Study On Gene Inactivation Of Acetate Kinase And Dextransucrase In Leuconostoc

Leuconostoc is the predominant bacterial genus in fermented vegetables, including kimchi, sauerkraut and pickles. It is particularly regarded as good hosts for heterologous protein expression since they are generally recognized as safe (GRAS), do not produce endotoxin and secrete specific proteins such as dextransucrase without being digested by proteolytic enzymes. In the study, we inactivated acetate kinase gene and dextransucrase gene in Leuconostoc strains respectively. We compared physiological characterization of mutant strains with wild-type strain to study carbon metabolism and lay the foundation for genetic engineering breeding.Because of the poor reproducibility and the slightly low efficiency of transformation of Leuconostoc, we re-optimized the method for electrotransformation. Using plasmid pCW4as a vector and Leuconostoc as a host, we improved pH of PBS buffer, time of pre-treatment with lithium acetate (LiAc) and dithiothreitol (DTT), concentrations of lysozyme and sucrose in the LiAc-DTT to increase the transformation efficiency. To obtain a steady efficiency, precise inoculation amount and concentration of amp ic ill in were applied. An overnight culture of Leuconostoc was diluted with MRS to an optical density at600nm wavelength (OD600) of0.048and incubated until an OD600of0.5was reached. The cells were pre-treated with LiAc-DTT (0.5mol/L sucrose) supplemented with100U/mL lysozyme for20min. After pre-treatment, PBS of pH6.9was used to wash and resuspend the cells. Using this protocol, we achieved a higher transformation efficiency of2.47x105CFU/μg DNA.To reveal carbon and energy flux distributions in the metabolic network of L. mesenteroides CGMCC1.10327, we obstruct the formation of acetic acid by inactivating the ack gene encoding acetate kinase. Upsteam and downsteam fragments of ack gene are amplified by PCR A homologous recombination vector with the tetracycline marker flanked by the upsteam and downsteam fragments is constructed. Then the vector is transferred to L. mesenteroides and ack gene is inactivated by double-crossover gene replacement. Finally we detect the physiological characteristics of the mutant and the original strain. PCR analysis show that a fragment of ack gene in the mutant is1200bp longer than that in the original strain. Compared with the original strain, the yield of acetic acid in the mutant is decreased by49.1%, the yield of ethanol yield is increased by8.3%and the yield of diacetyl is dropped by16.3%.Leuconotoc mesenteroides, isolated from kimchi, is a group of hetero-fermentative lactic acid bacteria. They can produce mannitol which has various applications in the food and medical industries. Sucrose is utilized in the microorganism to produce mannitol and dextran. To improve mannitol yield, we hindered the dextran biosynthetic pathway by inactivating dts and studied effects on mannitol production. The gene encoding dextransucrase from L. mesenteroides CGMCC1.10327was cloned and sequenced. We constructed a homologous recombination vector by using the method of gene splicing by overlap extension. The vector was transferred to L. mesenteroides and the dextransucrase gene(dst) was inactivated by double-crossover gene replacement. A dst inactivated mutant strain without antibiotic resistant marker was obtained. Fermentation results showed that yield of dextran in the mutant was decreased by28.8%and mannitol yield was increased by15.3%.