Effect of LysR family transcriptional regulatory factors on the biosynthesis of butenyl-spinosyn

doi:10.11949/0438-1157.20250345

CIESC Journal ›› 2025, Vol. 76 ›› Issue (12): 6551-6561.DOI: 10.11949/0438-1157.20250345

• Biochemical engineering and technology • Previous Articles Next Articles

Effect of LysR family transcriptional regulatory factors on the biosynthesis of butenyl-spinosyn

Xia CHEN¹^,²(), Chao GUO², Xinying LI²^,³, Chao WANG²(), Chun LI¹^,³^,⁴()

^1.School of Chemistry and Chemical Engineering, Shihezi University/State Key Laboratory Incubation Base for Green Processing of Chemical Engineering, Shihezi 832003, Xinjiang, China
^2.Academy of National Food and Strategic Reserves Administration, Beijing 100037, China
^3.Key Laboratory of Medicinal Molecule Science and Pharmaceutical Engineering of 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 of Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China

Received:2025-04-07 Revised:2025-06-17 Online:2026-01-23 Published:2025-12-31
Contact: Chao WANG, Chun LI

LysR家族转录调控因子对丁烯基多杀菌素生物合成的影响

陈霞¹^,²(), 郭超², 李新颖²^,³, 王超²(), 李春¹^,³^,⁴()

^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信部重点实验室，北京 100081
^4.清华大学化学工程系工业生物催化教育部重点实验室，北京 100084

通讯作者: 王超,李春
作者简介:陈霞（1999—），女，硕士研究生， cx991009@163.com
基金资助:
十四五重点研发计划项目(2023YFA0914700);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);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(2024003)

Abstract

Abstract:

Butenyl spinosyns are new green biopesticides produced by Saccharopolyspora pogona. However, due to their complex metabolic network, unclear genetic background and inadequately analyzed regulatory mechanisms, their synthesis efficiency is low and it is difficult to meet actual needs. In this study, comparative transcriptomics analysis of S. pogona ASAGF58 (wild-type) and its high-yielding mutant ASAGF40 led to the identification of LysR family transcriptional regulators associated with secondary metabolism and butenyl-spinosyn biosynthesis. Engineered strains were subsequently constructed to validate their regulatory roles. Among them, RS18275 significantly enhanced butenyl-spinosyn production to 67.1 mg/L, achieving a yield 2.3-fold higher than the parental strain ASAGF58, accompanied by a 30.63% enhancement in biomass and accelerated glucose consumption. Comparative transcriptomic analysis demonstrated that RS18275 promotes precursor supply (e.g., acetyl-CoA) by regulating primary metabolic pathways and increases product biosynthetic flux by upregulating the expression of key genes involved in the butenyl-spinosyn biosynthetic pathway. Based on comparative transcriptomics analysis and verification, the physiological mechanism of RS18275 as a global regulatory factor to achieve efficient synthesis of target compounds by regulating primary metabolism and secondary metabolism was preliminarily analyzed, providing theoretical and engineering strategies for the subsequent efficient synthesis of butenyl spinosad.

Key words: butenyl-spinosyn, Saccharopolyspora pogona, transcriptional regulatory factors, comparative transcriptomics

摘要：

丁烯基多杀菌素（butenyl-spinosyn）是由须糖多孢菌（Saccharopolyspora pogona）产生的新型绿色生物杀虫剂，然而，由于其代谢网络复杂、遗传背景不清晰、调控机制未得到充分解析，导致其合成效率低，难以满足实际需求。本研究基于对S. pogona ASAGF58和S. pogona ASAGF40转录组学数据分析，挖掘参与次级代谢调控、影响丁烯基多杀菌素合成的LysR家族转录调控因子并构建相关工程菌株。结果显示，RS18275使丁烯基多杀菌素产量达到67.1 mg/L，为出发菌ASAGF58的2.3倍，同时促进细胞生长，菌体生物量提高30.63%，并加快了底物葡萄糖的消耗。比较转录组学分析表明，RS18275通过调控初级代谢途径增强了乙酰辅酶A等前体供应，上调丁烯基多杀菌素生物合成途径关键基因提高了产物合成通量。基于比较转录组学分析和验证，初步解析了RS18275作为全局调控因子，通过调控初级代谢与次级代谢实现目标化合物高效合成的生理机制，为后续丁烯基多杀菌素的高效合成提供理论与工程化策略。

关键词: 丁烯基多杀菌素, 须糖多孢菌, 转录调控因子, 比较转录组学

CLC Number:

Q 814

Xia CHEN, Chao GUO, Xinying LI, Chao WANG, Chun LI. Effect of LysR family transcriptional regulatory factors on the biosynthesis of butenyl-spinosyn[J]. CIESC Journal, 2025, 76(12): 6551-6561.

陈霞, 郭超, 李新颖, 王超, 李春. LysR家族转录调控因子对丁烯基多杀菌素生物合成的影响[J]. 化工学报, 2025, 76(12): 6551-6561.

Figures/Tables 9

References 35

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质粒和菌株	描述	来源
质粒
pSET159	整合型质粒，含启动子ermEp，抗性：aac(3)-IV*	实验室保藏
pSET159-RS18275	pSET159连接RS18275基因	本研究构建
菌株
E. coli DH5α	重组质粒构建时大肠转化	实验室保藏
ET12567/pUZ8002	接合转移供体菌，抗性：cat、aph(3')-IIa	实验室保藏
S. pogona ASAGF58	野生型须糖多孢菌（NZ_CP040605.1）	实验室保藏
TF23645	ASAGF58: attB:: pSET159, ermEp -RS23645*	本研究构建
TF28350	ASAGF58: attB:: pSET159, ermEp -RS28350*	本研究构建
TF34545	ASAGF58: attB:: pSET159, ermEp -RS34545*	本研究构建
TF18275	ASAGF58: attB:: pSET159, ermEp -RS18275*	本研究构建
TF26805	ASAGF58: attB:: pSET159, ermEp -RS26805*	本研究构建
TF19675	ASAGF58: attB:: pSET159, ermEp -RS19675*	本研究构建
TF31170	ASAGF58: attB:: pSET159, ermEp -RS31170*	本研究构建
TF35325	ASAGF58: attB:: pSET159, ermEp -RS35325*	本研究构建
TF30620	ASAGF58: attB:: pSET159, ermEp -RS30620*	本研究构建
TF27790	ASAGF58: attB:: pSET159, ermEp -RS27790*	本研究构建
TF18300	ASAGF58: attB:: pSET159, ermEp -RS18300*	本研究构建
TF23375	ASAGF58: attB:: pSET159, ermEp -RS23375*	本研究构建
TF17855	ASAGF58: attB:: pSET159, ermEp -RS17855*	本研究构建
TF16865	ASAGF58: attB:: pSET159, ermEp -RS16865*	本研究构建
TF25420	ASAGF58: attB:: pSET159, ermEp -RS25420*	本研究构建
TF33530	ASAGF58: attB:: pSET159, ermEp -RS33530*	本研究构建
TF06895	ASAGF58: attB:: pSET159, ermEp -RS06895*	本研究构建
TF27320	ASAGF58: attB:: pSET159, ermEp -RS27320*	本研究构建
TF27710	ASAGF58: attB:: pSET159, ermEp -RS27710*	本研究构建
TF31575	ASAGF58: attB:: pSET159, ermEp -RS31575*	本研究构建

质粒和菌株	描述	来源
质粒
pSET159	整合型质粒，含启动子ermEp，抗性：aac(3)-IV*	实验室保藏
pSET159-RS18275	pSET159连接RS18275基因	本研究构建
菌株
E. coli DH5α	重组质粒构建时大肠转化	实验室保藏
ET12567/pUZ8002	接合转移供体菌，抗性：cat、aph(3')-IIa	实验室保藏
S. pogona ASAGF58	野生型须糖多孢菌（NZ_CP040605.1）	实验室保藏
TF23645	ASAGF58: attB:: pSET159, ermEp -RS23645*	本研究构建
TF28350	ASAGF58: attB:: pSET159, ermEp -RS28350*	本研究构建
TF34545	ASAGF58: attB:: pSET159, ermEp -RS34545*	本研究构建
TF18275	ASAGF58: attB:: pSET159, ermEp -RS18275*	本研究构建
TF26805	ASAGF58: attB:: pSET159, ermEp -RS26805*	本研究构建
TF19675	ASAGF58: attB:: pSET159, ermEp -RS19675*	本研究构建
TF31170	ASAGF58: attB:: pSET159, ermEp -RS31170*	本研究构建
TF35325	ASAGF58: attB:: pSET159, ermEp -RS35325*	本研究构建
TF30620	ASAGF58: attB:: pSET159, ermEp -RS30620*	本研究构建
TF27790	ASAGF58: attB:: pSET159, ermEp -RS27790*	本研究构建
TF18300	ASAGF58: attB:: pSET159, ermEp -RS18300*	本研究构建
TF23375	ASAGF58: attB:: pSET159, ermEp -RS23375*	本研究构建
TF17855	ASAGF58: attB:: pSET159, ermEp -RS17855*	本研究构建
TF16865	ASAGF58: attB:: pSET159, ermEp -RS16865*	本研究构建
TF25420	ASAGF58: attB:: pSET159, ermEp -RS25420*	本研究构建
TF33530	ASAGF58: attB:: pSET159, ermEp -RS33530*	本研究构建
TF06895	ASAGF58: attB:: pSET159, ermEp -RS06895*	本研究构建
TF27320	ASAGF58: attB:: pSET159, ermEp -RS27320*	本研究构建
TF27710	ASAGF58: attB:: pSET159, ermEp -RS27710*	本研究构建
TF31575	ASAGF58: attB:: pSET159, ermEp -RS31575*	本研究构建

引物	序列 (5′-3′)	描述
159-F	gtcacgacgttgtaaaacgacgg	扩增ermE*p-pSET159线性化载体
e159-R	taatttctcctctttaatgttaaacaaaattatttctag	扩增ermE*p-pSET159线性化载体
T159-F	atagctgcgccgatggtt	重组质粒验证
Y2-R	gccgcggatcctctagagtcga	重组质粒验证
T-F	ccgcttactttcaaccgaggcg	接合子验证
T-R	cttctgctcgccctcgtagg	接合子验证
18275-F	gtttaacattaaagaggagaaattaatgcgctacgacctggacgacc	RS18275基因扩增
18275-R	ggccgtcgttttacaacgtcgtgactcagccgtcgtggcgggga	RS18275基因扩增

引物	序列 (5′-3′)	描述
159-F	gtcacgacgttgtaaaacgacgg	扩增ermE*p-pSET159线性化载体
e159-R	taatttctcctctttaatgttaaacaaaattatttctag	扩增ermE*p-pSET159线性化载体
T159-F	atagctgcgccgatggtt	重组质粒验证
Y2-R	gccgcggatcctctagagtcga	重组质粒验证
T-F	ccgcttactttcaaccgaggcg	接合子验证
T-R	cttctgctcgccctcgtagg	接合子验证
18275-F	gtttaacattaaagaggagaaattaatgcgctacgacctggacgacc	RS18275基因扩增
18275-R	ggccgtcgttttacaacgtcgtgactcagccgtcgtggcgggga	RS18275基因扩增

引物	序列 (5′-3′)	引物	序列 (5′-3′)
bldD-F	CACTACCTCCCGACTGGCTC	busL-F	CGGACGGTTTCTTTCACGC
bldD-R	TCGTCGGGTCCTATGAGCG	busL-R	GCCAGGTTCCAGGACTCGGT
whiA-F	CCGACGGGCTGAGGTTTC	busM-F	CCGGGAGCTGAGCGATTT
whiA-R	GTGCCCGAACAGCTCGTG	busM-R	TGGTAGACGAGCGTTGGGAC
busA-F	CATGCCTTGGGTCTTGTCGG	busN-F	CTGCTGCTGCGGGAGAATT
busA-R	GCAGTGCGGATCGAGTGGTC	busN-R	TCCGAACACGGCGACGAAC
busB-F	GGGGAGCGTCTTGGTTGTG	busO-F	CGGTGGATGACTTTGGGACA
busB-R	CGGTGCTTCCAGGGTTAGTTC	busO-R	CACAAGCTATGCAGGGAGGC
busC-F	GGAAGGGCTGTGGAAACTGG	busP-F	TGACCAAGCGGCAGCAGA
busC-R	ACCACGGTCAGCGGGAAA	busP-R	CGGACAGCGTGGCGATAA
busD-F	GCGGTTACGGTGGATACGG	busQ-F	GACCGGCAGTCTGGAGTTGG
busD-R	CGGAACGCAACGACTGACA	busQ-R	CCTCTTGCGGCAGGTGTTGT
busE-F	TCGTTCTTCGTGCTCTTCTCCT	busR-F	CAGCAGGCGCAGGGAAAT
busE-R	CGCCGCGTAGTTTCCTTG	busR-R	GGCACATCGGAAAGCAACC
busF-F	CGCCATCCACCACGGCTACT	busS-F	CAACCGAGGCCGAGGAAA
busF-R	TACCGCACCCCACATCCAG	busS-R	CAGGCGATGTCGAGTAGGGA
busG-F	GCCACCAGAACCTCGTGTCC	gdh-F	CGGCTTCATCGGCTCGCACTA
busG-R	TCGTTCCCTTGCCCTATCC	gdh-R	GTGAGCTTGTCGAGCACAACC
busH-F	TCCACCACCCAGACGGTACG	kre-F	CCGGCCTACTCGGTCCTGT
busH-R	GCGGTTCTCCAGGCATTCG	kre-R	TCGCCGTCTTTCTCGAAGGC
busI-F	CTGGTACACCGAACACTACGAGC	gtt-F	GGCTGCTCGGGGACGGGTCACAG
busI-R	CGGAAATACCGCTGCCACAT	gtt-R	CTCGGCGTACTCGATCTGGATG
busJ-F	CGGAATCCACGCAACTCG	epi-F	TTGAGATCACGGGTGCATAC
busJ-R	GTAGCCTGCTTGGCGGTGT	epi-R	GCCGAGACGCTGTGATTGG
busK-F	TCGCTGCGGATCGGTTAC	16S -F	CCGCAAGGCTAAAACTCAAAG
busK-R	GGTGGTTCGGAGGAAGTTGG	16S -R	CTAGTGCATGTCAAACCCAGGTAA

Effect of LysR family transcriptional regulatory factors on the biosynthesis of butenyl-spinosyn

LysR家族转录调控因子对丁烯基多杀菌素生物合成的影响

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