摘　要：合成生物学在高效底盘细胞构建、活性天然产物挖掘以及代谢途径优化改造等方面能全力推进目标天然产物高效生物制造。然而，在刺糖多孢菌合成生物学研究中，由于对调控多杀菌素生物合成的重要功能基因及其作用机制了解甚少，难以通过“设计-构建-测试-学习”策略构建高效细胞工厂以大幅提高多杀菌素产量。为解决该问题，本研究在通过人工诱变获得一株具有多杀菌素产量提高、生长速率增快以及胞外葡萄糖摄取能力增强的优良底盘菌株CW-12基础上，对其进行了比较蛋白质组学分析。研究结果发现，与细胞内碳代谢、脂肪酸代谢、氨基酸生物合成、三羧酸循环等相关的代谢途径的增强是诱变株CW-12多杀菌素生物合成能力提高的重要原因，并筛选到668个表达水平上调的蛋白。随后，我们选择3-羟脂酰辅酶A脱氢酶（SS_2202）和乙酰辅酶A乙酰基转移酶（SS_2203）进行了作用机制分析，明确其过表达能有效促进多杀菌素生物合成。该研究对如何挖掘调控多杀菌素生物合成的重要功能基因及其作用机制解析具有一定的指导意义，为后续通过合成生物学策略构建多杀菌素高效生物合成细胞工厂奠定了重要基础。
关键词：刺糖多孢菌；多杀菌素；合成生物学；比较蛋白质组分析；基因编辑
中图分类号：Q93                                文献标志码：ADOI：10.3969/j.issn.1007-7146.2024.05.007

Abstract: Synthetic biology can fully promote the efficient bio-manufacturing of target natural products in the aspects of efficient chassis cell construction, active natural product mining and metabolic pathway optimization. However, in the study of synthetic biology of Saccharopolyspora spinosa, due to the limited understanding of the important functional genes regulating spinosyn biosynthesis and their action mechanisms, it is difficult to construct efficient cell factories to significantly increase the spinosyn production through the “design-build-test-learn” strategy. In order to solve this problem, an excellent chassis strain CW-12 with high spinosyn production, fast growth rate and strong extracellular glucose uptake ability was obtained by artificial mutagenesis, and comparative proteomic analysis was performed. The results showed that the enhancement of metabolic pathways related to intracellular carbon metabolism, fatty acid metabolism, amino acid biosynthesis, and TCA cycle was an important reason for the improvement of spinosyn biosynthesis of mutant CW-12, and 668 up-regulated proteins were screened. Subsequently, we selected 3-hydroxyacyl-CoA dehydrogenase (SS_2202) and acetyl-CoA acetyltransferase (SS_2203) to analyze their action mechanisms, confirming that their overexpression can effectively promote the spinosyn biosynthesis. This study has certain guiding significance on how to explore the important functional genes regulating spinosyn biosynthesis and the analysis of their action mechanisms, and lays an important foundation for the subsequent construction of spinosyn efficient biosynthesis cell factories through synthetic biology strategies.
Key word: Saccharopolyspora spinosa; spinosyn; synthetic biology; comparative proteomic analysis; gene editing
（Acta Laser Biology Sinica, 2024, 33(5): 450-460）