Biocatalytic Production Of D-phenyllactic Acid Using Whole Cells Of Engineered E. Coli

Phenyllactic acid(PLA), which is a naturally organic acid widely exiting in lactic acid bacteria fermented food and honey. It has been obtaining increasing interest due to it has broad and effective antimicrobial activity against both bacteria and fungi and therefore can be used as a new type of antimicrobial preservative in food area s. Besides, it also plays an important role in pharmaceutical industry, animal husbandry and cosmetic industry.Biotechnological routes, which include enzymatic catalysis, microbial fermentation and whole cells biotransformation, have been developed for PLA production. Both enzymatic catalysis and microorganism fermentation strategies have some disadvantages, such as the former needs mild conditions and not stable in out surroundings, the latter needs longer fermentation period, insufficient of substrate usability and more difficult for product separation and purification. In this study, in order to obtain high yield of D-PLA with high optical purity, a whole cells biotransformation was developed by expression of lactate dehydrogenase and its variants in engineered E.coli, cofactor regeneration systems were also investigated through heterologous co-expression of an NAD+ or NADP+-dependent dehydrogenases. The main results were as follows:(1) The genomic DNA of Lactobacillus plantarum was obtained and used for D-ldh amplification. Purified PCR products were ligated into p ET-28 a, resulting in recombinant plasmid p ET-28a-ldh and then transformed into host strain. The expression of recombinant D-LDH proteins were induced by 1 mmol/L IPTG and checked using SDS-PAGE, which showed protein bands with a molecular mass of approximately 41 k Da, which indicated that D-ldh was successfully cloned and expressed in engineered E. coli. Compared with control, the recombinant strain remarkable increased lactate dehydrogenase activity and bioconversion performance. The specific activity of crude recombinant protein extracts for PPA was 6.3 U/mg, D-PLA production yield 5.01 g/L with 100% optical purity.(2) To enhance wide type lactate dehydrogenase catalytic activity and whole cells biotransformation, the Tyr52 residue of D-lactate dehydrogenase(D-LDH) was replaced with small hydrophobic residues(LDHY52L, LDHY52 A and LDHY52V) and overexpressed in E. coli BL21(DE3). Among the three mutants, compared to that of E. coli p ET-28a-ldh, E. coli p ET-28a-ldhY52 V showed the highest enzyme activity for PPA(17.8 times higher) and as a result, the highest PLA yield(improved by 21%). We further optimized biotransformation conditions in flasks, results showed that the optimized induction conditions for D-ldh expression were IPTG 0.2 mmol/L and inducing for 4 h at 25℃ when OD600=1.2, the optimized batch reaction conditions in phosphate buffer(p H 7.0) were: 8 g/L PPA, 10 g/L glucose, 1% Triton X-100 and 1 mmol/L Mg2+, the reaction was carried out in p H 7.0 phosphate buffer(0.1 M) at 37℃. Based on the above optimized conditions, fed-batch fermentation was conducted by intermittent feeding PPA and glucose. E. coli p ET-28a-ldh produced 12.16 g/L PLA in 3 h, with a molar conversion rate of 61%, while E. coli p ET-28a-ldhY52 V produced 15.56 g/L PLA, with a molar conversion rate of 77%. This study demonstrated the feasibility of using engineered E. coli for PLA production from PPA and showed that site-directed mutagenesis of D-ldh markedly improved PLA yield and substrate conversion rate.(3) In this chapter, cofactor engineering was applied to improve D-PLA production by overexpressing two cofactor regeneration enzymes, formate dehydrogenase(FDH) and glucose-6-phosphate dehydrogenase(ZWF), which were introduced into E. coli and co-expressed with D-ldh, respectively. Introduction of FDH results in higher D-PLA concentration from PPA, surprisingly, ZWF cofactor regeneration system didn’t improved products yield, on the contrary, only nearly 50% of the control. Thus, E. coli p ET-28a-ldhY52 V was chosen for further fed-batch bioconversion. Under optimized conditions with p H control, E. coli p ET-28a-ldhY52 V produced 18.15 g/L PLA, with productivity and molar conversion rate reached 4.54 g/L/h and 94%, respectively. This paper not only obtained a high level of D-PLA production with high conversion rate, but laid a foundation for valuable and optical purity compounds production using engineered E. coli.