Breeding Of Lactate Tolerant Lactobacillus Rhamnosus By Global Transcription Machinery Engineering

Lactic acid and its derivants were widely applicated in the fields of food,pharmaceutical, light industries, agriculture etc. Lactobacillus rhamnosus was an opticallypure lactic acid producing strain. It was attracting widespread interest as for the functionof adjusting the bacterial community in human intestine, prevention and treatment ofdiarrhea and prevention of decayed tooth. However, reports on the breeding of L.rhamnosus with genetic engineering modification were rare. So a stable genetictransformation system was necessary to establish. During the lactic acid fermentation,product inhibition was existed generally, which limited the development of lactic acidindustry.In this study, global transcription machinery engineering was utilized and thetranscriptional mutant library was constructed via error-prone PCR. After that, differentstress screening methods were adopted to separate the lactate tolerant L. rhamnosus.(1)The genetic engineering system of L. rhamnosus was constructed. In the plasmidextracting, the cell wall could be removed after30mg/ml lysozyme treatment for30minunder37℃. The electroporation condition was as follows: preliminary weakening with2%Gly, adding ampicillin when OD600value was between0.2and0.3to make the finalconcentration was12μg/mL, recollect the cells when OD600value was0.4to prepare thecompetent cells, the electroporation electric field intensity and time were8.5kv/cm and4ms respectively.(2) Three kinds of high throughput screening methods were adopted to obtain the lactatetolerant L. rhamnosus LR-LAS4, the the maximum biomass nearly doubled in comparisonof the wild type under high concentration of lactate.(3)Then the sequence analysis and3-D structure analysis of the modified rpoD wascarried out. There were two mutant points, they were A29S and S334R. It might lead aresult of affecting the formation of σ70factor’s3-D structure and the binding strength withthe-10element and-35element of the promoter. That might be the main reason for theproperty of lactate tolerance.