School of Marine Sciences, Ningbo University, 818 Fenghua Road, Ningbo City, Zhejiang Province, 315211, People's Republic of China.
State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People's Republic of China.
Appl Microbiol Biotechnol. 2017 May;101(10):4247-4257. doi: 10.1007/s00253-017-8165-0. Epub 2017 Feb 25.
Giant linear plasmids, which replicate independently of the chromosomes, widely exist in actinobacteria. Previous studies mostly focused on the replication and evolution of the linear plasmids or the secondary metabolite gene clusters and the resistance gene clusters therein. However, the relationships of the linear plasmids to the productivities of secondary metabolites have not been studied. In this work, we developed a method to eliminate the indigenous linear plasmid pSHJG1 in Streptomyces hygroscopicus var. jinggangensis, and validamycin A titer increased by 12.5% (from 19.16 ± 1.93 to 21.56 ± 2.25 g/L) in the high-yielding strain TL01 and 43.7% (from 4.67 ± 0.05 to 6.71 ± 0.21 g/L) in the wild-type strain 5008, whereas the cellular growth of the plasmid-cured mutant was reduced. Subsequently, the plasmid-cured mutant was complemented with three structure genes involved in cellular growth in pSHJG1 under the control of a strong PvalA promoter. Among them, the complementation of genes pSHJG1.069 and pSHJG1.072, encoding a putative hydrolase and putative P-loop ATPase, respectively, resulted in the restoration of cellular growth and validamycin A titer. Furthermore, the elimination of indigenous linear plasmid pHZ228 in the candicidin producer Streptomyces sp. FR008 also led to enhanced candicidin production and reduced cellular growth. Because of the wide distribution of indigenous linear plasmids in actinobacteria, the engineering strategy described here could be implemented in a variety of strains for the overproduction of various natural products.
大型线性质粒独立于染色体复制，广泛存在于放线菌中。以前的研究主要集中在线性质粒的复制和进化，以及其中的次级代谢产物基因簇和抗性基因簇。然而，线性质粒与次级代谢产物生产力的关系尚未得到研究。在这项工作中，我们开发了一种消除吸水链霉菌井冈变种中本土线性质粒 pSHJG1 的方法，在高产菌株 TL01 中，有效霉素 A 的产量增加了 12.5%（从 19.16±1.93 增至 21.56±2.25 g/L），在野生型菌株 5008 中增加了 43.7%（从 4.67±0.05 增至 6.71±0.21 g/L），而质粒消除突变体的细胞生长减少。随后，在 pSHJG1 中，通过强 PvalA 启动子控制，将与细胞生长有关的三个结构基因插入到质粒消除突变体中进行互补。其中，分别编码假定水解酶和假定 P 环 ATP 酶的基因 pSHJG1.069 和 pSHJG1.072 的互补，导致细胞生长和有效霉素 A 产量得到恢复。此外，在棘白菌素产生菌链霉菌 sp. FR008 中消除本土线性质粒 pHZ228 也导致棘白菌素产量增加和细胞生长减少。由于本土线性质粒在放线菌中的广泛分布，这里描述的工程策略可以在各种菌株中实施，以实现各种天然产物的过量生产。
