AUTHOR=Zhang Dong , Zhang Ting , Lei Yuqing , Lin Wenqian , Chen Xingyi , Wu Minchen TITLE=Enantioselective Biosynthesis of L-Phenyllactic Acid From Phenylpyruvic Acid In Vitro by L-Lactate Dehydrogenase Coupling With Glucose Dehydrogenase JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=10 YEAR=2022 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2022.846489 DOI=10.3389/fbioe.2022.846489 ISSN=2296-4185 ABSTRACT=

As a valuable versatile building block, L-phenyllactic acid (L-PLA) has numerous applications in the fields of agriculture, pharmaceuticals, and biodegradable plastics. However, both normally chemically synthesized and naturally occurring PLA are racemic, and the production titer of L-PLA is not satisfactory. To improve L-PLA production and reduce the high cost of NADH, an in vitro coenzyme regeneration system of NADH was achieved using the glucose dehydrogenase variant LsGDH^D255C and introduced into the L-PLA production process. Here an NADH-dependent L-lactate dehydrogenase-encoding variant gene (L-Lcldh1^Q88A/I229A) was expressed in Pichia pastoris GS115. The specific activity of L-LcLDH1^Q88A/I229A (Pp) was as high as 447.6 U/mg at the optimum temperature and pH of 40°C and 5.0, which was 38.26-fold higher than that of wild-type L-LcLDH1 (Pp). The catalytic efficiency (k_cat/K_m) of L-LcLDH1^Q88A/I229A (Pp) was 94.3 mM⁻¹ s⁻¹, which was 67.4- and 25.5-fold higher than that of L-LcLDH1(Pp) and L-LcLDH1^Q88A/I229A (Ec) expressed in Escherichia coli, respectively. Optimum reactions of L-PLA production by dual-enzyme catalysis were at 40°C and pH 5.0 with 10.0 U/ml L-LcLDH1^Q88A/I229A (Pp) and 4.0 U/ml LsGDH^D255C. Using 0.1 mM NAD⁺, 400 mM (65.66 g/L) phenylpyruvic acid was completely hydrolyzed by fed-batch process within 6 h, affording L-PLA with 90.0% yield and over 99.9% ee_p. This work would be a promising technical strategy for the preparation of L-PLA at an industrial scale.