解淀粉芽孢杆菌分子遗传操作及其应用研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2021-0447

摘要/Abstract

摘要：

解淀粉芽孢杆菌是FDA认定的安全级（generally recognized as safe,GRAS）菌株,在工业酶制剂、高分子聚合物、大宗化学品、绿色生物农药等方面的生产具有突出的优势。近年来,随着解淀粉芽孢杆菌的分子遗传操作技术越来越成熟,对利用该菌开发成微生物发酵平台化菌株用于合成生物学制造领域提出了更迫切的需求。文中围绕解淀粉芽孢杆菌的遗传操作工具、代谢改造应用和未来发展前景等方面进行了详细综述,为进一步推动解淀粉芽孢杆菌合成生物学技术的创新与发展提供借鉴和参考。

关键词: 解淀粉芽孢杆菌, 底盘细胞, 分子遗传操作, 遗传改造, 代谢工程

Abstract:

Bacillus amyloliquefaciens is a Generally Recognized as Safe（GRAS）strain recognized by the FDA. It has outstanding advantages in the production of industrial enzyme preparations,high molecular polymers,bulk chemicals,and green biological pesticides. In recent years,as the molecular genetic manipulation technology of B. amyloliquefaciens has become more and more mature,there has been a more urgent need for the use of the bacteria to develop microbial fermentation platform strains for use in the field of synthetic biology manufacturing. In this paper,the genetic manipulation tools,metabolic modification applications and future development prospects of B. amyloliquefaciens are reviewed in detail,which will provide reference for further promoting the innovation and development of B. amyloliquefaciens on synthetic biology technology.

Key words: Bacillus amyloliquefaciens, chassis cells, molecular genetic manipulation, genetic modification, metabolic engineering

邱益彬, 马艳琴, 沙媛媛, 朱逸凡, 苏二正, 雷鹏, 李莎, 徐虹. 解淀粉芽孢杆菌分子遗传操作及其应用研究进展[J]. 生物技术通报, 2022, 38(2): 205-217.

QIU Yi-bin, MA Yan-qin, SHA Yuan-yuan, ZHU Yi-fan, SU Er-zheng, LEI Peng, LI Sha, XU Hong. Research Progress in Molecular Genetic Manipulation Technology of Bacillus amyloliquefaciens and Its Application[J]. Biotechnology Bulletin, 2022, 38(2): 205-217.

图/表 6

参考文献 62

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Plasmid	Promoter	Terminator	Replicon	Reference
pUBXC	PxylA	t0	repB	[2]
pDR	PHpaII	Tfd	repF	[3]
pMA5	PHpaII	Tfd	repB	[3]
pNX01	P43	Tamy	Ori-rep（p2Sip）	[4]
pWH1520	PxylA		pBC16 ori	[5]
pKSV7	P43		Temperature-sensitive replication from pE194ts	[5]
pNW33N	ctaB、qcr、qox、resD promoter		repB	[6]
pLY-3	Promoter from amyE of B. subtilis 168			[7]
pLakr	PQ			[8]
pHT01	Pgrac		Theta replicon	[9]

Plasmid	Promoter	Terminator	Replicon	Reference
pUBXC	PxylA	t0	repB	[2]
pDR	PHpaII	Tfd	repF	[3]
pMA5	PHpaII	Tfd	repB	[3]
pNX01	P43	Tamy	Ori-rep（p2Sip）	[4]
pWH1520	PxylA		pBC16 ori	[5]
pKSV7	P43		Temperature-sensitive replication from pE194ts	[5]
pNW33N	ctaB、qcr、qox、resD promoter		repB	[6]
pLY-3	Promoter from amyE of B. subtilis 168			[7]
pLakr	PQ			[8]
pHT01	Pgrac		Theta replicon	[9]

Strain	Transformation method	Concrete operation	Transformation plasmid	Transformation efficiency /（CFU·μg^-1）
B. amyloliquefaciens TA20^8[22]	Electroproration	Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium	pUB110	（1.13 ± 0.34）×10⁷
B. amyloliquefaciens TA20^8[22]	Electroproration	Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium;heat shock treatment	pHCMC02	（8.94 ± 0.77）×10⁵
B. amyloliquefaciens NB^[3]	Electroproration	Demethylation	pDR	4.94 ± 0.42）×10⁴
B. amyloliquefaciens NB^[3]	Electroproration	Demethylation	pMA5	（6.15 ± 0.19）×10³
B. amyloliquefaciens LL3^[5]	Electroproration	Demethylation	pWH1520	7.6×10²
B. amyloliquefaciens^[2]	Chemical transformation	Overexpressing ComK regulator to induce the formation of competent	pUBXC	[（129 ± 20.6）- （1.7 ± 0.1）]×10⁵
B. amyloliquefaciens^[2]	Chemical transformation		PCR fragment-mediated knockout	[（3.2 ± 0.76）- （3.5 ± 0.42）]×10⁴
B. amyloliquefaciens^[23]	Transformation of protoplasts	Mediating by polyethylene glycol	pUB110	（2-4）×10⁵

Strain	Transformation method	Concrete operation	Transformation plasmid	Transformation efficiency /（CFU·μg^-1）
B. amyloliquefaciens TA20^8[22]	Electroproration	Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium	pUB110	（1.13 ± 0.34）×10⁷
B. amyloliquefaciens TA20^8[22]	Electroproration	Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium;heat shock treatment	pHCMC02	（8.94 ± 0.77）×10⁵
B. amyloliquefaciens NB^[3]	Electroproration	Demethylation	pDR	4.94 ± 0.42）×10⁴
B. amyloliquefaciens NB^[3]	Electroproration	Demethylation	pMA5	（6.15 ± 0.19）×10³
B. amyloliquefaciens LL3^[5]	Electroproration	Demethylation	pWH1520	7.6×10²
B. amyloliquefaciens^[2]	Chemical transformation	Overexpressing ComK regulator to induce the formation of competent	pUBXC	[（129 ± 20.6）- （1.7 ± 0.1）]×10⁵
B. amyloliquefaciens^[2]	Chemical transformation		PCR fragment-mediated knockout	[（3.2 ± 0.76）- （3.5 ± 0.42）]×10⁴
B. amyloliquefaciens^[23]	Transformation of protoplasts	Mediating by polyethylene glycol	pUB110	（2-4）×10⁵

Products		Strategies	Yield	Reference
γ-PGA		Knock out genes involved in by-products,degrading enzymes and optimize the endogenous glutamate synthesis	20.3 g/L	[60]
		Modification of substrate inulin utilization,sugar metabolism and by-product pathways	32.14 g/L	[13]
		Using the CRISPRi system’s multiple sgRNA combination strategy to control the expression of degrading enzymes,that achieve multiple molecular weights of γ-PGA	Different molecular weights of γ-PGA High（>800 kD）、middle（400-600 kD）、low（50-100 kD）;25-27 g/L	[21]
		To control the stereochemical configuration	Low-molecular-weight（<10 kD）γ-PGA;28.35 g/L	[34]
EPS		Optimization of fermentation conditions	4.46 g/L	[61]
Levan		Removing six protease-related genes,the biofilm matrix protein TasA and γ-PGA synthase genes	31.1 g/L	[40]
Levan		Optimization of levansucrase expression based on promoters and signal peptides	102 g/L	[9]
Enzymes production	α-amylase	Optimization of the promoters and host	2714 U/mL	[26]
	Pullulanase	Cloning and expression optimization	2.8 ASPU/mL	[27]
	Keratinase	Cloning and expression optimization	1 361.54 U/mL	[28]
	lytic polysaccharide monooxygenase	Establishing a high-throughput screening system for expression and secretion	12.17 U/g	[7]
Nucleotides	Guanosine	Relieving the purine operon of the purine biosynthetic pathway and optimizing the energy of the respiratory chain	19 g/L	[44]
Nucleotides	Inosine	Protoplast fusion	6 g/L	[46]
Antimicrobial lipopeptides	Surfactin	Using nano iron particles to improve the permeability of cell membranes	7.15 g/L	[49]
	Iturin A	Overexpression of the iturin A biosynthesis genes	37.35 mg/L	[50]
	Fengycin	Genome shuffling	450.51 mg/L	[52]
	Bacillomycin D	Knocking out of the gene rapC	（360.8±30.7）mg/L	[62]
Bulk chemicals	Acetoin	Compound mutagenesis	85.2 g/L	[57]
Bulk chemicals	2, 3-butanediol	Overexpression of glyceraldehyde-3-phosphate dehydrogenase and 2, 3-butanediol dehydrogenase	132.9 g/L	[59]