核糖体蛋白RpsL调控须糖多孢菌中丁烯基多杀菌素的合成

doi:10.3969/j.issn.1674-7968.2025.01.016

Abstract
Figure/Table
References
Related Citation (4)

Download: PDF (2997 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Butenyl-spinosyn, a secondary metabolite produced by fermentation of Saccharopolyspora pogona, is a green bio-insecticide. However, due to the low yield of fermentation and can not meet the needs of industrial production, the breeding of strains with high production performance of butenyl-spinosyn is the key to realize industrial production. In this study, ribosomal protein S12 (RpsL) and RpsL mutant gene overexpression engineering strains Q8 (rpsl)、W43 (rpsl^K88R)、W44 (rpsl^R86P)、W45 (rpsl^K88E)和W46 (rpsl^K43N) were constructed were constructed using Saccharopolyspora pogona as the base cells. The effect of RpsL on the synthesis of butenyl-spinosyn was studied by shaking flask fermentation and comparison of RpsL protein structure. The results showed that the engineered strains Q8, W43, W44, W45, W46 increased the yield of butenyl-spinosyn to 275%, 146%, 111%, 122% and reduced to 75%, respectively. The overexpression of rpsl and rpsl^K88R genes increased the transcription level of the synthetic pathway genes, and thus significantly increased the yield of butenyl-spinosyn. At the same time, homologous modeling of the two was conducted, and it was found that RpsL^K88R mutant structure had a larger deflection of the side chain compared with RpsL, which affected the binding effect to mRNA and thus reduced the ribosome translation efficiency, so its yield extraction effect was slightly lower than RpsL. This study provides a reference for protein engineering RpsL to improve the yield of butenyl-spinosyn.

Key words： Butenyl-spinosyn Ribosomal protein Saccharopolyspora pogona Gene mutation

Received: 08 January 2024

ZTFLH:	S482.3
	Q785

Corresponding Authors: * wc@ags.ac.cn

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	SU Chang
	WANG Jing-Nan
	GUO Chao
	XU Zhou-Qin
	LI Chun
	ZHANG Gen-Lin
	WANG Chao

Cite this article:

SU Chang,WANG Jing-Nan,GUO Chao, et al. Ribosomal Protein RpsL Regulates Butenyl-spinosyn Synthesis in Saccharopolyspora pogona[J]. 农业生物技术学报, 2025, 33(1): 179-189.

URL:

https://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2025.01.016 OR https://journal05.magtech.org.cn/Jwk_ny/EN/Y2025/V33/I1/179

[1] 曹明坤. 2022. 我国杀虫剂产业发展应用现状与方略[J].安徽化工, 48(03): 1-10.
(Cao M K.2022. Development and application status and strategy of pesticide industry in China[J]. Anhui Chemical Industry, 48(03): 1-10.)
[2] 陈骁昱, 吴忧, 雷佳乐, 等. 2022. 核糖体工程技术选育优质菌株的研究进展[J]. 发酵科技通讯, 51(03): 169-175.
(Chen X Y, Wu Y, Lei J L, et al.2022. Advance in screening robust mutant strains using ribosome engineering techniques[J]. Bulletin of Fermentation Science and Technology, 51(03): 169-175.)
[3] 陈园. 2013. 多杀菌素高产菌株的诱变选育及快速筛选方法的研究[D]. 硕士学位论文, 华南理工大学, 导师: 熊犍,张晓琳, pp. 19-31.
(Chen Y.2013. Mutation breeding of spinosad high-producing strain and study of rapid screening method[D]. Thesis for M.S., South China University of Technology, Supervisor: Xiong J, Zhang X L,pp. 19-31.)
[4] 崔佳佳, 张雪洪. 2021. 微生物源农用抗生素的研发与高产策略[J]. 生物工程学报, 37(03): 1032-1041.
(Cui J J,Zhang X H.2021. Development and high yield strategies of microbial-derived antibiotics inagriculture[J]. Journal of Bioengineering, 37(03): 1032-1041.)
[5] 郭超, 王超, 郭伟群. 2019. 一种多杀菌素糖苷配基半抗原及其制备方法和应用. 中国, CN112390776A[P].
(Guo C,Wang C,Guo W Q.2019. The invention relates to a spinosyn aglycone hapten, a preparation method and application thereof. China, CN112390776A[P])
[6] 李光月, 胡文锋, 李雪玲. 2023. 复合诱变改善枯草芽胞杆菌表面活性素的抗菌能力[J]. 农业生物技术学报, 31(07): 1488-1500.
(Li G Y, Hu W F, Li X L.2023. Compound mutagenesis to improve the antibacterial ability of surfactin from Bacillus subtilis[J]. Journal of Agricultural Biotechnology, 31(07): 1488-1500.)
[7] 李晓晨. 2023. 多杀菌素异源生物合成体系优化研究[D].博士学位论文, 山东大学, 导师: 张友明, 王海龙, pp. 33-34.
(Li X C.2023. Optimization of spinosad heterologous biosynthesis system[D]. Thesis for Ph. D., ShanDong University, Supervisor: Zhang Y P, Wang H L, pp. 33-34.)
[8] 罗林根, 杨燕, 魏慧, 等. 2016. 须糖多孢菌Saccharopolyspora pogona的核糖体工程改造对丁烯基多杀菌素合成的影响[J]. 生物工程学报, 1(02): 259-263.
(Luo L G, Yang Y, Wui H, et al.2016. Effect of ribosome engineering on butenyl-spinosyns synthesis of Saccharopolyspora pogona[J]. Chinese Journal of Biotechnology, 1(02): 259-263.)
[9] 马正, 施越, 章金垚, 等. 2022. 链霉菌沉默基因簇激活在天然产物生物合成中的研究进展[J]. 中国计量大学学报, 33(02): 272-280.
(Ma Z, Shi Y, Zhang J Y, et al.2022. Advances in the activation of cryptic biosynthetic gene clusters of Streptomyces in biosynthesis of natural products[J]. Journal of China Jiliang University, 33(02): 272-280.)
[10] 孙子龙, 扶教龙, 王月, 等. 2023. ε-聚赖氨酸生产菌育种研究进展[J/OL]. 食品科学, 44(23): 1-14.
(Sun Z L, Fu J L, Wang Y, et al.2023. Advances in strain improvement for the production of ε-Poly-L-lysine[J/OL]. Food Science, 44(23): 1-14.)
[11] 王靖楠, 庞建, 秦磊, 等. 2022. 丁烯基多杀菌素高产菌株的选育和改造策略[J]. 化工学报, 73(02): 566-576.
(Wang J N, Pang J, Qin L, et al.2022. Breeding and modification strategies of butenyl-spinosyn high-yield strains[J]. Journal of Chemical Engineering, 73(02): 566-576.)
[12] 王欣荣, 张爱雪, 唐智超, 等. 2020. 产多杀菌素刺糖多孢菌的诱变选育[J]. 中国抗生素杂志, 45(09): 873-877.
(Wang X R, Zhang A X, Tang Z C, et al.2020. Mutagenesis and breeding of spinosad-producing strains of Saccharopolyspora spinosa[J]. Chinese Journal of Antibiotics, 45(09): 873-877.)
[13] 邬洋, 徐妙, 罗林根, 等. 2015. 丁烯基多杀菌素高产菌株的巴龙霉素抗性筛选[J]. 中国生物防治学报, 31(1): 106-114.
(Wu Y, Xu M, Luo L G, et al.2015. Screening of butenyl-spinosyn high-yield strains by paromomycin resistance[J]. Chinese Journal of Biological Control, 31(1): 106-114.)
[14] 谢运昌, 姚仕杰, 李炜, 等. 2022. 放线菌核糖体工程的发展与应用[J]. 生物工程学报, 38(02): 546-564.
(Xie Y C,Yao S J, Li W, et al.2022. Development and application of ribosomal engineering in actinomycetes[J]. Chinese Journal of Biotechnology, 38(2): 546-564.)
[15] 徐周钦, 郭超, 李金萍, 等.2023. 60Co-NTG复合诱变选育丁烯基多杀菌素高产菌株及其杀虫活性[J/OL]. 中国生物防治学报, 10.16409/j.cnki.2095-039x.2023.01.027.
(Xu Z Q, Guo C, Li J P, et al.2023. Breeding of butenyl-spinosyns high yielding strain by 60Co-NTG compound mutation and lts insecticidal activity[J/OL]. Chinese Journal of Biological Control, 10.16409/j.cnki.2095-039x.2023.01.027.)
[16] 薛正莲, 王珊, 孙俊峰, 等. 2021. 链霉菌形态分化与次级代谢产物合成的研究进展[J]. 微生物学报, 61(12): 3870-3886.
(Xue Z L, Wang S, Sun J F, et al.2021. Research progress on morphological differentiation and secondary metabolite biosynthesis of Streptomyces[J]. Acta Microbiologica Sinica, 61(12): 3870-3886.)
[17] 张逍遥, 郭超, 王靖楠, 等. 2022. 常压室温等离子体诱变选育丁烯基多杀菌素高产菌株及培养基优化[J]. 粮油食品科技, 30(01): 174-181.
(Zhang X Y, Guo C, Wang J N, et al.2022. Breeding of butenyl-spinosyns producing strain by atmospheric room temperature plasma and optimization of culture medium[J]. Science and Technology of Cereals, Oils and Foods, 30(01): 174-181.)
[18] 周淑芳. 2010. 刺糖多孢菌基因组重排育种及快速筛选模型的研究[D]. 硕士学位论文, 浙江工业大学, 导师: 裘娟萍, pp. 16-23.
(Genome shuffling breeding of Saccharopolyspora spinosa and study of rapid screening model[D]. Thesis for M.S., Zhejiang University of Technology,Supervisor: Qiu J P, pp. 16-23)
[19] Bustin S A, Vladimir B, Garson J A, et al.2009. The MIQE guidelines: Minimum information for publication of Quantitative real-time PCR experiments[J]. Clinical Chemistry, 1(4): 611.
[20] Den Hengst C D, Tran N T, Bibb M J, et al.2010. Genes essential for morphological development and antibiotic production in Streptomyces coelicolor are targets of BldD during vegetative growth[J]. Molecular Microbiology, 78(2): 361-379.
[21] Guo C, Guo W Q, Liu Y C, et al.2020. Complete genome sequence of butenyl-spinosyn-producing Saccharopolyspora strain ASAGF58[J]. Annals of Microbiology, 70(46): 1-6.
[22] Lan J Q, Zhao H W, Jin X T, et al.2019. Development of a monoclonal antibody-based immunoaffinity chromatography and a sensitive immunoassay for detection of spinosyn A in milk, fruits, and vegetables[J]. Food Control, 95: 196-205.
[23] Lopatniuk M, Myronovskyi M, Nottebrock N, et al.2019. Effect of 'ribosome engineering' on the transcription level and production of S. albus indigenous secondary metabolites[J]. Applied Microbiology and Biotechnology, 103: 7097-7110
[24] Tamehiro N, Hosaka T, Xu J, et al.2003. Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus[J]. Applied and Environmental Microbiology, 69(11): 6412-6417.
[25] Tanaka Y, Komatsu M, Okamoto S,et al.2009. Antibiotic overproduction by rpsL and rsmG mutants of various Actinomycetes[J]. Applied and Environmental Microbiology,75(14): 4919-4922.
[26] Wang Q L, Zhang D, Li Y D, et al.2014. Genome shuffling and ribosome engineering of Streptomyces actuosus for high-yield nosiheptide production[J]. Applied Biochemistry and Biotechnology, 173(6): 1553-1563.
[27] Wang L, Li S, Zhao J J, et al.2019. Efficiently activated ε-poly-L-lysine production by multiple antibitic-resistance mutations and acidic pH shock optimization in Streptomyces albulus[J]. Microbiologyopen, 8(5): e728.
[28] Wang H, Xue W, He Y M, et al.2015. Improvement of the ability to produce spinosad in Saccharopolyspora spinosa through the acquisition of drug resistance and genome shuffling[J]. Annals of Microbiology, 65(2): 771-777.
[29] Yang G J, He Y P, Jiang Y, et al.2016. A new medium for improving spinosad production by Saccharopolyspora spinosa[J]. Jundishapur Journal of Microbiology, 9(6): 1-5.
[30] Zhang Q, Ren J W, Wang W S, et al.2020. A versatile transcription-translation in one approach for activation of cryptic biosynthetic gene clusters[J]. ACS Chemical Biology, 15(9): 2551-2557.
[31] Zhu S, Duan Y, Huang Y.2019. The application of ribosome engineering to natural product discovery and yield improvement in Streptomyces[J]. Antibiotics, 8(3): 133-133.
[32] Zhuang Z, Jiang C, Zhang F, et al.2019. Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D[J]. Biotechnology and Bioengineering, 116(6): 1304-1314.