阴沟肠杆菌乳酸脱氢酶基因缺失突变株的构建及其生物学特性

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

摘要/Abstract

摘要：

旨在应用自杀质粒重组技术,构建阴沟肠杆菌乳酸脱氢酶突变株,为进一步提高乙偶姻的产量和扩大菌株选择范围奠定基础。利用双酶切的方法将同源片段插入到自杀质粒pKR6K中,构建出ldh基因敲除质粒,然后利用细菌接合的方法敲除E. cloacae的ldh基因。成功克隆出两段E. cloacae乳酸脱氢酶基因的同源序列,长度分别为526 bp,通过序列比对分析,E. cloacae乳酸脱氢酶基因序列相似性为100%。通过对E. cloacae进行乳酸脱氢酶基因的敲除,成功构建一株ldh缺失重组菌株E. cloacae△ldh,同时2,3-丁二醇提高6.8%,乙酸提高了24.3%。E. cloacae乳酸脱氢酶缺失工程菌株构建成功,对利用微生物法工业化生产乙偶姻奠定基础。

关键词: 阴沟肠杆菌, 乳酸脱氢酶, 自杀质粒, 同源重组, 基因敲除

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

中图分类号:

S182

石会玲, 周宇航, 何平, 黄蒙蒙, 邵帅, 葛菁萍, 凌宏志. 阴沟肠杆菌乳酸脱氢酶基因缺失突变株的构建及其生物学特性[J]. 中国农学通报, 2021, 37(23): 29-37.

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.

图/表 14

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

表1. 引物序列

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	—

表2. ldh1和ldh2 PCR反应体系组分

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

表3. ldh1和ldh2 PCR反应程序

图1. 基因敲除质粒pKR6K-△L完整构建策略

图2. E. cloacae基因组DNA M:DNA Marker DL15000;1:E. cloacae基因组DNA

图3. ldh1和ldh2片段扩增结果 M:DNA Marker DL2000;1:以E. cloacae的基因组为模板扩增ldh1结果;2:以E. cloacae的基因组为模板扩增ldh2结果

图4. HindIII酶切结果 a:pT-ldh1酶切结果;b:pT-ldh2酶切结果;M:DNA Marker DL15000;1:阳性克隆子提取的原始质粒;2:酶切结果

图5. EcoRI和BamHI酶切结果 a:pT-ldh1酶切结果,M:DNA Marker DL15000,1:阳性克隆子提取的原始质粒pT-ldh1,2:pT-ldh1酶切结果;b:pKR6K酶切结果;M:DNA Marker DL15000,1:pKR6K酶切结果

图6. XbaI和SalI酶切结果 a:pT-ldh2酶切结果;M:DNA Marker DL15000;1:阳性克隆子提取的原始质粒pT-ldh1;3:pT-ldh2酶切结果;b:pKR6K-ldh1酶切结果（XbaI和SalI）;M:DNA Marker DL15000;1:阳性克隆子提取的原始质粒pKR6K-ldh1;2:pKR6K-ldh1酶切结果

图7. E. cloacae中ldh基因单交换菌株的筛选

图8. E. cloacae△ldh突变株筛选结果 M:DNA Marker DL2000;1-4:PCR扩增结果;5:以原始菌株为模板作对照

图9. E. cloacae与 E. cloacae△ldh的生长情况

图10. E. cloacae与E. cloacae△ldh的发酵产物及底物消耗情况结果 a:E. cloacae与E. cloacae△ldh的乙偶姻、2,3-丁二醇和葡萄糖浓度变化情况;b:E. cloacae与E. cloacae△ldh的乳酸和丁二酸浓度变化情况

产物浓度/(g/L)	菌株		变化情况
产物浓度/(g/L)	E. cloacae	E. cloacae△ldh	变化情况
乙偶姻	2.83±0.48a(48 h)	3.05±0.27a(48 h)	—
乳酸	2.85±0.21a(12 h)	0.01±0.01b(48h)	↓
2,3-BD	17.11±0.51b(12 h)	18.28±0.42a(12 h)	↑
丁二酸	2.08±0.24b(48 h)	2.46±0.10a(24 h)	↑
乙酸	2.92±0.20b(48 h)	3.63±0.31a(48 h)	↑
乙醇	2.81±0.11a(24 h)	3.17±0.31a(24 h)	—

表4. E. cloacae和E. cloacae△ldh的摇瓶发酵产物的比较

参考文献 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