Research Progress in Molecular Genetic Manipulation Technology of Bacillus amyloliquefaciens and Its Application

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

Abstract

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

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.

Figures/Tables 6

References 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]