Lactic Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae: Construction and Biological Characteristics

doi:10.11924/j.issn.1000-6850.casb2020-0837

Abstract

Abstract:

The aims are to construct a lactic dehydrogenase mutant of Enterobacter cloacae by suicide plasmid recombination technique, and lay a foundation for further increasing acetoin yield and expanding the range of strain selection. The homologous fragment was inserted into the suicide plasmid pKR6K by double restriction endonuclease digestion, and the ldh gene knockout plasmid was constructed. Then the ldh gene of E.cloacae was knocked out by bacterial conjugation. Two homologous sequences of E.cloacae lactate dehydrogenase gene were successfully cloned with a length of 526 bp. Sequence alignment analysis showed that the sequence similarity of E.cloacae lactate dehydrogenase gene was 100%. By knocking out the lactate dehydrogenase gene of E.cloacae, a ldh deletion recombinant strain E.cloacae△ldh, was successfully constructed, while 2,3-butanediol and acetic acid were increased by 6.8% and 24.3%, respectively. The E.cloacae lactic dehydrogenase deletion engineering strain was successfully created, which lays a foundation for the industrial production of acetoin by the microbial method.

Key words: Enterobacter cloacae, lactate dehydrogenase, suicide plasmid, homologous recombination, gene knockout

CLC Number:

S182

Shi Huiling, Zhou Yuhang, He Ping, Huang Mengmeng, Shao Shuai, Ge Jingping, Ling Hongzhi. Lactic Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae: Construction and Biological Characteristics[J]. Chinese Agricultural Science Bulletin, 2021, 37(23): 29-37.

Figures/Tables 14

References 28

[1]	Xiao Z, Lu J R. Generation of Acetoin and Its Derivatives in Foods[J]. Journal of Agricultural & Food Chemistry, 2014, 62(28):6487-97.
[2]	Xiao Z, Xu P. Acetoin metabolism in bacteria[J]. Critical Reviews in Microbiology, 2007, 33(2):127-140. doi: 10.1080/10408410701364604 URL
[3]	刘晓霏, 付晶, 霍广鑫, 等. 生物法制备平台化合物乙偶姻的最新研究进展[J]. 中国生物工程杂志, 2015, 35(10):91-99.
[4]	张小舟, 曾崇余, 任晓乾. 乙偶姻合成工艺[J]. 南京化工大学学报:自然科学版, 2001.
[5]	胡明一, 王中. 食用香料乙偶姻[J]. 精细与专用化学品, 2002, 10(1):20-21.
[6]	Odile M, M B, Pascal L, Patrick A D. GRIMONT. Taxonomic Diversity of the D-Glucose Oxidation Pathway in the Enterobacteriaceae[J]. International Journal of Systematic Bacteriology, 1989, 39(1):61-67. doi: 10.1099/00207713-39-1-61 URL
[7]	葛岚, 邵晓丛, 吴晓敏, 等. 工业化制备2,3-丁二醇的新途径[J]. 科技创新导报, 2009(33):106.
[8]	Choi E J, Kim J W, Kim S J, et al. Enhanced production of 2,3-butanediol in pyruvate decarboxylase-deficient Saccharomyces cerevisiae through optimizing ratio of glucose/galactose[J]. Biotechnol J, 2016, 11(11):1424-1432. doi: 10.1002/biot.v11.11 URL
[9]	Stefano R, Davide P, Giulio Z, et al. Effect of oxygen mass transfer rate on the production of 2,3-butanediol from glucose and agro-industrial byproducts by Bacillus licheniformis ATCC9789[J]. Biotechnology for Biofuels, 2018, 11(1).
[10]	Kim D K, Rathnasingh C, Song H, et al. Metabolic engineering of a novel Klebsiella oxytoca strain for enhanced 2,3-butanediol production[J]. Journal of Bioscience & Bioengineering, 2013, 116(2):186-192.
[11]	Birajdar S D, Rajagopalan S, Sawant J S, et al. Continuous predispersed solvent extraction process for the downstream separation of 2,3-butanediol from fermentation broth[J]. Separation & Purification Technology, 2015, 151:115-123.
[12]	Ji X J, Liu L G, Shen M Q, et al. Constructing a synthetic metabolic pathway inEscherichia colito produce the enantiomerically pure (R, R)-2,3-butanediol[J]. Biotechnology and Bioengineering, 2015, 112(5):1056-1059. doi: 10.1002/bit.v112.5 URL
[13]	Tong Y J, Ji X J, Shen M Q, et al. Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production[J]. Applied Microbiology & Biotechnology, 2016, 100(2).
[14]	Xu Y, Chu H, Gao C, et al. Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2,3-butanediol[J]. Metabolic Engineering, 2014, 23(5):22-33. doi: 10.1016/j.ymben.2014.02.004 URL
[15]	Yang Z, Zhang Z. Production of (2R,3R)-2,3-butanediol using engineered Pichia pastoris : strain construction, characterization and fermentation[J]. Biotechnology for Biofuels, 2018, 11(1):35. doi: 10.1186/s13068-018-1031-1 URL
[16]	Gao Y, Huang H, Chen S, et al. Production of optically pure 2,3-butanediol from Miscanthus floridulus hydrolysate using engineered Bacillus licheniformis strains[J]. World Journal of Microbiology & Biotechnology, 2018, 34(5):66. doi: 10.1007/s11274-018-2450-7 URL
[17]	王金星. B29菌株LPS合成基因缺失突变株的构建及分析[D]. 上海:上海交通大学, 2014.
[18]	He Y X, Hui X U, Fei Y E, et al. Constuction of suiside vector of aroA gene of Haemophilus parasuis[J]. Heilongjiang Animal Science and Veterinary Medicine, 2011(7):20-22.
[19]	于慧敏, 马玉超. 工业微生物代谢途径调控的基因敲除策略[J]. 生物工程学报, 2010, 26(9):1199-1208.
[20]	戴旭明, 薛红, 杨桦, 等. 基因打靶置换型载体的构建和应用研究[J]. 第二军医大学学报, 1998, 19(1):5-8.
[21]	王鸿姣. 基因敲除技术[J]. 农村科学实验, 2017(4).
[22]	Xiao Z J, Liu P H, Qin J Y, et al. Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate[J]. Applied Microbiology & Biotechnology, 2007, 74(1):61-68.
[23]	Bornstein N, Fleurette J. Acetoin Production in the Identification of Isolates as Members of Staphylococcus intermedius Hájek[J]. International Journal of Systematic Bacteriology, 1981, 31(3).
[24]	Zhang L, Liu Q, Ge Y, et al. Biotechnological production of acetoin, a bio-based platform chemical, from a lignocellulosic resource by metabolically engineered Enterobacter cloacae[J]. Green Chemistry, 2016, 18.
[25]	Hillman J D, Andrews S W, Dzuback A L. Acetoin production by wild-type strains and a lactate dehydrogenase-deficient mutant of Streptococcus mutans[J]. Infection & Immunity, 1987, 55(6):1399-1402.
[26]	Liu D, Chen Y, Ding F, et al. Simultaneous production of butanol and acetoin by metabolically engineered Clostridium acetobutylicum[J]. Metabolic Engineering, 2015. 27
[27]	饶志明, 包腾, 张显, 等. 加强表达枯草芽孢杆菌葡萄糖-6-磷酸脱氢酶提高乙偶姻产量[P]. 2015.
[28]	Xu Q M, Xie L X, Li Y Y, et al. Metabolic engineering of Escherichia coli for efficient production of (3R)-acetoin[EB/OJ]. Journal of Chemical Technology & Biotechnology, 2014.DOI 10.1002/jctb.4293. doi: 10.1002/jctb.4293

引物	序列5’-3’	酶切位点	用途
ldh1F	AATTxxxxxGAATTChhhhhACCGTGTTAAGTTCAAGCGCACCAA	EcoRI	克隆ldh基因上游片段526 bp
ldh1R	AATTxxxxxGAATTCGGATCChhhhhAAGACTTTCTCCAGTGATTTTACAT	EcoRI, BamHI	克隆ldh基因上游片段526 bp
ldh2F	AATTxxxxxTCTAGAhhhhhGCCGACATGCCGGGTGGCGGTTACG	XbaI	克隆ldh基因下游片段526 bp
ldh2R	AATTxxxxxGCATGCGTCGAChhhhhGGCGACGGTCATTATTTCGCAGGCG	SphI, SalI	克隆ldh基因下游片段526 bp
ldh-up	TTTTTGGCGCAACGGTTGACGGTGC	—	验证ldh基因敲除结果
ldh-down	ATGCGGGTCGCCGCCGCGCCTGCCA	—	验证ldh基因敲除结果
ldhF	CGGCTTAGACTATCTCGTTAGGACAC	—	克隆ldh基因
ldhR	GTCTTATGAAACTCGCGGTATATAGCAC	—	克隆ldh基因

引物	序列5’-3’	酶切位点	用途
ldh1F	AATTxxxxxGAATTChhhhhACCGTGTTAAGTTCAAGCGCACCAA	EcoRI	克隆ldh基因上游片段526 bp
ldh1R	AATTxxxxxGAATTCGGATCChhhhhAAGACTTTCTCCAGTGATTTTACAT	EcoRI, BamHI	克隆ldh基因上游片段526 bp
ldh2F	AATTxxxxxTCTAGAhhhhhGCCGACATGCCGGGTGGCGGTTACG	XbaI	克隆ldh基因下游片段526 bp
ldh2R	AATTxxxxxGCATGCGTCGAChhhhhGGCGACGGTCATTATTTCGCAGGCG	SphI, SalI	克隆ldh基因下游片段526 bp
ldh-up	TTTTTGGCGCAACGGTTGACGGTGC	—	验证ldh基因敲除结果
ldh-down	ATGCGGGTCGCCGCCGCGCCTGCCA	—	验证ldh基因敲除结果
ldhF	CGGCTTAGACTATCTCGTTAGGACAC	—	克隆ldh基因
ldhR	GTCTTATGAAACTCGCGGTATATAGCAC	—	克隆ldh基因

PCR反应体系组分	添加量/μL	终浓度
Template DNA	1	—
Forward primer (10 μmol/L)	1	0.2 μmol/L
Reverse primer (10 μmol/L)	1	0.2 μmol/L
TransStart® FastPfu DNA Polymerase	1	2.5 units
5× TransStart® FastPfu Buffer	10	1×
dNTPs (2.5 mmol/L)	4	0.2 mmol/L
ddH₂O	Up to 50	—

PCR反应体系组分	添加量/μL	终浓度
Template DNA	1	—
Forward primer (10 μmol/L)	1	0.2 μmol/L
Reverse primer (10 μmol/L)	1	0.2 μmol/L
TransStart® FastPfu DNA Polymerase	1	2.5 units
5× TransStart® FastPfu Buffer	10	1×
dNTPs (2.5 mmol/L)	4	0.2 mmol/L
ddH₂O	Up to 50	—

步骤	温度/℃	时间	循环数
预变性	95	2 min	1
变性	95	20 s	35
退火	55	20 s
延伸	72	15 s
终延伸	72	5 min	1