Zhang Kuipu, Mohsin Ali, Dai Yichen, Chen Zhongbing, Zhuang Yingping, Chu Ju, Guo Meijin
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Zhejiang Biok Biology Co., Ltd., Zhongguan Industrial Park, Zhejiang, China.
Front Bioeng Biotechnol. 2019 Sep 3;7:212. doi: 10.3389/fbioe.2019.00212. eCollection 2019.
Salinomycin, an important polyketide, has been widely utilized in agriculture to inhibit growth of pathogenic bacteria. In addition, salinomycin has great potential in treatment of cancer cells. Due to inherited characteristics and beneficial potential, its demand is also inclining. Therefore, there is an urgent need to increase the current high demand of salinomycin. In order to obtain a high-yield mutant strain of salinomycin, the present work has developed an efficient breeding process of by using atmospheric and room temperature plasma (ARTP) combined with ribosome engineering. In this study, we investigate the presented method as it has the advantage of significantly shortening mutant screening duration by using an agar block diffusion method, as compared to other traditional strain breeding methods. As a result, the obtained mutant TetChl with tetracycline and chloramphenicol resistance provided a salinomycin yield of 34,712 mg/L in shake flask culture, which was over 2.0-fold the parental strain S12. In addition, comparative transcriptome analysis of low and high yield mutants, and a parental strain revealed the mechanistic insight of biosynthesis pathways, in which metabolic pathways including butanoate metabolism, starch and sucrose metabolism and glyoxylate metabolism were closely associated with salinomycin biosynthesis. Moreover, we also confirmed that enhanced flux of glyoxylate metabolism via overexpression gene of isocitrate lyase () promoted salinomycin biosynthesis. Based on these results, it has been successfully verified that the overexpression of crotonyl-CoA reductase gene () and transcriptional regulator genes ( 3 and 15), located in salinomycin synthesis gene cluster, is possibly responsible for the increase in salinomycin production in a typical strain DSM41398. Conclusively, a tentative regulatory model of ribosome engineering combined with ARTP in is proposed to explore the roles of transcriptional regulators and stringent responses in the biosynthesis regulation of salinomycin.
盐霉素是一种重要的聚酮化合物，已在农业中广泛用于抑制病原菌的生长。此外，盐霉素在癌细胞治疗方面具有巨大潜力。由于其遗传特性和潜在益处，对它的需求也在增加。因此，迫切需要满足当前对盐霉素的高需求。为了获得盐霉素高产突变菌株，本研究开发了一种利用常压室温等离子体（ARTP）结合核糖体工程的高效育种方法。在本研究中，我们对所提出的方法进行了研究，因为与其他传统菌株育种方法相比，该方法具有使用琼脂块扩散法显著缩短突变体筛选时间的优点。结果，获得的具有四环素和氯霉素抗性的突变体TetChl在摇瓶培养中盐霉素产量为34,712 mg/L，是亲本菌株S12的2.0倍以上。此外，对高产和低产突变体以及亲本菌株进行的比较转录组分析揭示了生物合成途径的机制，其中包括丁酸代谢、淀粉和蔗糖代谢以及乙醛酸代谢在内的代谢途径与盐霉素生物合成密切相关。此外，我们还证实，通过过表达异柠檬酸裂解酶（）增强乙醛酸代谢通量可促进盐霉素生物合成。基于这些结果，已成功验证位于盐霉素合成基因簇中的巴豆酰辅酶A还原酶基因（）和转录调节基因（3和15）的过表达可能是典型菌株DSM41398中盐霉素产量增加的原因。最后，提出了一种ARTP结合核糖体工程在中的初步调控模型，以探索转录调节因子和严格反应在盐霉素生物合成调控中的作用。
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