基于前体供给途径遗传改造提高褐黄孢链霉菌纳他霉素的产量

Engineering the precursor supply pathway in Streptomyces gilvosporeus for overproduction of natamycin

摘要:

纳他霉素(natamycin)是一种高效、广谱、安全的抗真菌剂，广泛应用于食品防腐与医药领域。纳他霉素可由多种链霉菌发酵产生。它是以乙酰辅酶A、丙二酰辅酶A及甲基丙二酰辅酶A为前体经Ⅰ型聚酮合酶(polyketide synthase,PKS)催化合成的多烯大环内酯类化合物。本研究以纳他霉素产生菌——褐黄孢链霉菌为研究材料，分别对不同前体分子供给途径中的关键酶进行过表达，并确定影响纳他霉素产量的关键前体供给途径。研究结果发现：通过过表达乙酰辅酶A合成酶(acetyl-CoA synthase,ACS)加强乙酰辅酶A合成途径，以及通过过表达甲基丙二酰辅酶A变位酶(methylmalonyl-CoA mutase,MCM)加强甲基丙二酰辅酶A合成途径，重组菌株纳他霉素产量分别比野生型菌株提高了44.19%和20.51%。共过表达ACS和MCM，重组菌株纳他霉素产量获得进一步提升(达1123.34mg/L)，比野生型菌株提高了66.29%。上述发现为通过前体代谢工程的策略构建纳他霉素工业高产菌株提供了参考，也为其他聚酮类天然产物高产工程菌株的构建提供了借鉴。

关键词:褐黄孢链霉菌;纳他霉素;前体供给;乙酰辅酶A合成酶;甲基丙二酰辅酶A变位酶

Abstract:

Natamycin is a safe and efficient antimycotics which is widely used in food and medicine industry. The polyene macrolide compound, produced by several bacterial species of the genus Streptomyces, is synthesized by type Ⅰ polyketide synthases using acetyl-CoA, malonyl-CoA, and methylmalonyl-CoA as substrates. In this study, four pathways potentially responsible for the supply of the three precursors were evaluated to identify the effective precursor supply pathway which can support the overproduction of natamycin in Streptomyces gilvosporeus, a natamycin-producing wild-type strain. The results showed that over-expressing acetyl-CoA synthetase and methylmalonyl-CoA mutase increased the yield of natamycin by 44.19% and 20.51%, respectively, compared with the wild type strain under shake flask fermentation. Moreover, the yield of natamycin was increased by 66.29% compared with the wild-type strain by co-overexpression of acetyl-CoA synthetase and methylmalonyl-CoA mutase. The above findings will facilitate natamycin strain improvement as well as development of strains for producing other polyketide compounds.

Key words:Streptomyces gilvosporeus;natamycin;precursor supply;acetyl-CoA synthase;methylmalonyl-CoA mutase