Hunan Provincial Key Laboratory for Microbial Molecular Biology, State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha 410083, China.
ACS Synth Biol. 2021 Oct 15;10(10):2740-2752. doi: 10.1021/acssynbio.1c00344. Epub 2021 Oct 3.
Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes. can produce butenyl-spinosyn during aerobic fermentation, and its genome contains 32 different gene clusters. This suggests that there is a large amount of potential competitive metabolism in , which affects the biosynthesis of butenyl-spinosyn. By analyzing the genome of , six polyketide gene clusters were identified. From those, the complete deletion of clu13, a flaviolin-like gene cluster, generated a high butenyl-spinosyn-producing strain. Production of this strain was 4.06-fold higher than that of the wildtype strain. Transcriptome profiling revealed that butenyl-spinosyn biosynthesis was not primarily induced by the polyketide synthase RppA-like but was related to hypothetical protein Sp1764. However, the repression of was not enough to explain the enormous enhancement of butenyl-spinosyn yields in -Δclu13. After the comparative proteomic analysis of -Δclu13 and , two proteins, biotin carboxyl carrier protein (BccA) and response regulator (Reg), were investigated, whose overexpression led to great advantages of butenyl-spinosyn biosynthesis. In this way, we successfully discovered three key genes that obviously optimize the biosynthesis of butenyl-spinosyn. Gene cluster simplification performed in conjunction with multiomics analysis is of great practical significance for screening dominant chassis strains and optimizing secondary metabolism. This work provided an idea about screening key factors and efficient construction of production strains.
减少和优化微生物基因组是构建合成生物学底盘细胞和克服天然产物发现和生产障碍的重要策略。然而，由于放线菌基因组的复杂性，发现可删除和优化的靶基因具有很大的挑战性。在有氧发酵过程中可以产生丁烯基-spinosyn，其基因组包含 32 个不同的基因簇。这表明 中存在大量潜在的竞争代谢物，这会影响丁烯基-spinosyn 的生物合成。通过分析 的基因组，鉴定出六个聚酮基因簇。从这些基因簇中，完整删除 flaviolin 样基因簇 clu13 可产生高产丁烯基-spinosyn 的菌株。该菌株的产量比野生型菌株高 4.06 倍。转录组谱分析表明，丁烯基-spinosyn 的生物合成不是主要由聚酮合酶 RppA 样蛋白诱导的，而是与假定蛋白 Sp1764 有关。然而， 的抑制不足以解释 -Δclu13 中丁烯基-spinosyn 产量的巨大提高。在 -Δclu13 和 之间进行比较蛋白质组学分析后，研究了两种蛋白质，生物素羧基载体蛋白（BccA）和响应调节蛋白（Reg），它们的过表达导致丁烯基-spinosyn 生物合成具有巨大优势。通过这种方式，我们成功发现了三个明显优化丁烯基-spinosyn 生物合成的关键基因。与多组学分析相结合的基因簇简化对于筛选优势底盘菌株和优化次级代谢具有重要的实际意义。这项工作为筛选关键因素和高效构建生产菌株提供了思路。
