Abstract:Lactic acid bacteria （LAB）， as natural producers of the novel biopreservative phenyllactic acid（PLA）， are widely present in the nature. However， due to their thick cell walls and high mechanical strength，it is difficult to introduce exogenous genes into LAB. Compound mutagenesis was carried out for Pediococcus acidilactici GM， a strain screened out in the laboratory， for the efficient synthesis of PLA. The strain was first induced with atmospheric and room temperature plasma（ARTP） for 50 s and then with 0.8% diethyl sulfate（DES） for 25 min， and PLA was added to the culture medium to improve the strain resistance. A mutant strain with high vitality and a high yield of PLA was selected， and then shake flask fermentation and genetic stability validation were conducted. In view of the difficult introduction and transformation of exogenous DNA， the amino acids， buffer， lysozyme， bacterial growth status， and electroporation parameters were optimized by single factor experiments. The optimal electroporation conditions were obtained as follows. The bacterial cells were cultured in the MRS medium with 0.75% L-threonine to reach OD600 of 0.8， washed with washing buffer III （0.5 mol/L sucrose， 7 mmol/L Na2HPO4， 7 mmol/L NaH2PO4， and 20 mmol/L MgCl2）， and treated with lysozyme （30 mg/mL） at 37 ℃ for 30 min and then at the electric field intensity of 20 kV/cm. To further increase the production of PLA by LAB， genetic engineering was employed to replace the promoter P32 （low intensity）of the plasmid pMG36e with Pldh （high intensity） to construct a lactate dehydrogenase （LDH） overexpression plasmid， which was then transformed into P. acidilactici GM under the optimal electroporation conditions. RTReal-time quantitative polymerase chain reaction （RT-qPCR） results showed that the obtained mutant presented upregulated expression of the LDH gene ldh， and the yield of PLA was increased to 56.3 g/L and the conversion rate was increased to 94.8% after fermentation and culture in 5 L tanks with phenylpyruvic acid （PPA）.