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Lactic Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae: Construction and Biological Characteristics
Shi Huiling, Zhou Yuhang, He Ping, Huang Mengmeng, Shao Shuai, Ge Jingping, Ling Hongzhi
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Lactic Dehydrogenase Gene Deletion Mutant of Enterobacter cloacae: Construction and Biological Characteristics
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.
Enterobacter cloacae / lactate dehydrogenase / suicide plasmid / homologous recombination / gene knockout {{custom_keyword}} /
表1 引物序列 |
| 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 | |
| ldh2F | AATTxxxxxTCTAGAhhhhhGCCGACATGCCGGGTGGCGGTTACG | XbaI | 克隆ldh基因下游片段526 bp |
| ldh2R | AATTxxxxxGCATGCGTCGAChhhhhGGCGACGGTCATTATTTCGCAGGCG | SphI, SalI | |
| ldh-up | TTTTTGGCGCAACGGTTGACGGTGC | — | 验证ldh基因敲除结果 |
| ldh-down | ATGCGGGTCGCCGCCGCGCCTGCCA | — | |
| ldhF | CGGCTTAGACTATCTCGTTAGGACAC | — | 克隆ldh基因 |
| ldhR | GTCTTATGAAACTCGCGGTATATAGCAC | — |
| 注:下划线部分为酶切位点位置。 |
表2 ldh1和ldh2 PCR反应体系组分 |
| 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3 ldh1和ldh2 PCR反应程序 |
| 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4 E. cloacae和E. cloacae△ldh的摇瓶发酵产物的比较 |
| 产物浓度/(g/L) | 菌株 | 变化情况 | |
|---|---|---|---|
| E. cloacae | E. cloacae△ldh | ||
| 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) | — |
| 注:括号中时间为相应产物最大产量时的时间;↑:提高;↓:下降;—:不变。 |
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PDF(2033 KB)
表1 引物序列表2 ldh1和ldh2 PCR反应体系组分表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图3 ldh1和ldh2片段扩增结果图4 HindIII酶切结果图5 EcoRI和BamHI酶切结果图6 XbaI和SalI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表4 E. cloacae和E. cloacae△ldh的摇瓶发酵产物的比较/
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