Breeding and modification strategies of butenyl-spinosyn high-yield strains

doi:10.11949/0438-1157.20210898

Abstract

Abstract:

Butenyl-spinosyn is an insecticide produced by Saccharopolyspora pogona, which has both the safety of biological pesticides and the quick-acting properties of chemical pesticides. However, the low synthesis efficiency of butenyl-spinosyn by wild-type strains cannot meet the need of industrial production, obtaining high-yield strains is an urgent problem. At present, there are few related studies on butenyl-spinosyn. The spinosyn produced by Saccharopolyspora spinosa has a similar structural and biosynthetic pathway. This article describes the basic characteristics of them, draws on the research experience of spinosyn, summarizes the available strategies for breeding and modifying high-yield butenyl-spinosyn strains, including mutagenesis methods and precise genetic engineering methods such as metabolic flux regulation, pathway genes regulation, transcriptional regulation, heterologous expression, which may provide ideas for further research of butenyl-spinosyn.

Key words: biological engineering, molecular biology, synthetic biology, butenyl-spinosyn, spinosyn, Saccharopolyspora pogona, mutation breeding, genetic engineering

摘要：

丁烯基多杀菌素（butenyl-spinosyn）是一种由须糖多孢菌（Saccharopolyspora pogona）产生的杀虫剂，兼具生物农药的安全性和化学农药的速效性。当前，野生型须糖多孢菌合成丁烯基多杀菌素效率低，达不到工业生产要求，获得高产菌株是亟待解决的问题。目前关于丁烯基多杀菌素的相关研究较少，由刺糖多孢菌（Saccharopolyspora spinosa）产生的多杀菌素是丁烯基多杀菌素的结构类似物，具有相似的生物合成途径，本文介绍了两者的基本特性，借鉴多杀菌素的研究经验总结了丁烯基多杀菌素高产菌株已有及可用的选育和改造策略，包括传统的理化诱变方法以及代谢流调控、途径基因调控、转录调控、异源表达等更加精准的基因工程方法，以期为后续丁烯基多杀菌素的深入研究提供思路。

关键词: 生物工程, 分子生物学, 合成生物学, 丁烯基多杀菌素, 多杀菌素, 须糖多孢菌, 诱变选育, 基因工程

CLC Number:

TQ 458

Jingnan WANG, Jian PANG, Lei QIN, Chao GUO, Bo LYU, Chun LI, Chao WANG. Breeding and modification strategies of butenyl-spinosyn high-yield strains[J]. CIESC Journal, 2022, 73(2): 566-576.

王靖楠, 庞建, 秦磊, 郭超, 吕波, 李春, 王超. 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.

Figures/Tables 8

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分类	诱变技术	机理^[12]	产量变化
物理诱变	UV诱变	使嘧啶形成二聚体	4.84倍，从77 mg/L到373 mg/L^[13]
物理诱变	⁶⁰Co诱变	引起DNA单链或双链断裂	1.16倍，从404 mg/L到469 mg/L^[14]
化学诱变	NTG诱变	烷基取代核酸碱基氢原子引起碱基错配^[15]	1.44倍，从436 mg/L到628 mg/L^[16]
化学诱变	MNNG诱变	烷基取代核酸碱基氢原子引起碱基错配^[15]	2.28倍，从102 mg/L到232 mg/L^[15]
生物诱变	基因组重排	通过原生质体融合富集理化诱变得到的正向突变	6.60倍，从50 mg/L到332 mg/L^[17]
生物诱变	转座诱变	转座子片段随机插入到基因组产生突变	1.14倍，从129 mg/L到147 mg/L^[18]
新型诱变技术	ARTP诱变	激发态的自由基等活性粒子损伤DNA	1.32倍，从417 mg/L到550 mg/L^[16]
	航空搭载育种	太空环境的微重力等条件可能影响DNA	3.88倍，从38 mg/L到148 mg/L^[19]
	核糖体工程	核糖体蛋白突变影响次级代谢产物产生	1.24倍^[20]
复合诱变	MNNG、⁶⁰Co诱变	增加突变概率	1.87倍，从549 mg/L到1035 mg/L^[21]
	EMS及ARTP诱变、链霉素诱变	增加突变概率	7.71倍，从158 mg/L到1218 mg/L^[22]
	N⁺离子注入诱变、鼠李糖及多杀菌素耐受	能量沉积、粒子注入、动量传递等过程与生物体发生作用	1.44倍，从651 mg/L 到937 mg/L^[23]
	MPMS、核糖体工程	等离子体中的活性成分损伤生物大分子，微生物产生大量随机突变^[24]	1.29倍^[24]

分类	诱变技术	机理^[12]	产量变化
物理诱变	UV诱变	使嘧啶形成二聚体	4.84倍，从77 mg/L到373 mg/L^[13]
物理诱变	⁶⁰Co诱变	引起DNA单链或双链断裂	1.16倍，从404 mg/L到469 mg/L^[14]
化学诱变	NTG诱变	烷基取代核酸碱基氢原子引起碱基错配^[15]	1.44倍，从436 mg/L到628 mg/L^[16]
化学诱变	MNNG诱变	烷基取代核酸碱基氢原子引起碱基错配^[15]	2.28倍，从102 mg/L到232 mg/L^[15]
生物诱变	基因组重排	通过原生质体融合富集理化诱变得到的正向突变	6.60倍，从50 mg/L到332 mg/L^[17]
生物诱变	转座诱变	转座子片段随机插入到基因组产生突变	1.14倍，从129 mg/L到147 mg/L^[18]
新型诱变技术	ARTP诱变	激发态的自由基等活性粒子损伤DNA	1.32倍，从417 mg/L到550 mg/L^[16]
	航空搭载育种	太空环境的微重力等条件可能影响DNA	3.88倍，从38 mg/L到148 mg/L^[19]
	核糖体工程	核糖体蛋白突变影响次级代谢产物产生	1.24倍^[20]
复合诱变	MNNG、⁶⁰Co诱变	增加突变概率	1.87倍，从549 mg/L到1035 mg/L^[21]
	EMS及ARTP诱变、链霉素诱变	增加突变概率	7.71倍，从158 mg/L到1218 mg/L^[22]
	N⁺离子注入诱变、鼠李糖及多杀菌素耐受	能量沉积、粒子注入、动量传递等过程与生物体发生作用	1.44倍，从651 mg/L 到937 mg/L^[23]
	MPMS、核糖体工程	等离子体中的活性成分损伤生物大分子，微生物产生大量随机突变^[24]	1.29倍^[24]

调控	描述	基因编辑方法	产量变化	分析机理
过表达PNPase^[55]	转录因子，多核苷酸磷酸化酶，与氨基酸代谢、有机酸代谢及细胞生物合成相关酶等相关	同源重组- 单交换	1.96倍	减少菌丝体聚集，增加孢子产生，影响核苷酸代谢从而影响能量供应
过表达AfsR^[56]	与链霉菌AfsR同源，属于抗生素调节蛋白家族	同源重组- 单交换	1.17倍	减少菌丝体聚集，影响产孢，降低初级代谢提升次级代谢
敲除padR^[57]	普遍的调控因子，在链霉菌中与抗生素生物合成相关	同源重组- 双交换	1.27倍	促进转运相关蛋白的表达
过表达Sp1418^[58]	TetR家族的转录调控因子，与营养生长、菌丝分化及氧化应激相关	同源重组- 双交换	2.50倍	影响菌体氧化应激
过表达regX3^[59]	Sen X3-Reg X3双组分系统组分，与无机磷吸收相关	同源重组- 单交换	2.30倍	全局调控因子，无磷条件培养使得磷吸收最少
敲除lytS-L^[60]	LytTR家族双组分系统组分，传感器激酶基因，与改善营养环境相关	同源重组- 单交换	0.60倍	全局调控因子
敲除SP_1288^[61]	TetR家族调控蛋白	CRISPR-Cas9	3.10倍	全局调控因子

调控	描述	基因编辑方法	产量变化	分析机理
过表达PNPase^[55]	转录因子，多核苷酸磷酸化酶，与氨基酸代谢、有机酸代谢及细胞生物合成相关酶等相关	同源重组- 单交换	1.96倍	减少菌丝体聚集，增加孢子产生，影响核苷酸代谢从而影响能量供应
过表达AfsR^[56]	与链霉菌AfsR同源，属于抗生素调节蛋白家族	同源重组- 单交换	1.17倍	减少菌丝体聚集，影响产孢，降低初级代谢提升次级代谢
敲除padR^[57]	普遍的调控因子，在链霉菌中与抗生素生物合成相关	同源重组- 双交换	1.27倍	促进转运相关蛋白的表达
过表达Sp1418^[58]	TetR家族的转录调控因子，与营养生长、菌丝分化及氧化应激相关	同源重组- 双交换	2.50倍	影响菌体氧化应激
过表达regX3^[59]	Sen X3-Reg X3双组分系统组分，与无机磷吸收相关	同源重组- 单交换	2.30倍	全局调控因子，无磷条件培养使得磷吸收最少
敲除lytS-L^[60]	LytTR家族双组分系统组分，传感器激酶基因，与改善营养环境相关	同源重组- 单交换	0.60倍	全局调控因子
敲除SP_1288^[61]	TetR家族调控蛋白	CRISPR-Cas9	3.10倍	全局调控因子

宿主菌株	提产方法	产量提升	基因簇克隆方法
S. albus J1074^[65]	1.组学分析找到三个限速问题：鼠李糖生物合成不足、甲基转移酶活性不足以及聚酮合酶活性不足； 2.改进上述限速步骤	到1.4 mg/L	BAC文库
S. coelicolor M145^[66];S. lividans TK24^[66]	过表达鼠李糖合成基因	到1.0 mg/L；到1.5 mg/L	BAC文库
S. albus J1074^[67]	1.构建79 kb的人工基因簇，将通路分成7个操纵子； 2.每个操纵子都以一个强组成性启动子表达	到1.1 mg/L	ExoCET^[68]
Sa. erythraea^[69]	1.替换红色糖多孢菌的PKS基因簇； 2.将spnGF替换为eryAB； 3.引入sfp基因； 4.过表达鼠李糖合成基因	到830.0 mg/L	Cosmid文库