Advances in design, construction and applications of Bacillus subtilis chassis cells

doi:10.12211/2096-8280.2020-030

Abstract

Abstract:

As a model industrial host and an important generally recognized as safe microorganism, Bacillus subtilis has been used for a wide range of applications such as the industrial production of enzymes and nutraceuticals. In recent years, with the elucidation of the genetic regulation mechanism of B. subtilis, various research strategies and technologies have been designed and developed with this chassis, including gene editing, gene circuits, spatial biomolecular scaffold and cell-free expression systems. In this review, we start with systematic summaries on the construction of B. subtilis chassis based on gene editing systems and endogenous regulatory mechanisms. Then applications of B. subtilis cell factories are discussed for producing N-acetylglucosamine, menaquinone-7, riboflavin, hyaluronic acid and β-cyclodextrin glycosyltransferase. Finally, prospects for the design, construction and applications of engineered B. subtilis strains are commented, with an emphasis on improving genome editing efficiency, expanding responsive metabolite spectrum for genetic circuits, and rewiring the whole genome.

Key words: Bacillus subtilis, chassis cell, reduced genome, gene circuit, gene editing

摘要：

作为一种重要的模式工业微生物和食品安全微生物，枯草芽孢杆菌在工业酶和功能营养品的生产方面具有广泛应用。近年来，随着对枯草芽孢杆菌遗传调控机制的不断揭示，多种研究策略和技术，包括基因编辑系统、基因回路、空间支架、无细胞表达系统等，被用于设计和构建枯草芽孢杆菌底盘细胞高效合成各种生物化学品。本文首先对基于基因编辑和基于内源性调控系统的枯草芽孢杆菌底盘细胞设计与构建的策略进行了系统的概述。然后，以高产N-乙酰氨基葡萄糖、七烯甲萘醌、核黄素、透明质酸和β-环糊精糖基转移酶为例介绍了枯草芽孢杆菌底盘细胞工厂的应用。最后，从基因编辑效率提升、基因回路响应信号拓展和基因组设计与重构三方面对枯草芽孢杆菌底盘细胞的设计、构建与应用进行了展望。

关键词: 枯草芽孢杆菌, 底盘细胞, 基因组简化, 基因回路, 基因编辑

CLC Number:

Q819

LIN Lu, LV Xueqin, LIU Yanfeng, DU Guocheng, CHEN Jian, LIU Long. Advances in design, construction and applications of Bacillus subtilis chassis cells[J]. Synthetic Biology Journal, 2020, 1(2): 247-265.

林璐, 吕雪芹, 刘延峰, 堵国成, 陈坚, 刘龙. 枯草芽孢杆菌底盘细胞的设计、构建与应用[J]. 合成生物学, 2020, 1(2): 247-265.

Figures/Tables 8

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类别	构建方法	功能特性	文献
基因编辑	基因组简化	利用无痕基因敲除技术构建多种基因组简化菌株	[6]
	CRISPR/Cas9迭代系统	实现基因敲除、点突变和基因敲入，避免脱靶效应	[7]
	CRISPR-Cas9工具包	实现基因敲除、多基因敲入和转录抑制	[8]
	CAMERS-B系统	通过设计crRNA阵列实现多基因编辑和调控	[9]
	内源性启动子库	基于细胞生长构建的内源性启动子文库	[10]
	模块化操作单元	在转录翻译和蛋白水解水平上共同调节基因和蛋白的表达	[11]
	DNA支架	通过DNA支架调节途径酶的空间比例和方向	[12]
	RNA支架	通过RNA支架与特异性蛋白结合，转录抑制靶基因的表达	[13]
	内源性空间支架	通过FMMs空间支架构建FMMs-多酶复合物系统	[14]
	无细胞蛋白表达系统	以外源DNA为模板，利用细胞裂解物，可在体外表达蛋白质	[15]
基因回路	glmS核糖开关	基于GlcN6P响应的天然glmS核糖开关	[16]
	配体-适体生物传感器	基于配体凝血酶设计的双功能基因表达调控电路	[17]
	ADC系统	基于生物传感器、CRISPR和合成基因回路的代谢流量动态自发双重调控(autonomous dual control,ADC)系统	[18]
内源性调控系统	Sec分泌途径	通过改善Sec分泌途径，提高异源蛋白的分泌	[19,20]
	群体感应系统	细胞密度达到阈值，动态调节相关基因的表达	[21,22]
	I型毒素-抗毒素系统	通过抗毒素反义RNA与毒素mRNA互补配对，下调基因的表达	[23]
	碳分解代谢阻遏作用	解除碳分解代谢阻遏作用，细胞可同时利用多种碳源	[24]

类别	构建方法	功能特性	文献
基因编辑	基因组简化	利用无痕基因敲除技术构建多种基因组简化菌株	[6]
	CRISPR/Cas9迭代系统	实现基因敲除、点突变和基因敲入，避免脱靶效应	[7]
	CRISPR-Cas9工具包	实现基因敲除、多基因敲入和转录抑制	[8]
	CAMERS-B系统	通过设计crRNA阵列实现多基因编辑和调控	[9]
	内源性启动子库	基于细胞生长构建的内源性启动子文库	[10]
	模块化操作单元	在转录翻译和蛋白水解水平上共同调节基因和蛋白的表达	[11]
	DNA支架	通过DNA支架调节途径酶的空间比例和方向	[12]
	RNA支架	通过RNA支架与特异性蛋白结合，转录抑制靶基因的表达	[13]
	内源性空间支架	通过FMMs空间支架构建FMMs-多酶复合物系统	[14]
	无细胞蛋白表达系统	以外源DNA为模板，利用细胞裂解物，可在体外表达蛋白质	[15]
基因回路	glmS核糖开关	基于GlcN6P响应的天然glmS核糖开关	[16]
	配体-适体生物传感器	基于配体凝血酶设计的双功能基因表达调控电路	[17]
	ADC系统	基于生物传感器、CRISPR和合成基因回路的代谢流量动态自发双重调控(autonomous dual control,ADC)系统	[18]
内源性调控系统	Sec分泌途径	通过改善Sec分泌途径，提高异源蛋白的分泌	[19,20]
	群体感应系统	细胞密度达到阈值，动态调节相关基因的表达	[21,22]
	I型毒素-抗毒素系统	通过抗毒素反义RNA与毒素mRNA互补配对，下调基因的表达	[23]
	碳分解代谢阻遏作用	解除碳分解代谢阻遏作用，细胞可同时利用多种碳源	[24]

名称	类型	性质	文献
P₄₃	组成型	B. subtilis中cdd基因的启动子，能持续性高效表达基因	[38]
P _HpaII	组成型	来源于Staphylococcus aureus中的载体pUB110	[39]
P _laps	组成型	人工构建的双启动子，启动强度是P₄₃的13倍	[40]
P _grac	诱导型	IPTG诱导，由lac操纵子和groESL启动子融合而成	[41]
P _xyl	诱导型	受木糖诱导，且受葡萄糖抑制	[42]
P _sacB	诱导型	受蔗糖诱导	[43]
P _mtlA	诱导型	受甘露醇诱导	[44]
P _glv	诱导型	受麦芽糖诱导	[45]
P _amy	诱导型	受淀粉诱导，可以抵抗碳分解代谢物阻遏的启动子	[46]
P _ohrB	诱导型	受环境压力和葡萄糖匮乏诱导	[47]
P _gsiB	诱导性	受环境因素（热、盐、酸、乙醇、缺氧等）诱导	[48]
P _rpsF	时期特异型	在对数期被激活	[49]
P _srfA	时期特异型	在对数中后期被激活	[50]
P _aprE	时期特异型	在对数末期被激活	[51]
P _cry3Aa	时期特异型	在稳定期被激活	[52]
P _manP	自诱导型	适合于高密度发酵	[53]
P _bs	合成型	在E.coli、B. subtilis和S. cerevisiae均有活力的广谱启动子	[54]

名称	类型	性质	文献
P₄₃	组成型	B. subtilis中cdd基因的启动子，能持续性高效表达基因	[38]
P _HpaII	组成型	来源于Staphylococcus aureus中的载体pUB110	[39]
P _laps	组成型	人工构建的双启动子，启动强度是P₄₃的13倍	[40]
P _grac	诱导型	IPTG诱导，由lac操纵子和groESL启动子融合而成	[41]
P _xyl	诱导型	受木糖诱导，且受葡萄糖抑制	[42]
P _sacB	诱导型	受蔗糖诱导	[43]
P _mtlA	诱导型	受甘露醇诱导	[44]
P _glv	诱导型	受麦芽糖诱导	[45]
P _amy	诱导型	受淀粉诱导，可以抵抗碳分解代谢物阻遏的启动子	[46]
P _ohrB	诱导型	受环境压力和葡萄糖匮乏诱导	[47]
P _gsiB	诱导性	受环境因素（热、盐、酸、乙醇、缺氧等）诱导	[48]
P _rpsF	时期特异型	在对数期被激活	[49]
P _srfA	时期特异型	在对数中后期被激活	[50]
P _aprE	时期特异型	在对数末期被激活	[51]
P _cry3Aa	时期特异型	在稳定期被激活	[52]
P _manP	自诱导型	适合于高密度发酵	[53]
P _bs	合成型	在E.coli、B. subtilis和S. cerevisiae均有活力的广谱启动子	[54]

类别	产品	菌株	方法	产量	文献
生物化学品	七烯甲萘醌	B. subtilis	Phr60-Rap60-Spo0A群体响应调控系统	360 mg/L	[22]
	核黄素	B. subtilis RH33	定点诱变zwf、gnd	15.7 g/L	[88]
	鲨肌醇	B. subtilis KU303	敲除iolABCDEF、iolHIJ、iolX、iolR,过表达IolG、IolW、IolT、PntAB	27.6 g/L	[89]
	软骨素	B. subtilis E168H	表达kfoC-kfoA、kfiC-kfiA,上调tuaD	5.22 g/L	[90]
	鸟苷	BSK814G2	敲除purA被敲除,共表达PRS、purF、guaB	115.2 mg/L	[92]
	胸苷	BSK756T3	敲除tdk,过表达prs、ushA、thyA、dut、ndk	151.2 mg/L	[92]
	紫穗槐二烯	B. subtilis 1A1	过表达dxs、idi、ads	20 mg/L	[93]
	乙缩醛	B. subtilis BMN	敲除bdhA,过表达基因 nox	56.7 g/L	[94]
	2,3-丁二醇	B. subtilis BSF9	敲除upp、acoA、bdhA、pta、ldh,过表达alsS、alsD、budC、udhA	103.7 g/L	[95]
	异丁醇	BSUL08	敲除alsS、ldh、pdhC、pgi,过表达zwf、udhA	6.12 g/L	[96]
	二吡啶甲酸	B. subtilis OA105	敲除alsSD,过表达spoVGBA	2.3 g/L	[97]
	苹果酸	BSUPML	敲除ldh,过表达异源基因mdh	1.23 g/L	[98]
	透明质酸	B. subtilis E168TH	表达hasA、tuaD、gtaB、glmU、glmM、glmS	19.38 g/L	[99]
	N-乙酰氨基葡萄糖	BP18-afGNA1	重构中心碳和氧化还原代谢	24.5 g/L	[102]
典型化学品	α-淀粉酶	B. subtilis 1A237	共表达PrsA和DnaK蛋白	1352 U/mL	[19]
	纳豆激酶	BSG328	改造srfA启动子核心区	93.6 U/mL	[21]
	氨肽酶	BSG329	改造srfA启动子核心区	69.8 U/mL	[21]
	L-天冬酰胺酶	B. subtilis WB600	表达type Ⅱ ASN	407.6 U/mL	[91]
	β-环糊精糖基转移酶	BS5 ATCC 6051a	敲除srfC、spoIIAC、nprE、aprE、amyE	227.8 U/mL	[100]
	植酸酶	B. subtilis BD170	表达phyC	47.7 U/mL	[101]