Breeding Of Lactic Acid Tolerant Strain By Global Transcription Machinery Engineering.

Lactic acid, an important natural organic acid, are widely used in food, medicine, brewing, textiles and other fields. What's more, L-lactic acid as the raw material of synthesing polylactic acid is known as the most promising biodegradable high molecular material. Presently, there are some diffculties in Lactic acid bacteria traditional breeding, such as longer time in screening cycle, difficulties in constructing multivalent vector and obtaining global optimal target phenotype which limits the development of Lactic acid bacteria's genetical breeding. Therefore, based on the level of global transcription, the study is described as follows: Firstly, complete expression sequences ofσfactor, the primary subunit of RNA polymerase(RANP), was constructed by fusion PCR; Secondly, then transcriptional mutant library was constructed by error-prone PCR; Lastly, destinated strains with significantly improved cell phenotype were selected.(1)Theσfactor and native promoter region was amplified from Lactobacillus rhamnosus; Fusion primers were designed to obtain the complete expression sequences ofσfactor by fusion PCR, and the EcoRI restriction site inσfactor was mutated in the premise of unchanging the open reading frame; Then the complete expression sequence ofσfactor was subjected to error-prone PCR and inserted into the plasmid pMG36e which is the Lactic acid bacteria constitutive expression vector, and then transformed into Escherichia coli DH5αcompetent cells to obtain the mutant library.(2)It showed that Lactobacillus amylovorus B0112 was the best recipient strain; Highest electrotransformation efficiency of 10⁴ transformants could be obtained when Lactobacillus amylovorus B0112 was pretreated under 5% of glycine/0.5 mol/L sucrose, and washed three times with electrotransformation buffer of EB2, as well as the 1.5 kV of field strength and one time of pulse with the OD₆₀₀ nm value of 0.6～0.65.(3)LA5 strain was obtained under the pressure of Lactic acid, of which the growth rate faster than control 1 and control 2 strains. It is speculated that substitution of amino acids and the lost of 27 amino acids ofσfactor in C terminal led to a change in spatial structure, and resulted in a certain regulatory effect.