食品与发酵工业 >

2025 , Vol. 51 >Issue 16: 43 - 50

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.040863

研究报告

鼠李糖甲基化相关基因的组合表达调控丁烯基多杀菌素的合成

  • 苏畅 ,
  • 郭超 ,
  • 李新颖 ,
  • 陈霞 ,
  • 庞建 ,
  • 王超 ,
  • 张根林
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  • 1(石河子大学 化学化工学院,化工绿色过程省部共建国家重点实验室培育基地,新疆 石河子,832003)
    2(国家粮食和物资储备局科学研究院,北京,100037)
    3(北京理工大学 化学与化工学院,生物化工研究所,医药分子科学与制剂工程工信部重点实验室,北京,100081)
    4(北京农学院,农业农村部华北都市农业重点实验室,北京,102206)
第一作者:硕士研究生(张根林教授和王超副研究员为共同通信作者,E-mail:zhgl_food@sina.com;wc@ags.ac.cn)

收稿日期: 2024-08-27

  修回日期: 2024-11-26

  网络出版日期: 2025-08-29

基金资助

十四五重点研发计划项目(2023YFA0914700);中央公益性科研院所专项课题(JY2311);工业生物催化教育部重点实验室(清华大学)开放基金资助项目(2023003);国家自然科学青年基金项目(22108153)

收起

The combined expression of rhamnose methylation related genes regulates synthesis of butenyl-spinosyn

  • SU Chang ,
  • GUO Chao ,
  • LI Xinying ,
  • CHEN Xia ,
  • PANG Jian ,
  • WANG Chao ,
  • ZHANG Genlin
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  • 1(School of Chemistry and Chemical Engineering, Shihezi University, State Key Laboratory Incubation Base for Green Processing of Chemical Engineering, Shihezi 832003, China)
    2(Academy of National Food and Strategic Reserves Administration, Beijing 100037, China)
    3(Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China)
    4(Key Laboratory for Northern Urban Agriculture of Ministry of Agriculture and Rural Affairs, Beijing University of Agriculture, Beijing 102206, China)

Received date: 2024-08-27

  Revised date: 2024-11-26

  Online published: 2025-08-29

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摘要

绿色生物杀虫剂丁烯基多杀菌素具有广谱高效的杀虫活性,在农作物生产、粮食贮藏等方面有着极大的应用前景,高生产性能菌株的选育是实现工业化生产的关键所在。在丁烯基多杀菌素生物合成过程中S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)作为甲基供体,在3种不同的O-甲基转移酶作用下对鼠李糖基团进行甲基化修饰,O-甲基转移酶及SAM供给充足,对丁烯基多杀菌素的合成极为重要。该研究通过外源添加SAM发酵实验、内源SAM合酶挖掘与活性表征、metK和O-甲基转移酶基因协同表达来探究鼠李糖甲基化及相关基因对丁烯基多杀菌素合成的影响。结果表明,metK和O-甲基转移酶基因的组合过表达工程菌株发酵验证获得3株高产工程菌株I-1、I-2和H-1,产量分别提高3.17、2.21、1.43倍。菌株I-1中busI和metK1的过表达调控促进了菌株的生长代谢和底物的消耗,从而更高效合成丁烯基多杀菌素。该研究阐明了鼠李糖甲基化相关基因对丁烯基多杀菌素合成的影响,为基因工程改造须糖多孢菌促进目标产物合成提供了参考。

关键词： 须糖多孢菌; 丁烯基多杀菌素; 粮食贮藏; 虫害防治; O-甲基转移酶; S-腺苷甲硫氨酸

本文引用格式

苏畅 , 郭超 , 李新颖 , 陈霞 , 庞建 , 王超 , 张根林 . 鼠李糖甲基化相关基因的组合表达调控丁烯基多杀菌素的合成[J]. 食品与发酵工业, 2025 , 51(16) : 43 -50 . DOI: 10.13995/j.cnki.11-1802/ts.040863

Abstract

The green biological insecticide butenyl-spinosyn has a broad spectrum and high-efficiency insecticidal activity and has great market prospects in crop production and grain storage, the breeding of strains with high production performance of butenyl-spinosyn is the key to realizing industrial production.In the biosynthesis of butenyl-spinosyn, S-adenosylmethionine (SAM) serves as a methyl donor and methylation rhamnose under the action of three different O-methyltransferases.The adequacy of O-methyltransferase and SAM supply is crucial for butenyl-spinosyn synthesis.Study investigated the impact of rhamnose methylation and related genes on butenyl-spinosyn synthesis by exogenously adding SAM, identifying and characterizing endogenous SAM synthase, metK, and O-methyltransferase gene cooperative expression.Results showed that the combined overexpression of metK and O-methyltransferase genes obtained three high-yielding engineering strains I-1, I-2, and H-1, and the yield was increased by 3.17, 2.21, and 1.43 times, respectively.In strain I-1, the overexpression of busI and metK1 regulated and promoted the strain’s growth metabolism and substrate consumption, thereby synthesizing butenyl-spinosyn more efficiently.The study provides a reference for the genetic engineering modification of Saccharopolyspora pogona to promote the synthesis of target products.

Key words： Saccharopolyspora pogona; butenyl-spinosyn; grain storage; pest control; O-methyltransferase; S-adenosylmethionine

参考文献

[1] 寿佳丽, 裘娟萍.新型生物农药-丁烯基多杀菌素[J].农药, 2011, 50(4):239-243;272.
SHOU J L, QIU J P.A new type of biological pesticide:Butenyl-spinosyns[J].Agrochemicals, 2011, 50(4):239-243;272.
[2] 唐建立. 须糖多孢菌丁烯基多杀菌素生物合成调控的几个关键基因的功能研究[D].长沙:湖南师范大学, 2021.
TANG J L.The function study of several key genes regulating butenyl-spinosyn biosynthesis in Saccharopolyspora pogona[D].Changsha:Hunan:Hunan Normal University, 2021.
[3] 田博宇, 高爽, 刘阳星月, 等.微生物源杀虫剂对储粮害虫的防治应用与展望[J].粮食与油脂, 2021, 34(5):7-9;16.
TIAN B Y, GAO S, LIU Y X Y, et al.Application and prospect of microbial insecticides in the control of stored grain pests[J].Cereals & Oils, 2021, 34(5):7-9;16.
[4] 孙艳敏, 韩锦峰, 陈小丽, 等.减施化学农药防治植物病害措施的研究进展[J].贵州农业科学, 2021, 49(5):58-66.
SUN Y M, HAN J F, CHEN X L, et al.Advances in measures of reduction of chemical pesticides to control plant diseases[J].Guizhou Agricultural Sciences, 2021, 49(5):58-66.
[5] 何昊城. 丁烯基多杀菌素合成关键调控基因及代谢网络优化研究[D].长沙:湖南师范大学, 2021.
He H C.Optimization of key regulatory genes and metabolic networks of butenyl-spinosyn biosynthesis[D].Changsha:Hunan Normal University, 2021.
[6] RANG J, ZHU Z R, LI Y L, et al.Identification of a TetR family regulator and a polyketide synthase gene cluster involved in growth development and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona[J].Applied Microbiology and Biotechnology, 2021, 105(4):1519-1533.
[7] 彭胜男, 何昊城, 苑爽芹, 等.fcl基因对须糖多孢菌丁烯基多杀菌素生物合成及生长发育的影响[J].生物工程学报, 2019, 35(9):1662-1675.
PENG S N, HE H C, YUAN S Q, et al.Effect of fcl gene for butenyl-spinosyn biosynthesis and growth of Saccharopolyspora pogona[J].Chinese Journal of Biotechnology, 2019, 35(9):1662-1675.
[8] 王靖楠, 庞建, 秦磊, 等.丁烯基多杀菌素高产菌株的选育和改造策略[J].化工学报, 2022, 73(2):566-576.
WANG J N, PANG J, QIN L, et al.Breeding and modification strategies of butenyl-spinosyn high-yield strains[J].CIESC Journal, 2022, 73(2):566-576.
[9] HUANG K X, XIA L Q, ZHANG Y M, et al.Recent advances in the biochemistry of spinosyns[J].Applied Microbiology and Biotechnology, 2009, 82(1):13-23.
[10] LEWER P, HAHN D R, KARR L L, et al.Discovery of the butenyl-spinosyn insecticides:Novel macrolides from the new bacterial strain Saccharopolyspora pogona[J].Bioorganic & Medicinal Chemistry, 2009, 17(12):4185-4196.
[11] ZHAO X Q, GUST B, HEIDE L.S-Adenosylmethionine (SAM) and antibiotic biosynthesis:Effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces[J].Archives of Microbiology, 2010, 192(4):289-297.
[12] RANG J, HE H C, YUAN S Q, et al.Deciphering the metabolic pathway difference between Saccharopolyspora pogona and Saccharopolyspora spinosa by comparative proteomics and metabonomics[J].Frontiers in Microbiology, 2020, 11:396.
[13] OCHI K, SAITO Y, UMEHARA K, et al.Restoration of aerial Mycelium and antibiotic production in a Streptomyces griseoflavus arginine auxotroph[J].Journal of General Microbiology, 1984, 130(8):2007-2013
[14] HUH J H, KIM D J, ZHAO X Q, et al.Widespread activation of antibiotic biosynthesis by S-adenosylmethionine in Streptomycetes[J].FEMS Microbiology Letters, 2004, 238(2):439-447.
[15] 梁剑光, 黄鹏, 徐正军.阿维菌素生物合成及其代谢途径研究进展[J].常熟理工学院学报, 2007, 21(4):65-70.
LIANG J G, HUANG P, XU Z J.New research progress of avermectin biosynthesis pathway and the metabolic engineering[J].Journal of Changshu Institute of Technology, 2007, 21(4):65-70.
[16] YOON G S, KO K H, KANG H W, et al.Characterization of S-adenosylmethionine synthetase from Streptomyces avermitilis NRRL8165 and its effect on antibiotic production[J].Enzyme and Microbial Technology, 2006, 39(3):466-473.
[17] SHIN S K, XU D L, KWON H J, et al.S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus[J].FEMS Microbiology Letters, 2006, 259(1):53-59.
[18] OKAMOTO S, LEZHAVA A, HOSAKA T, et al.Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2)[J].Journal of Bacteriology, 2003, 185(2):601-609.
[19] ZHANG X C, FEN M Q, SHI X L, et al.Overexpression of yeast S-adenosylmethionine synthetase metK in Streptomyces actuosus leads to increased production of nosiheptide[J].Applied Microbiology and Biotechnology, 2008, 78(6):991-995.
[20] KIM D J, HUH J H,YANG Y Y, et al.Accumulation of S-adenosyl-L-methionine enhances production of actinorhodin but inhibits sporulation in Streptomyces lividans TK23[J].Journal of Bacteriology, 2003, 185(2):592-600.
[21] GUO C, GUO W Q, LIU Y C, et al.Complete genome sequence of butenyl-spinosyn producing Saccharopolyspora strain ASAGF58[J].Annals of Microbiology, 2020, 70(1):46.
[22] PANG J, LI X Y, GUO C, et al.Exploring a high-efficiency genetic transformation system for engineering Saccharopolyspora pogona ASAGF58 to improve butenyl-spinosyn production[J].ACS Agricultural Science & Technology, 2023, 3(2):203-210.
[23] 张逍遥, 郭超, 王靖楠, 等.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, 2022, 30(1):174-181.
ZHANG X Y, GUO C, WANG J N, et al.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, 2022, 30(1):174-181.
[24] XU Y R, TAN G Q, KE M L, et al.Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis[J].Journal of Industrial Microbiology & Biotechnology, 2018, 45(5):345-355.
[25] 李晓晨. 多杀菌素异源生物合成体系优化研究[D].济南:山东大学, 2023.
LI X C.Optimization of spinosad heterologous biosynthesis system[D].Jinan:ShanDong University, 2023.
[26] OH T J, NIRAULA N P, LIOU K, et al.Identification of the duplicated genes for S-adenosyl-l-methionine synthetase (metK1-sp and metK2-sp) in Streptomyces peucetius var.caesius ATCC 27952[J].Journal of Applied Microbiology, 2010, 109(2):398-407.
[27] 徐周钦, 郭超, 李金萍, 等.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].中国生物防治学报, 2024, 40(2):299-309.
XU Z Q, GUO C, LI J P, et al.Breeding of butenyl-spinosyns high yielding strain by 60Co-NTG compound mutation and its insecticidal activity[J].Chinese Journal of Biological Control, 2024, 40(2):299-309.
[28] BUSTIN S A, BENES V, GARSON J A, et al.The MIQE guidelines:Minimum information for publication of quantitative real-time PCR experiments[J].Clinical Chemistry, 2009, 55(4):611-622.
[29] KIM H J, WHITE-PHILLIP J A, OGASAWARA Y, et al.Biosynthesis of spinosyn in Saccharopolyspora spinosa:Synthesis of permethylated rhamnose and characterization of the functions of SpnH, SpnI, and SpnK[J].Journal of the American Chemical Society, 2010, 132(9):2901-2903.
[30] 薛正莲, 王珊, 孙俊峰, 等.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, 2021, 61(12):3870-3886.
XUE Z L, WANG S, SUN J F, et al.Research progress on morphological differentiation and secondary metabolite biosynthesis of Streptomyces[J].Acta Microbiologica Sinica, 2021, 61(12):3870-3886.
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