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

Lu LIN, Xueqin LV, Yanfeng LIU, Guocheng DU, Jian CHEN, Long LIU. 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 20

References 111

1	LEE J W,NA D,PARK J M,et al. Systems metabolic engineering of microorganisms for natural and non-natural chemicals [J]. Nature Chemical Biology,2012,8(6): 536-546.
2	LEE S,MATTANOVICH D,VILLAVERDE A. Systems metabolic engineering,industrial biotechnology and microbial cell factories [J]. Microbial Cell Factories,2012,11(1): 156.
3	GOEL A,WORTEL M T,MOLENAAR D,et al. Metabolic shifts: a fitness perspective for microbial cell factories [J]. Biotechnology Letters,2012,34(12): 2147-2160.
4	LIU L,LIU Y,SHIN H D,et al. Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology [J]. Applied Microbiology and Biotechnology,2013,97(14): 6113-6127.
5	JUHAS M,REU D R,ZHU B,et al. Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering [J]. Microbiology,2014,160(11): 2341-2351.
6	李杨. 基因组简化枯草芽孢杆菌的构建及应用 [D]. 天津：天津大学,2017.
	LI Y. Construction of genome reduced Bacillus subtilis and their application in production of bio-based chemicals [D]. Tianjin: Tianjin University,2017.
7	SO Y,PARK S Y,PARK E H,et al. A highly efficient CRISPR-Cas9-Mediated large genomic deletion in Bacillus subtilis [J]. Frontiers in Microbiology,2017,8: a1167.
8	WESTBROOK A W,YOUNG M,CHOU C P. Development of a CRISPR-Cas9 tool kit for comprehensive engineering of Bacillus subtilis [J]. Applied and Environmental Microbiology,2016,82(16): 4876-4895.
9	WU Y,LIU Y,LV X,et al. CAMERS-B: CRISPR/Cpf1 Assisted multiple-genes editing and regulation system for Bacillus subtilis [J]. Biotechnology and Bioengineering,2020. DOI: org/10.1002/bit.27322. DOI URL
10	SONG Y,NIKOLOFF J M,FU G,et al. Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications [J]. PLoS One,2016,11(7): E158447.
11	GUIZIOU S,SAUVEPLANE V,CHANG H J,et al. A part toolbox to tune genetic expression in Bacillus subtilis [J]. Nucleic Acids Research,2016,44(15): 7495-7508.
12	LIU Y,ZHU Y,MA W,et al. Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by Bacillus subtilis [J]. Metabolic Engineering,2014,24: 61-69.
13	LIU Y,ZHU Y,LI J,et al. Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production [J]. Metabolic Engineering,2014,23: 42-52.
14	LV X,WU Y,TIAN R,et al. Synthetic metabolic channel by functional membrane microdomains for compartmentalized flux control [J]. Metabolic Engineering,2020,59: 106-118.
15	KELWICK R,WEBB A J,MACDONALD J T,et al. Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements [J]. Metabolic Engineering,2016,38: 370-381.
16	NIU T,LIU Y,LI J,et al. Engineering a glucosamine-6-phosphate responsive glmS ribozyme switch enables dynamic control of metabolic flux in Bacillus subtilis for overproduction of N-acetylglucosamine [J]. ACS Synthetic Biology,2018,7(10): 2423-2435.
17	DENG J,CHEN C,GU Y,et al. Creating an in vivo bifunctional gene expression circuit through an aptamer-based regulatory mechanism for dynamic metabolic engineering in Bacillus subtilis [J]. Metabolic Engineering,2019,55: 179-190.
18	WU Y,CHEN T,LIU Y,et al. Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis [J]. Nucleic Acids Research,2020,48(2): 996-1009.
19	CHEN J,FU G,GAI Y,et al. Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression [J]. Microbial Cell Factories,2015,14(1): 92.
20	FERREIRA L C S,FERREIRA R C C,SCHUMANN W. Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors [J]. Anais Da Academia Brasileira De Ciências,77(1): 113-124.
21	GUAN C,CUI W,CHENG J,et al. Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis [J]. Microbial Cell Factories,2016,15(1): 66.
22	CUI S,LV X,WU Y,et al. Engineering a bifunctional Phr60-Rap60-Spo0A quorum-sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis [J]. ACS Synthetic Biology,2019,8(8): 1826-1837.
23	YANG S,WANG Y,WEI C,et al. A new sRNA-mediated posttranscriptional regulation system for Bacillus subtilis [J]. Biotechnology and Bioengineering,2018,115(12): 2986-2995.
24	REU D R,RATH H,RMER A TH,et al. Changes of DNA topology affect the global transcription landscape and allow rapid growth of a Bacillus subtilis mutant lacking carbon catabolite repression [J]. Metabolic Engineering,2018,45: 171-179.
25	ARA K,OZAKI K,NAKAMURA K,et al. Bacillus minimum genome factory: effective utilization of microbial genome information [J]. Biotechnology and Applied Biochemistry,2007,46(3): 169-178.
26	WESTERS H,DORENBOS R,DIJL J M VAN,et al. Genome engineering reveals large dispensable regions in Bacillus subtilis [J]. Molecular Biology and Evolution,2003,20(12): 2076-2090.
27	TAKUYA M,RYOSUKE K,KEIJI E,et al. Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis [J]. DNA Research,2008,15(2): 73-81.
28	JAKOČIŪNAS T,JENSEN M K,KEASLING J D. CRISPR/Cas9 advances engineering of microbial cell factories [J]. Metabolic Engineering,2016,34: 44-59.
29	DOUDNA J A,CHARPENTIER E. The new frontier of genome engineering with CRISPR-Cas9 [J]. Science,2014,346(6213): 1258096.
30	BIKARD D,JIANG W,SAMAI P,et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system [J]. Nucleic Acids Research,2013,41(15): 7429-7437.
31	JEONG D E,PARK S H,PAN J G,et al. Genome engineering using a synthetic gene circuit in Bacillus subtilis [J]. Nucleic Acids Research,2014,43(6): e42.
32	ISAACS F J,DWYER D J,DING C,et al. Engineered riboregulators enable post-transcriptional control of gene expression [J]. Nature Biotechnology,2004,22(7): 841-847.
33	KIM J Y H,CHA H J. Down-regulation of acetate pathway through antisense strategy in Escherichia coli: improved foreign protein production [J]. Biotechnology and Bioengineering,2003,83(7): 841-853.
34	MAN S,CHENG R,MIAO C,et al. Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria [J]. Nucleic Acids Research,2011,39(8): e50.
35	DOMINGUEZ A A,LIM W A,QI L S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation [J]. Nature Reviews Molecular Cell Biology,2016,17(1): 5-15.
36	DONG C,FONTANA J,PATEL A,et al. Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria [J]. Nature Communications,2018,9(1): 2489.
37	ZHU H,LIANG C. CRISPR D T: designing gRNAs for the CRISPR-Cpf1 system with improved target efficiency and specificity [J]. Bioinformatics,2019,35(16): 2783-2789.
38	ZHANG X Z,CUI Z L,HONG Q,et al. High level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800 [J]. Applied and Environmental Microbiology,2005,71(7): 4101.
39	ZYPRIAN E V A,MATZURA H. Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system [J]. DNA,1986,5(3): 219-225.
40	YANG M,ZHANG W,JI S,et al. Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system [J]. PLoS One,2013,8(2): E56321.
41	PHAN T T P,NGUYEN H D,SCHUMANN W. Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements [J]. Journal of Biotechnology,2012,157(1): 167-172.
42	KIM L,MOGK A,SCHUMANN W. A xylose-inducible Bacillus subtilis integration vector and its application [J]. Gene,1996,181(1): 71-76.
43	YE R,KIM J H,KIM B G,et al. High-level secretory production of intact,biologically active staphylokinase from Bacillus subtilis [J]. Biotechnology and Bioengineering,1999,62(1): 87-96.
44	MORABBI H K,WENZEL M,ALTENBUCHNER J. Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors [J]. Microbial Cell Factories,2011,10(1): 83.
45	MING Y M,WEI Z W,LIN C Y,et al. Development of a Bacillus subtilis expression system using the improved P_glv promoter [J]. Microbial Cell Factories,2010,9(1): 55.
46	NAGARAJAN D R,KRISHNAN C. Use of a new catabolite repression resistant promoter isolated from Bacillus subtilis KCC103 for hyper-production of recombinant enzymes [J]. Protein Expression and Purification,2010,70(1): 122-128.
47	PANAHI R,VASHEGHANI-FARAHANI E,SHOJAOSADATI S A,et al. Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation [J]. Annals of Microbiology,2014,64(2): 879-882.
48	MITTENHUBER G. A phylogenomic study of the general stress response sigma factor sigmaB of Bacillus subtilis and its regulatory proteins [J]. J. Mol. Microbiol. Biotechnol.,2002,4(4): 427-452.
49	NIJLAND R,LINDNER C,HARTSKAMP M VAN,et al. Heterologous production and secretion of Clostridium perfringens β-toxoid in closely related Gram-positive hosts [J]. Journal of Biotechnology,2007,127(3): 361-372.
50	WILLENBACHER J,MOHR T,HENKEL M,et al. Substitution of the native srfA promoter by constitutive P_veg in two B. subtilis strains and evaluation of the effect on Surfactin production [J]. Journal of Biotechnology,2016,224: 14-17.
51	OGURA M,SHIMANE K,ASAI K,et al. Binding of response regulator DegU to the aprE promoter is inhibited by RapG,which is counteracted by extracellular PhrG in Bacillus subtilis [J]. Molecular Microbiology,2003,49: 1685-1697.
52	LEE S J,PAN J G,PARK S H,et al. Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis [J]. Journal of Biotechnology,2010,149(1): 16-20.
53	WENZEL M,LLER A,SIEMANN H M,et al. Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation [J]. Applied and Environmental Microbiology,2011,77(18): 6419.
54	YANG S,LIU Q,ZHANG Y,et al. Construction and characterization of broad-spectrum promoters for synthetic biology [J]. ACS Synthetic Biology,2018,7(1): 287-291.
55	SIU K H,CHEN R P,SUN Q,et al. Synthetic scaffolds for pathway enhancement [J]. Current Opinion in Biotechnology,2015,36: 98-106.
56	CHANDRASEKARAN A R. Programmable DNA scaffolds for spatially-ordered protein assembly [J]. Nanoscale,2016,8(8): 4436-4446.
57	PORTER E B,POLASKI J T,MORCK M M,et al. Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors [J]. Nature Chemical Biology,2017,13(3): 295-301.
58	CONRADO R J,WU G C,BOOCK J T,et al. DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency [J]. Nucleic Acids Research,2011,40(4): 1879-1889.
59	DELEBECQUE C J,SILVER P A,LINDNER A B. Designing and using RNA scaffolds to assemble proteins in vivo [J]. Nature Protocols,2012,7(10): 1797-1807.
60	KONERMANN S,BRIGHAM M D,TREVINO A E,et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex [J]. Nature,2015,517(7536): 583-588.
61	PFEIFFER V,PAPENFORT K,LUCCHINI S,et al. Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing downstream of translational initiation [J]. Nature Structural and Molecular Biology,2009,16(8): 840-846.
62	LEE M J,MANTELL J,HODGSON L,et al. Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm [J]. Nature Chemical Biology,2018,14(2): 142-147.
63	BRAMKAMP M,LOPEZ D. Exploring the existence of lipid rafts in bacteria [J]. Microbiology and Molecular Biology Reviews,2015,79(1): 81.
64	LV X,JIN K,WU Y,et al. Enzyme assembly guided by SPFH-induced functional inclusion bodies for enhanced cascade biocatalysis [J]. Biotechnology and Bioengineering, 2020. DOI: org/10. 1002/bit.27304. DOI URL
65	ANESIADIS N,KOBAYASHI H,CLUETT W R,et al. Analysis and design of a genetic circuit for dynamic metabolic engineering [J]. ACS Synthetic Biology,2013,2(8): 442-452.
66	ZHANG J,JENSEN M K,KEASLING J D. Development of biosensors and their application in metabolic engineering [J]. Current Opinion in Chemical Biology,2015,28:1-8.
67	MANDAL M,BREAKER R R. Gene regulation by riboswitches [J]. Nature Reviews Molecular Cell Biology,2004,5(6): 451-463.
68	BERENS C,GROHER F,SUESS B. RNA aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression [J]. Biotechnology Journal,2015,10(2): 246-257.
69	PEREZ J G,STARK J C,JEWETT M C. Cell-free synthetic biology: engineering beyond the cell [J]. Cold Spring Harbor Perspectives in Biology,2016,8(12): A023853.
70	HONG S H,KWON Y C,JEWETT M C. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis [J]. Frontiers in Chemistry,2014,2: A34.
71	SHRESTHA P,HOLLAND T M,BUNDY B C. Streamlined extract preparation for Escherichia coli based cell-free protein synthesis by sonication or bead vortex mixing [J]. BioTechniques,2012,53(3): 163-174.
72	KARIM A S,JEWETT M C. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery [J]. Metabolic Engineering,2016,36: 116-126.
73	NGUYEN H D,PHAN T T P,SCHUMANN W. Analysis and application of Bacillus subtilis sortases to anchor recombinant proteins on the cell wall [J]. AMB Express,2011,1(1): 22.
74	CASCHERA F,NOIREAUX V. A cost-effective polyphosphate-based metabolism fuels an all Escherichia coli cell-free expression system [J]. Metabolic Engineering,2015,27: 29-37.
75	SONG Y,NIKOLOFF J M,ZHANG D. Improving protein production on the level of regulation of both expression and secretion pathways in Bacillus subtilis [J]. Journal of Microbiology Biotechnology,2015,25(7): 963-977.
76	WATERS C M,BASSLER B L. Quorum sensing: Cell-to-cell communication in bacteria [J]. Annual Review of Cell and Developmental Biology,2005,21(1): 319-346.
77	MILLER M B,BASSLER B L. Quorum sensing in bacteria [J]. Annual Review of Microbiology,2001,55(1): 165-199.
78	LÓPEZ D,KOLTER R. Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis [J]. FEMS Microbiol Review,2010,34(2): 134-149.
79	SCHULTZ D,WOLYNES P G,JACOB E B,et al. Deciding fate in adverse times: sporulation and competence in Bacillus subtilis [J]. PNAS,2009,106(50): 21027-21034.
80	HAMOEN L W,VENEMA G,KUIPERS O P. Controlling competence in Bacillus subtilis: shared use of regulators [J]. Microbiology,2003,149(1): 9-17.
81	SHANK E A,KOLTER R. Extracellular signaling and multicellularity in Bacillus subtilis [J]. Current Opinion in Microbiology,2011,14(6): 741-747.
82	BOGUSLAWSKI K M,HILL P A,GRIFFITH K L. Novel mechanisms of controlling the activities of the transcription factors Spo0A and ComA by the plasmid-encoded quorum sensing regulators Rap60-Phr60 in Bacillus subtilis [J]. Molecular Microbiology,2015,96(2): 325-348.
83	DURAND S,JAHN N,CONDON C,et al. Type I toxin-antitoxin systems in Bacillus subtilis [J]. RNA Biology,2012,9(12): 1491-1497.
84	JAHN N,PREIS H,WIEDEMANN C,et al. BsrG/SR4 from Bacillus subtilis-the first temperature dependent type I toxin-antitoxin system [J]. Molecular Microbiology,2012,83(3): 579-598.
85	GÖRKE B,STÜLKE J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients [J]. Nature Reviews Microbiology,2008,6(8): 613-624.
86	SINGH K D,SCHMALISCH M H,STÜLKE J,et al. Carbon catabolite repression in Bacillus subtilis: quantitative analysis of repression exerted by different carbon sources [J]. Journal of Bacteriology,2008,190(21): 7275-7284.
87	应明，班睿. 枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响[J].微生物学报,2006，46（1）：23-27
	YING M,BAN R. Knockout of the ccpA gene in Bacillus subtilis and influence on riboflavin production [J]. Acta Microbiologica Sinica,2006,46(1): 23-27.
88	WANG Z,CHEN T,MA X,et al. Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum [J]. Bioresource Technology,2011,102(4): 3934-3940.
89	TANAKA K,TAKANAKA S,YOSHIDA K I. A second-generation Bacillus cell factory for rare inositol production [J]. Bioengineered,2014,5(5): 331-334.
90	JIN P,ZHANG L,YUAN P,et al. Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis [J]. Carbohydrate Polymers,2016,140: 424-432.
91	FENG Y,LIU S,JIAO Y,et al. Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy [J]. Applied Microbiology and Biotechnology,2017,101(4): 1509-1520.
92	LI Y,ZHU X,ZHANG X,et al. Characterization of genome-reduced Bacillus subtilis strains and their application for the production of guanosine and thymidine [J]. Microbial Cell Factories,2016,15(1): 94.
93	ZHOU K,ZOU R,ZHANG C,et al. Optimization of amorphadiene synthesis in Bacillus subtilis via transcriptional,translational,and media modulation [J]. Biotechnology and Bioengineering,2013,110(9): 2556-2561.
94	ZHANG X,ZHANG R,BAO T,et al. The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis [J]. Metabolic Engineering,2014,23: 34-41.
95	FU J,HUO G,FENG L,et al. Metabolic engineering of Bacillus subtilis for chiral pure meso-2,3-butanediol production [J]. Biotechnology for Biofuels,2016,9(1): 90.
96	QI H,LI S,ZHAO S,et al. Model-driven redox pathway manipulation for improved isobutanol production in Bacillus subtilis complemented with experimental validation and metabolic profiling analysis [J]. PLoS One,2014,9(4): e93815.
97	TOYA Y,HIRASAWA T,ISHIKAWA S,et al. Enhanced dipicolinic acid production during the stationary phase in Bacillus subtilis by blocking acetoin synthesis [J]. Bioscience,Biotechnology,and Biochemistry,2015,79(12): 2073-2080.
98	MU L,WEN J. Engineered Bacillus subtilis 168 produces L-malate by heterologous biosynthesis pathway construction and lactate dehydrogenase deletion [J]. World Journal of Microbiology and Biotechnology,2013,29(1): 33-41.
99	JIN P,KANG Z,YUAN P,et al. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168 [J]. Metabolic Engineering,2016,35: 21-30.
100	ZHANG K,DUAN X,WU J. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system [J]. Scientific Reports,2016,6(1): 27943.
101	VUOLANTO A,WEYMARN N VON,KEROVUO J,et al. Phytase production by high cell density culture of recombinant Bacillus subtilis [J]. Biotechnology Letters,2001,23(10): 761-766.
102	GU Y,LV X,LIU Y,et al. Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis [J]. Metabolic Engineering,2019,51: 59-69.
103	LIU Y,LIU L,SHIN H D,et al. Pathway engineering of Bacillus subtilis for microbial production of N-acetylglucosamine [J]. Metabolic Engineering,2013,19: 107-115.
104	LIU Y,LINK H,LIU L,et al. A dynamic pathway analysis approach reveals a limiting futile cycle in N-acetylglucosamine overproducing Bacillus subtilis [J]. Nature Communications,2016,7(1): 11933.
105	CUI S,XIA H,CHEN T,et al. Cell membrane and electron transfer engineering for improved synthesis of menaquinone-7 in Bacillus subtilis [J]. iScience,2020,23(3): 100918.
106	LIU S,HU W,WANG Z,et al. Production of riboflavin and related cofactors by biotechnological processes [J]. Microbial Cell Factories,2020,19(1): 31.
107	SHI S,SHEN Z,CHEN X,et al. Increased production of riboflavin by metabolic engineering of the purine pathway in Bacillus subtilis [J]. Biochemical Engineering Journal,2009,46(1): 28-33.
108	SHI T,WANG Y,WANG Z,et al. Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis [J]. Microbial Cell Factories,2014,13(1): 101.
109	SHI S,CHEN T,ZHANG Z,et al. Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production [J]. Metabolic Engineering,2009,11(4): 243-252.
110	LIU L,LIU Y,LI J,et al. Microbial production of hyaluronic acid: current state,challenges,and perspectives [J]. Microbial Cell Factories,2011,10(1): 99.
111	WESTERS L,WESTERS H,QUAX W J. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism [J]. Biochimica Biophysica Acta,1694(3): 299-310.

[1]	Meili SUN, Kaifeng WANG, Ran LU, Xiaojun JI. Rewiring and application of Yarrowia lipolytica chassis cell [J]. Synthetic Biology Journal, 2023, 4(4): 779-807.
[2]	Jicong LIN, Gen ZOU, Hongmin LIU, Yongjun WEI. Application of CRISPR/Cas genome editing technology in the synthesis of secondary metabolites of filamentous fungi [J]. Synthetic Biology Journal, 2023, 4(4): 738-755.
[3]	Ke LIU, Guihong LIN, Kun LIU, Wei ZHOU, Fengqing WANG, Dongzhi WEI. Mining, engineering and functional expansion of CRISPR/Cas systems [J]. Synthetic Biology Journal, 2023, 4(1): 47-66.
[4]	Liya LIANG, Rongming LIU. Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems [J]. Synthetic Biology Journal, 2023, 4(1): 86-101.
[5]	Jiaxin LIU, Chi CHENG, Xinqi LI, Chaojun WANG, Ying ZHANG, Chuang XUE. Recent progress in the molecular genetic modification tools of Clostridium [J]. Synthetic Biology Journal, 2022, 3(6): 1201-1217.
[6]	Qi LIU, Zhilan QIAN, Lili SONG, Chaoying YAO, Mingqiang XU, Yanna REN, Menghao CAI. Rewiring and application of Pichia pastoris chassis cell [J]. Synthetic Biology Journal, 2022, 3(6): 1150-1173.
[7]	Jiayu LIU, Zhihan YANG, Lei YANG, Liying ZHU, Zhengming ZHU, Ling JIANG. Advances in the development of Clostridium tyrobutyricum cell factories driven by synthetic biotechnology [J]. Synthetic Biology Journal, 2022, 3(6): 1174-1200.
[8]	Fei TAO, Tao SUN, Yu WANG, Ting WEI, Jun NI, Ping XU. Challenges and opportunities in the research of Synechococcus chassis under the context of carbon peak and neutrality [J]. Synthetic Biology Journal, 2022, 3(5): 932-952.
[9]	Runtao ZHU, Chao ZHONG, Zhuojun DAI. Biofilm matrixes-from soft matters to engineered materials [J]. Synthetic Biology Journal, 2022, 3(4): 626-637.
[10]	Fei SONG, Yuchen LIU, Zhiming CAI, Weiren HUANG. Construction of tumor gene circuits using CRISPR/Cas tool and their applications [J]. Synthetic Biology Journal, 2022, 3(1): 53-65.
[11]	Jiacheng BI, Zhigang TIAN. Synthetic immunology and future NK cell immunotherapy [J]. Synthetic Biology Journal, 2022, 3(1): 22-34.
[12]	Han XIAO, Yixin LIU. Progress and challenge of the CRISPR-Cas system in gene editing for filamentous fungi [J]. Synthetic Biology Journal, 2021, 2(2): 274-286.
[13]	Yang LI, Xiaolin SHEN, Xinxiao SUN, Qipeng YUAN, Yajun YAN, Jia WANG. Advances of CRISPR gene editing in microbial synthetic biology [J]. Synthetic Biology Journal, 2021, 2(1): 106-120.
[14]	Yongfu YANG, Binan GENG, Haoyue SONG, Qiaoning HE, Mingxiong HE, Jie BAO, Fengwu BAI, Shihui YANG. Progress and perspectives on developing Zymomonas mobilis as a chassis cell [J]. Synthetic Biology Journal, 2021, 2(1): 59-90.
[15]	Ke XU, Jingnan WANG, Chun LI. Intelligent microbial cell factory with tolerance for green biological manufacturing [J]. Synthetic Biology Journal, 2020, 1(4): 427-439.