Production Of Optical Pure L-phenyllactic Acid By Whole Cells Of Recombinant Escherichia Coli Strains

L-phenyllactic acid(L-PLA) is a naturally low-molecular-weight organic acid with important physiological functions and pharmacological properties. As a chiral intermediate, it has been widely used for the synthesis of various valuable pharmaceuticals and biorefinery chemicals. Recently, L-PLA has been identified as a novel antimicrobial compound with broad-spectrum and effective inhibitory activity against a variety of food spoilage bacteria and fungi. Therefore, it also has wide application perspective and practicability in food and feed industries.In this study, in order to obtain high yield of L-PLA with high optical purity, a whole-cell biotransformation from PPA was developed by overexpressing L-lactate dehydrogenase in engineered E.coli. NADH cofactor regeneration system was also investigated through heterologous coexpression of glucose dehydrogenase and L-lactate dehydrogenase. The main results were as follows:E.coli BL21(DE3)/pET28a-Lpldh and E.coli BL21(DE3)/pET28a-Bmldh were constructed to overexpress L-lactate dehydrogenase Lpldh from Lactobacillus plantarum subsp. plantarum ATCC14917 and Bmldh from Bacillus megaterium Z2013513, respectively. SDS-PAGE showed protein bands with a molecular mass of approximately 40 kDa for both LpLDH and BmLDH. In crude recombinant protein extracts, the specific activity of of LpLDH and BmLDH for PPA was 154.02 U/mg and 3.40 U/mg, respectively. Whole cells of E. coli BL21(DE3)/pET28a-Lpldh have much higher conversion ability than that of E.coli BL21(DE3)/pET28a-Bmldh. During 150 min fed-batch biotransformation by E. coli BL21(DE3)/pET28a-Lpldh whole cells, 77.26 mM L-PLA was accumulated with a productivity of 30.90 mM/h and a high optical purity of 97%, suggesting that recombinant E. coli overexpressing Lpldh possesses high stereoselectivity and delivers a high yield of L-PLA.E. coli BL21(DE3)/pETDuet-Lpldh-gdh was constructed to coexpress L-lactate dehydrogenase gene Lpldh from Lactobacillus plantarum subsp. plantarum ATCC14917 and glucose dehydrogenase gene gdh from Bacillus megaterium Z2013513. SDS-PAGE showed protein bands with the molecular masses of approximately 40 kDa and 27 kDa for L-lactate dehydrogenase LpLDH and glucose dehydrogenase GDH, respectively. In crude recombinant protein extracts, the specific activity of of LpLDH for PPA was 9.48 U/mg, and the specific activity of of GDH for glucose was 5.37 U/mg. L-PLA yield produced by E. coli BL21(DE3)/pETDuet-Lpldh-gdh was 16.79% higher than E. coli BL21(DE3)/pETDuet-Lpldh, and PPA molar conversion ratio was up to 70.08%, suggesting the enhancement of L-PLA production by a cofactor regeneration system.Based on optimized conditions of whole-cell biotransformation, 108.51 mM L-PLA was accumulated with a productivity of 72.34 mM/h and a very high optical purity(>99%) after 90 min fed-batch by E. coli BL21(DE3)/pETDuet-Lpldh-gdh, suggesting that the cofactor regeneration system contributed to a rise of both yield and enantiomeric excess. The strain coexpressing L-lactate dehydrogenase and glucose dehydrogenase obtained a high level of L-PLA production with a high conversion rate, which might be a promising candidate for use in large-scale production facilities and in fermented foods.