Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.

中文翻译：

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C10-脱氧庚烷NPP B2经由自发假性心动过速调节基因的过度表达刺激生物合成。

多烯大环内酯类，如制霉菌素A1，两性霉素B和NPP A1，属于一大类有价值的抗真菌聚酮化合物，通常由土壤放线菌产生。以前，NPP B1是一种具有庚烯核心结构的新型NPP A1衍生物，它是通过在拟南芥自养假单胞菌NPP A1聚酮合酶的模块5的假定的NADPH结合基团的烯丙基还原酶结构域的NADPH结合基序中引入两个氨基酸取代而产生的。该衍生物显示出优于NPP A1的抗真菌活性。在这项研究中，开发了另一种称为NPP B2的新型衍生物，该衍生物由于P450羟化酶NppL的位点特异性基因破坏而在C10位置缺少羟基。为了刺激NPP B2生物合成途径基因的极低表达，32-kb NPP特异的调控基因簇通过位点特异性染色体整合而过表达。六个NPP特异调控基因的额外拷贝导致NPP B2产量从0.19 mg / L显着增加，这是自养毕赤酵母菌株实现NPP B2产量的最高水平。随后的体外抗真菌活性和毒性研究表明NPP B2表现出相似的抗真菌活性，但溶血毒性明显低于NPP B1。这些结果表明，NPP生物合成途径的重构及其途径特异性调控基因的过表达是一种刺激极低水平代谢产物（如途径工程的稀有放线菌菌株中的NPP B2）产生的有效方法。六个NPP特异调控基因的额外拷贝导致NPP B2产量从0.19 mg / L显着增加，这是自养毕赤酵母菌株实现NPP B2产量的最高水平。随后的体外抗真菌活性和毒性研究表明NPP B2表现出相似的抗真菌活性，但溶血毒性明显低于NPP B1。这些结果表明，NPP生物合成途径的重构及其途径特异性调控基因的过表达是一种刺激极低水平代谢产物（如途径工程的稀有放线菌菌株中的NPP B2）产生的有效方法。六个NPP特异调控基因的额外拷贝导致NPP B2产量从0.19 mg / L显着增加，这是自养毕赤酵母菌株实现NPP B2产量的最高水平。随后的体外抗真菌活性和毒性研究表明NPP B2表现出相似的抗真菌活性，但溶血毒性明显低于NPP B1。这些结果表明，NPP生物合成途径的重构及其途径特异性调控基因的过表达是一种刺激极低水平代谢产物（如途径工程的稀有放线菌菌株中的NPP B2）产生的有效方法。这是自养毕赤酵母菌株有史以来最高的NPP B2产量。随后的体外抗真菌活性和毒性研究表明NPP B2表现出相似的抗真菌活性，但溶血毒性明显低于NPP B1。这些结果表明，NPP生物合成途径的重构及其途径特异性调控基因的过表达是一种刺激极低水平代谢产物（如途径工程的稀有放线菌菌株中的NPP B2）产生的有效方法。这是自养毕赤酵母菌株有史以来最高的NPP B2产量。随后的体外抗真菌活性和毒性研究表明NPP B2表现出相似的抗真菌活性，但溶血毒性明显低于NPP B1。这些结果表明，NPP生物合成途径的重构及其途径特异性调控基因的过表达是一种刺激极低水平代谢产物（如途径工程的稀有放线菌菌株中的NPP B2）产生的有效方法。