The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, PR China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, PR China.
The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, PR China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, PR China.
Microbiol Res. 2021 Jan;242:126623. doi: 10.1016/j.micres.2020.126623. Epub 2020 Oct 13.
The antifungal agent amphotericin B (AmB) is a polyketide produced by Streptomyces nodosus. The synthetic precursors of the amphotericin macrolactone skeleton are acetyl-CoA, malonyl-CoA and methylmalonyl-CoA. The genome sequence of the wild type S. nodosus ATCC14899 revealed a type II polyketide synthase (PKS) competing for malonyl-CoA. The same competitive branch was sequenced and verified in a mutant named S. nodosus ZJB2016050 (S. nodosus N3) screened in our lab. The transcriptome of the secondary metabolic synthetic gene cluster comparisons suggested that type II PKS (PKS5) competition is a factor in low production. The deletion of the PKS5 gene led to the titer of AmB improved from 5.01 g/L to 6.32 g/L while the by-product amphotericin A (AmA) reduced from 0.51 g/L to 0.12 g/L. A sequence of genes including PKS amphA, acc1, mme and mcm were overexpressed in a ΔPKS5 mutant, resulting in improved production AmB from 5.01 g/L to 7.06 g/L in shake flasks at 96 h. The yield of AmB and AmA in a 5 L bioreactor at 144 h was 15.6 g/L and 0.36 g/L, respectively. The intracellular reducibility of the wild type, mutagenesis type and genetically engineered type were detected, which was first found to be related to the by-product AmA. The increment of skeleton biosynthesis may consume more NADPH and reduces AmphC ER5 domain reduction. This study can be implemented for other polyketides in industrial production.
抗真菌药物两性霉素 B（AmB）是由游动放线菌产生的聚酮化合物。两性霉素大环内酯骨架的合成前体是乙酰辅酶 A、丙二酰辅酶 A 和甲基丙二酰辅酶 A。野生型游动放线菌 ATCC14899 的基因组序列显示出一种竞争丙二酰辅酶 A 的 II 型聚酮合酶（PKS）。在我们实验室筛选的突变株游动放线菌 ZJB2016050（游动放线菌 N3）中，测序并验证了相同的竞争分支。次级代谢合成基因簇比较的转录组表明，II 型 PKS（PKS5）竞争是产量低的一个因素。删除 PKS5 基因导致 AmB 的效价从 5.01 g/L 提高到 6.32 g/L，而副产物两性霉素 A（AmA）从 0.51 g/L 降低到 0.12 g/L。在一个ΔPKS5 突变体中过表达包括 PKS amphA、acc1、mme 和 mcm 的基因序列，导致摇瓶中 96 h 时 AmB 的产量从 5.01 g/L 提高到 7.06 g/L。在 144 h 的 5 L 生物反应器中，AmB 和 AmA 的产量分别为 15.6 g/L 和 0.36 g/L。检测了野生型、诱变型和基因工程型的细胞内还原性，这是首次发现与副产物 AmA 有关。骨架生物合成的增加可能消耗更多的 NADPH，并减少 AmphC ER5 结构域的还原。这项研究可以在工业生产中应用于其他聚酮类化合物。
