ldhL-ldb0094基因敲除对保加利亚乳杆菌产L-乳酸的影响

Effect of ldhL Gene Knock out Mutant on Lactobacillus delbrueckii subsp.blgaricus Producing L-lactic Acid

以保加利亚乳杆菌Lactobacillus delbrueckii subsp. bulgaricus CICC21101为出发菌株,利用PCR扩增L-乳酸脱氢酶(ldhL)基因上下游序列ldhL1、ldhL2,获得ldhL基因缺失且包含上下游序列的片段,连接到乳酸菌专用温敏性基因敲除质粒pGhost4,将构建好的敲除载体电转入保加利亚乳杆菌CICC21101,低温筛选。结果表明,成功获得敲除ldhL基因的敲除突变株,敲除后的工程菌D-乳酸产量由30. 5g/L降为4. 8g/L,L-乳酸的产量由25. 4g/L增至58. 3g/L,光学纯度由54. 56%增至90%。同时发现ldhL-ldb0094基因的敲除致使ldhL-ldb1020表达的上调,D-乳酸脱氢酶(ldbD)基因表达量没有变化,ldhL基因敲除株的成功构建将为进一步研究该基因在保加利亚乳杆菌中的功能及后续高光学活性D-乳酸工程菌构建奠定基础。

To construct ldhL gene deletion strain in Lactobacillus delbrueckii subsp. bulgaricus. Bulgaria lactobacillus CICC21101 was employed as the original strain,amplified the upstream sequence ldhL1 and downstream sequence ldhL2 by PCR,obtained successfully ldhL gene deletion fragment which includes both upstream and downstream sequence,then connected to knockout plasmid pGhost4,electricity to Bulgaria lactobacillus CICC21101,screening at low temperature. The results indicated that the ldhL gene knocked out deficient mutant was obtained. By gene knockout,the yield of D-lactic acid decreased from 30. 5 g/L to 4. 8 g/L,the yield of L-lactic acid increased from 25. 4 g/L to 58. 3 g/L,and the optical purity increased from 54. 56% to90%. It was a foundation to study the detail functions of ldh in Lactobacillus delbrueckii subsp. bulgaricus,it will lay the foundation of constuction on D-lactic acid engineering bacteria.