Knock-out Of L-lactate Dehydrogenase Gene From Bacillus Subtilis And Production Of Alanine

Bacillus subtilis is a Gram-positive bacterium which belongs to Bacillus with the ability of producing spores. Wild Bacillus subtilis doesn’t generate hazardous substances for human beings and contains no endotoxin on its cell wall, so Bacillus subtilis has been approved as a generally-recognized-as-safe (GRAS) microorganism by FDA. Bacillus subtilis can metabolize an extensive range of sugars, and efficiently synthesize a wide variety of proteinases and transport them out of the cell via its secretion systems. Now the applications of Bacillus subtilis mainly concentrates on the production of alpha-amylase and neutral protease. Alanine, a non-essential amino acid in the mammalian, belongs to non-polar aliphatic amino acid. Alanine plays an important role in the metabolism of organisms. In addition, alanine has great significances in the food industry and medicine. L-lactate dehydrogenase is an enzyme in a branch metabolism for alanine synthesis. This study aims to improve the yield of alamine by knocking-out L-lactate dehydrogenase gene (ldh) from Bacillus subtilis IBL23chromosome and to investigate the effect of ldh deletion on the yield of alanine. Besides, the effect of exogenous glucose on the yield of alanine was studied.Ldh gene was amplified by PCR from the genome of IBL23. The gene ldh was ligated to a pTG19-T vector and transformed to E.coli IBL15. Recombinants with plasmid pTG19-T-ldh werer selected by a blue-white screening method and then the recombinant plasmid pTG10-T-ldh was extracted. Cm gene was amplified by PCR from plasmid pKD3, digested with Nde I and EcoR V, and ligated to recombinant plasmid pTG19-T-ldh and transformed to E.coli IBL15. Recombinants with plasmid pTG19-T-ldh::cm were selected by plate method with chloramphenicol and the recombinant plasmid pTG19-T-ldh::cm was recoveried. The recombinant plasmid pTG19-T-ldh::cm was transformed to B. subtilis IBL23by the method of Anagnostopoulos and Spizizen for transformation. Finally, ldh gene deletion mutant IBL23△ldh was successfully obtained.The main morphological difference between strain IBL23△ldh and IBL23is that some of IBL23Aldh were elliptical, but most of IBL23were rod-shaped. In the caltivation process of72hours of these two strains in LB medium, the cell concentration of IBL23reached the highest with a value of OD6004.62±0.23at36h and had a decreased value being OD6003.70±0.03at48h, however, the cell concentration of IBL23△ldh reached the highest with a value of OD600is4.45±0.07at12h and had a decreased value being OD600is1.36±0.10at72h. The Final pHs of strains IBL23and IBL23△ldh were9.44±0.01and9.71±0.01, respectively. The yields of of strains IBL23and IBL23△ldh were0.087g/L and0.212g/L, a2.43times increase after deletion of ldh gene.In the studies of the effect of exogenous glucose on the yield of alanine, the yields of alanine by IBL23without and with glucose addation were respectively0.094g/L and0.143g/L, a1.5times increase after adding glucose.The yields of alanine by IBL23△ldh without and with glucose addation were respectively0.204g/L and0.674g/L, a3.3times increase after adding glucose.