
中科院分区：
文献类型：
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作者：
Ji Zhang;Jing An;Ji-Jia Wang;Yi-Jun Yan;Hai-Rong He;Xiang-Jing Wang;Wen-Sheng Xiang
Milbemycins A3/A4是重要的16元大环内酯类化合物，已商品化，广泛用作农药和兽药。然而，与其他米尔贝霉素产生菌类似，冰城链霉菌产生米尔贝霉素A3/A4时，通常伴随着C5-O-甲基米尔贝霉素B2/B3(α级)和β1/β2(β级)等不良副产物与南昌霉素一起产生。为了获得以产霉菌素A3/A4为主要成分的高产霉菌素A3/A4菌株，对冰城链霉菌C5-O-甲基转移酶基因MIRED进行了异源表达的生物学功能研究。酶分析表明，MILD能催化α类(A3/A4)和β类Milbemycins(β11)分别生成C5-O-甲基Milbemycins B2/B3和β1，表明C6和C8a之间形成的呋喃环对MILD催化的C5-O-甲基化反应几乎没有影响。突变株MilD基因的缺失导致C5-O-甲基Milbemycins B2/B3和β1/β2的缺失，同时提高了Milbemycins A3/A4的产量。进一步破坏了负责南昌霉素生物合成的聚酮合成酶的基因组编码加载模块，导致菌株BCJ36取消了南昌霉素的生产。重要的是，突变菌株BCJ36(∆Almand∆NanLD)产生的主要次生代谢产物为米尔贝菌素A3/A4，产量为2312 ± 47μg/ml，比出发菌株BC-109-6(1326 ± 37μg/ml)高出约74%。
Milbemycins A3/A4 are important 16-membered macrolides which have been commercialized and widely used as pesticide and veterinary medicine. However, similar to other milbemycin producers, the production of milbemycins A3/A4 in Streptomyces bingchenggensisis usually accompanied with undesired by-products such as C5-O-methylmilbemycins B2/B3 (α-class) and β1/β2 (β-class) together with nanchangmycin. In order to obtain high yield milbemycins A3/A4-producing strains that produce milbemycins A3/A4 as main components,milD, a putative C5-O-methyltransferase gene ofS.bingchenggensis, was biofunctionally investigated by heterologous expression inEscherichia coli. Enzymatic analysis indicated that MilD can catalyze both α-class (A3/A4) and β-class milbemycins (β11) into C5-O-methylmilbemycins B2/B3 and β1, respectively, suggesting little effect of furan ring formed between C6 and C8a on the C5-O-methylation catalyzed by MilD. Deletion ofmilDgene resulted in the elimination of C5-O-methylmilbemycins B2/B3 and β1/β2 together with an increased yield of milbemycins A3/A4 in disruption strain BCJ13. Further disruption of the genenanLDencoding loading module of polyketide synthase responsible for the biosynthesis of nanchangmycin led to strain BCJ36 that abolished the production of nanchangmycin. Importantly, mutant strain BCJ36 (∆milD∆nanLD) produced milbemycins A3/A4 as main secondary metabolites with a yield of 2312 ± 47 μg/ml, which was approximately 74 % higher than that of the initial strainS.bingchenggensisBC-109-6 (1326 ± 37 μg/ml).