基因组精简和启动子工程相结合的策略提升福莱菌肽产量

doi:10.16409/j.cnki.2095-039x.2026.02.003

摘要/Abstract

摘要： 布达佩斯致病杆菌X.budapestensis XBD8产生的抗菌天然产物福莱菌肽（Fabclavines），具有广谱抑菌活性，在农业病害防治中具有广阔的应用前景。然而，福莱菌肽产量低，应用成本高，限制了其产业化开发。本研究旨在通过基因组精简和启动子工程相结合的策略，构建高产福莱菌肽的工程菌株。通过生物信息学方法预测出菌株XBD8基因组中除福莱菌肽以外的11个次生代谢产物生物合成基因簇，对其核心生物合成基因进行多轮叠加敲除，成功构建了基因组精简的底盘菌株FRT12，并在此过程中发现距离福莱菌肽生物合成基因簇较远的一个非核糖体肽合成酶基因gene2204也参与福莱菌肽合成。利用启动子工程策略替换基因fclC和gene2204的启动子，成功构建了双启动子替换菌株MPW9，福莱菌肽产量从73.4 mg/L提高到了121.3 mg/L。在底盘菌株FRT12中构建双启动子替换菌株MPW11，福莱菌肽摇瓶发酵产量达到了142.6 mg/L，为其产业化开发奠定了基础。

关键词: 福莱菌肽, 布达佩斯致病杆菌, 基因组精简, 启动子工程

Abstract: Fabclavines produced by Xenorhabdus budapestensis strain XBD8 exhibit broad-spectrum antimicrobial activity, showing significant potential for application in the control of plant disease. However, their low yield and high application costs have hindered industrial development. This study aimed to construct high-yield Fabclavine-producing strains through a combined strategy of genome reduction and promoter engineering. Genome mining of Xenorhabdus budapestensis strain XBD8 using the bioinformatics tool antiSMASH identified 11 secondary metabolite biosynthetic gene clusters(BGCs) in addition to the Fabclavine BGC. Iterative knockout of core biosynthetic genes from these BGCs generated a streamlined chassis strain, designated FRT12. Notably, gene2204, involved in the biosynthesis of Fabclavines Fcl-7 and Fcl-8, was identified during this genome reduction process. Promoter engineering strategy was applied to replace the promoters of genes fclC and gene2204, leading to the successful construction of the dual-promoter replacement strain MPW9. The yield of Fabclavines was increased from 73.4 mg/L to 121.3 mg/L. By swapping these two promoters in the streamlined chassis strain FRT12, the constructed strain MPW11 demonstrated further improved Fabclavines production,reaching 142.6 mg/L in shake-flask fermentation. These findings establish a foundation for industrial-scale production of Fabclavines.

Key words: fabclavines, Xenorhabdus budapestensis, genome streamlining, promoter engineering

中图分类号:

S476

王玉乐, 尹长琰, 贾丰莲, 任杰, 李广悦. 基因组精简和启动子工程相结合的策略提升福莱菌肽产量[J]. 中国生物防治学报, 2026, 42(3): 600-610.

WANG Yule, YIN Changyan, JIA Fenglian, REN Jie, LI Guangyue. Combining Genome Streamlining and Promoter Engineering Strategies to Construct High-Yield Strain for Fabclavines[J]. Chinese Journal of Biological Control, 2026, 42(3): 600-610.

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参考文献

[1] Savary S, Willocquet L, Pethybridge S J, et al. The global burden of pathogens and pests on major food crops[J]. Nature Ecology&Evolution, 2019, 3(3):430-439.
[2] Carvajal-Yepes M, Cardwell K, Nelson A, et al. A global surveillance system for crop diseases[J]. Science, 2019, 364(6447):1237-1239.
[3] 王晓杰,甘鹏飞,汤春蕾,等.植物抗病性与病害绿色防控:主要科学问题及未来研究方向[J].中国科学基金, 2020, 34(4):381-392.
[4] 何亚文,李广悦,谭红,等.我国生防微生物代谢产物研发应用进展与展望[J].中国生物防治学报, 2022, 38(3):537-548.
[5] Fuchs S W, Sachs C C, Kegler C, et al. Neutral loss fragmentation pattern based screening for arginine-rich natural products in Xenorhabdus and Photorhabdus[J]. Analytical Chemistry, 2012, 84(16):6948-6955.
[6] Fodor A, Gualtieri M, Zeller M, et al. Type strains of entomopathogenic nematode-symbiotic bacterium species, Xenorhabdus szentirmaii(EMC)and X.budapestensis(EMA), are exceptional sources of non-ribosomal templated, large-target-spectral, thermotolerant-antimicrobial peptides(by both), and iodinin(by EMC)[J]. Pathogens, 2022, 11(3):29.
[7] Li B, Yuan B, Duan J, et al. Identification of Fcl-29 as an effective antifungal natural product against Fusarium graminearum and combinatorial engineering strategy for improving its yield[J]. Journal of Agricultural and Food Chemistry, 2023, 71(14):5554-5564.
[8] Abebew D, Sayedain F S, Bode E, et al. Uncovering nematicidal natural products from Xenorhabdus bacteria[J]. Journal of Agricultural and Food Chemistry, 2022, 70(2):498-506.
[9] Böszörményi E, Ersek T, Fodor A, et al. Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae[J]. Journal of Applied Microbiology, 2009, 107(3):746-759.
[10] Shi Y M, Bode H B. Chemical language and warfare of bacterial natural products in bacteria-nematode-insect interactions[J]. Natural Product Reports,2018, 35(4):309-335.
[11] Sajnaga E, Kazimierczak W, KaraśM A, et al. Exploring Xenorhabdus and Photorhabdus nematode symbionts in search of novel therapeutics[J].Molecules, 2024, 29(21):5151.
[12] Fuchs S W, Grundmann F, Kurz M, et al. Fabclavines:bioactive peptide-polyketide-polyamino hybrids from Xenorhabdus[J]. ChemBioChem, 2014,15(4):512-516.
[13] Wenski S L, Kolbert D, Grammbitter G L C, et al. Fabclavine biosynthesis in X. szentirmaii:shortened derivatives and characterization of the thioester reductase FclG and the condensation domain-like protein FclL[J]. Journal of Industrial Microbiology&Biotechnology, 2019, 46(3-4):565-572.
[14] Wenski SL, Cimen H, Berghaus N, et al. Fabclavine diversity in Xenorhabdus bacteria[J]. Beilstein Journal of Organic Chemistry, 2020, 16:956-965.
[15] Yuan B, Li B, Shen H, et al. Identification of fabclavine derivatives, Fcl-7 and Fcl-8, from Xenorhabdus budapestensis as major antifungal natural products against Rhizoctonia solani[J]. Journal of Applied Microbiology, 2023, 134(9):lxad190.
[16] Duan J, Yuan B, Jia F, et al. Development of an efficient and seamless genetic manipulation method for Xenorhabdus and its application for enhancing the production of fabclavines[J]. Journal of Agricultural and Food Chemistry, 2024, 72(1):274-283.
[17] Sengupta A, Bandyopadhyay A, Sarkar D, et al. Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973[J]. mBio, 2024, 15(3):e0353023.
[18] Leprince A, van Passel MW, dos Santos VA. Streamlining genomes:toward the generation of simplified and stabilized microbial systems[J]. Current Opinion in Biotechnology, 2012, 23(5):651-658.
[19] Chen X R, Cui Y Z, Li B Z, et al. Genome engineering on size reduction and complexity simplification:A review[J]. Journal of Advanced Research, 2023,60:159-171.
[20] Ma S, Su T, Lu X, et al. Bacterial genome reduction for optimal chassis of synthetic biology:A review[J]. Critical Reviews in Biotechnology, 2024, 44(4):660-673.
[21] Shen X, Wang Z, Huang X, et al. Developing genome-reduced Pseudomonas chlororaphis strains for the production of secondary metabolites[J]. BMC Genomics, 2017, 18(1):715.
[22] Liu J, Zhou H, Yang Z, et al. Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria[J]. Nature Communications, 2021, 12(1):4347.
[23] Mizoguchi H, Mori H, Fujio T. Escherichia coli minimum genome factory[J]. Biotechnology and Applied Biochemistry, 2007, 46:157-167.
[24] Myronovskyi M, Luzhetskyy A Native and engineered promoters in natural product discovery[J]. Natural Product Reports, 2016, 33(8):1006-1019.
[25] Siegl T, Tokovenko B, Myronovskyi M, et al. Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes[J]. Metabolic Engineering, 2013, 19:98-106.
[26] Chutrakul C, Panchanawaporn S, Jeennor S, et al. Promoter exchange of the cryptic nonribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae[J]. Journal of Microbiology, 2022, 60(1):47-56.
[27] Li Z, Huang P, Wang M, et al. Stepwise increase of thaxtomins production in Streptomyces albidoflavus J1074 through combinatorial metabolic engineering[J]. Metabolic Engineering, 2021, 68:187-198.
[28] Horbal L, Marques F, Nadmid S, et al. Secondary metabolites overproduction through transcriptional gene cluster refactoring[J]. Metabolic Engineering,2018, 49:299-315.
[29] Blin K, Shaw S, Augustijn H E, et al. antiSMASH 7.0:new and improved predictions for detection, regulation, chemical structures and visualisation[J].Nucleic Acids Research, 2023, 51(W1):W46-W50.
[30] Li S, Wang J, Xiang W, et al. An autoregulated fine-tuning strategy for titer improvement of secondary metabolites using native promoters in Streptomyces[J]. ACS Synthetic Biology, 2018, 7(2):522-530.
[31] Donmez Ozkan H, Cimen H, Ulug D et al. Nematode-associated bacteria:production of antimicrobial agent as a presumptive nominee for curing endodontic infections caused by Enterococcus faecalis[J]. Frontiers in Microbiology, 2019, 10:2672.
[32] Qin Y, Jia F, Li X, et al. Improving the yield of xenocoumacin 1 by P BAD promoter replacement in Xenorhabdus nematophila CB6[J]. Agriculture, 2021,11(12):1251.
[33] Bode E, Brachmann A O, Kegler C, et al. Simple"on-demand"production of bioactive natural products[J]. ChemBioChem, 2015, 16(7):1115-1119.
[34] 李杨,陈涛,赵学明.微生物基因组简化的研究进展[J].生命科学, 2011, 23(9):838-843.
[35] Staunton J, Weissman K J. Polyketide biosynthesis:a millennium review[J]. Natural Product Reports, 2001, 18(4):380-416.
[36] 王晓宇,蔡朝辉,乔长晟,等.基于转录组学分析刺糖多孢菌多杀菌素合成代谢途径关键酶基因挖掘[J].微生物学报, 2024, 64(10):3762-3779.
[37] 张慧,许宁,曹丽茹,等.我国微生物农药的研发与应用研究进展[J].农药学学报, 2023, 25(4):769-778.
[38] Brophy J A N, Triassi A J, Adams B L, et al. Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria[J]. Nature Microbiology, 2018, 3(9):1043-1053.