Department of Biological Engineering, Inha University, Incheon, 22212, Korea.
J Ind Microbiol Biotechnol. 2019 May;46(5):649-655. doi: 10.1007/s10295-019-02149-7. Epub 2019 Feb 23.
Pseudonocardia autotrophica was previously identified to produce a toxicity-reduced and solubility-improved disaccharide-containing anti-fungal compound belonging to the tetraene-family, Nystatin-like Pseudonocardia Polyene A1 (NPP A1). Subsequently NPP B1, a novel derivative harboring a heptaene core structure, was produced by a pathway-engineered Pseudonocardia strain through inactivation of the specific enoly reductase gene domain in the NPP biosynthetic gene cluster. Although in vitro and in vivo efficacy and toxicity studies indicate that NPP B1 is a promising lead antifungal compound, further improvement is required to increase the extremely low production yield in the pathway-engineered strain. To overcome this challenge, we performed the N-methyl-N'-nitro-N-nitrosoguanidine (NTG) iterative random mutagenesis, followed by zone-of-inhibition agar plug assay. After three rounds of the mutagenesis-and-screening protocol, the production yield of NPP B1 increased to 6.25 mg/L, which is more than an eightfold increase compared to the parental strain. The qRT-PCR analysis revealed that transcripts of the NPP B1 biosynthetic genes were increased in the mutant strain. Interestingly, an endogenous 125-kb plasmid was found to be eliminated through this mutagenesis. To further improve the NPP B1 production yield, the 32-kb NPP-specific regulatory gene cluster was cloned and overexpressed in the mutant strain. The chromosomal integration of the extra copy of the six NPP-specific regulatory genes led to an additional increase of NPP B1 yield to 31.6 mg/L, which is the highest production level of NPP B1 ever achieved by P. autotrophica strains. These results suggest that a synergistic combination of both the traditional and genetic strain improvement approaches is a very efficient strategy to stimulate the production of an extremely low-level metabolite (such as NPP B1) in a pathway-engineered rare actinomycetes strain.
先前鉴定出假诺卡氏菌（Pseudonocardia autotrophica）能够产生一种毒性降低、溶解度提高的含二糖抗真菌化合物，属于四烯家族，即类似制霉菌素的假诺卡氏多烯 A1（NPP A1）。随后，通过对 NPP 生物合成基因簇中特定烯醇还原酶基因结构域的失活，利用经过途径工程改造的假诺卡氏菌菌株产生了一种新型衍生物 NPP B1，该衍生物含有七烯核心结构。尽管体外和体内功效和毒性研究表明 NPP B1 是一种很有前途的先导抗真菌化合物，但需要进一步改进以提高途径工程改造菌株中极低的生产产量。为了克服这一挑战，我们进行了 N-甲基-N'-硝基-N-亚硝基胍（NTG）迭代随机诱变，然后进行抑菌琼脂塞法筛选。经过三轮诱变和筛选方案，NPP B1 的产量增加到 6.25mg/L，比亲本菌株增加了 8 倍以上。qRT-PCR 分析显示，突变株中 NPP B1 生物合成基因的转录物增加。有趣的是，通过这种诱变消除了一个内源性 125-kb 质粒。为了进一步提高 NPP B1 的产量，克隆并在突变株中过表达了 32-kb 的 NPP 特异性调控基因簇。染色体整合这六个 NPP 特异性调控基因的额外拷贝导致 NPP B1 产量额外增加到 31.6mg/L，这是假诺卡氏菌菌株生产 NPP B1 的最高水平。这些结果表明，传统和遗传菌株改进方法的协同组合是一种非常有效的策略，可以刺激途径工程化稀有放线菌菌株中极低水平代谢物（如 NPP B1）的产生。
