Hunan Provincial Key Laboratory for Microbial Molecular Biology, State Key Laboratory of Development Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China.
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (MOE of China), National & Local Joint Engineering Laboratory for New Petro-chemical Materials and Fine Utilization of Resources, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410081, China.
Microb Biotechnol. 2021 Nov;14(6):2369-2384. doi: 10.1111/1751-7915.13694. Epub 2020 Oct 31.
Butenyl-spinosyn, a promising biopesticide produced by Saccharopolyspora pogona, exhibits stronger insecticidal activity and a broader pesticidal spectrum. However, its titre in the wild-type S. pogona strain is too low to meet the industrial production requirements. Deletion of non-target natural product biosynthetic gene clusters resident in the genome of S. pogona could reduce the consumption of synthetic precursors, thereby promoting the biosynthesis of butenyl-spinosyn. However, it has always been a challenge for scientists to genetically engineer S. pogona. In this study, the Latour gene knockout system (linear DNA fragment recombineering system) was established in S. pogona. Using the Latour system, a hybrid NRPS-T1PKS cluster (˜20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. pogona. The resultant mutant S. pogona-Δura4-Δc14 exhibited an extended logarithmic phase, increased biomass and a lower glucose consumption rate. Importantly, the production of butenyl-spinosyn in S. pogona-Δura4-Δc14 was increased by 4.72-fold compared with that in the wild-type strain. qRT-PCR analysis revealed that phthoxazolin biosynthetic gene cluster deletion could promote the expression of the butenyl-spinosyn biosynthetic gene cluster. Furthermore, a TetR family transcriptional regulatory gene that could regulate the butenyl-spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster. Because dozens of natural product biosynthetic gene clusters exist in the genome of S. pogona, the strategy reported here will be used to further promote the production of butenyl-spinosyn by deleting other secondary metabolite synthetic gene clusters.
丁烯基多杀菌素是一种由多孢菌产生的有前景的生物农药，具有更强的杀虫活性和更广泛的杀虫谱。然而，其在野生型多孢菌菌株中的产量太低，无法满足工业生产的要求。删除基因组中驻留的非目标天然产物生物合成基因簇可以减少合成前体的消耗，从而促进丁烯基多杀菌素的生物合成。然而，对科学家来说，基因工程多孢菌一直是一个挑战。在本研究中，建立了多孢菌的拉图尔基因敲除系统（线性 DNA 片段重组系统）。利用拉图尔系统，有效地敲除了负责 phthoxazolin 生物合成的杂合 NRPS-T1PKS 簇（˜20 kb）。所得突变株 S. pogona-Δura4-Δc14 表现出延长的对数生长期、增加的生物量和降低的葡萄糖消耗率。重要的是，与野生型菌株相比，S. pogona-Δura4-Δc14 中丁烯基多杀菌素的产量增加了 4.72 倍。qRT-PCR 分析表明，phthoxazolin 生物合成基因簇的缺失可以促进丁烯基多杀菌素生物合成基因簇的表达。此外，从 phthoxazolin 生物合成基因簇中鉴定出一种可以调节丁烯基多杀菌素生物合成的 TetR 家族转录调节基因。因为在多孢菌的基因组中存在数十个天然产物生物合成基因簇，所以这里报道的策略将用于通过删除其他次级代谢产物合成基因簇来进一步提高丁烯基多杀菌素的产量。
