Study On Gene Inactivation Of Acetaldehyde Dehydrogenase In Leuconostoc

Leuconostoc strains are Gram-positive, facultatively anaerobic, heteromorphosis lacticacid bacteria (LAB). Leuconostoc spp. is generally regarded as safe (GRAS) organisms bythe U.S. FDA. Leuconostoc strains own the ability to metabolize dextran, mannitol, diacetyland other substances, thereby, they are widely used in the fields of functional foods anddairy products. This subject specificlly inactivated the gene of ALDH in Leuconostocbacteria to obtain the mutant strain and compared the physiological and biochemicalcharacteristics between the mutant strain and the original strain, laid the foundation forgenetic engineering and breeding.The electrotransformation efficiency directly affects the probability of homologousrecombination. This study optimized the electrotransformation conditions of Leuconostocfrom some factors. Our result indicated that using MRS medium with0.7μg/mL ampicillinto culture leuconastoc till OD600up to appromix0.5and harvest competent cells by usingPBS buffer, then mixing the competent cells and10μL DNA to electrotransform under theconduction which is12KV/cm electric field strength,200Ω resistance and25μFcapacitance, after adding MRS culture medium containing80mmol/L MgCl2to develop for3h, we got the highest transformation efficiency which is approach1.75×105CFU/μg DNA.As for gene inactivation technology, the construction of homologous recombinationvector is important. In this study, the erythromycin resistance was used as a selectionmarker, its gene was connected to pTA2to construct the carrier which namedpTA2-ErmAM, then the right and left arm of ALDH gene were connected to the left andright side of pTA2-ErmAM respectively. After that, the homologous recombination vectorpTA2-ErmAM-Aldh was obtained.After electrotransformation, strains were coated on a flat plate containingerythromycin and ampicillin respectively, only erythromycin-resistant strains were obtained.Further amplified the homologous recombination gene, we got the mutationstrain-LeuALDH-. Then compared the original strain and mutant strains, the result showedthat, the mannitol and diacetyl production of mutant strain were improved significantly.