1. Efficient synthesis of d-phenyllactic acid by a whole-cell biocatalyst co-expressing glucose dehydrogenase and a novel d-lactate dehydrogenase from Lactobacillus rossiae
Yongqian Fu, Yingying Zhang, Longfei Yin, Weilong Zheng, Xi Luo 3 Biotech . 2020 Jan;10(1):14. doi: 10.1007/s13205-019-2003-2.
d-Phenyllactic acid is a versatile natural organic acid, which is used as an antiseptic agent, monomer of the biodegradable material poly-phenyllactic acid and in the synthesis chiral intermediate of pharmaceuticals. In this report, the novel NADH-dependent d-lactate dehydrogenaseLrLDH was identified by screening a shotgun genome ofLactobacillus rossiae. To improve cofactor regeneration, theExiguobacterium sibiricumglucose dehydrogenaseEsGDH was overexpressed together withLrLDH inE. coliBL21(DE3)-pCDFDuet-1-gdh-ldh. The total enzyme activity in the fermentation broth ofE. coliBL 21(DE3)-pCDFDuet-1-gdh-ldhpeaked at 2359.0 U l-1when induced by 10 g l-1lactose at 28 °C and 150 rpm for 14 h. The biocatalytic reduction of sodium phenylpyruvate to d-phenyllactic acid was successfully carried out using whole cells of the engineeredE. coli. Under the optimized biocatalysis conditions, 50 g l-1sodium phenylpyruvate was completely converted to d-phenyllactic acid with a space-time yield and enantiomeric excess of 262.8 g l-1day-1and > 99.5%, respectively. To our best knowledge, it is the highest productivity reported to date, with great potential for the mass production of d-phenyllactic acid.
2. Characterization of D-lactate dehydrogenase producing D-3-phenyllactic acid from Pediococcus pentosaceus
Bo Jiang, Wanmeng Mu, Houyi Jiang, Shuhuai Yu Biosci Biotechnol Biochem . 2012;76(4):853-5. doi: 10.1271/bbb.110955.
D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s(-1), and 100 (mmol/L)(-1) s(-1) respectively.
3. Heterologous expression of a novel d‑lactate dehydrogenase from Lactobacillus sp. ZX1 and its application for d‑phenyllactic acid production
Jiangfeng Ma, Min Jiang, Weiliang Dong, Wenming Zhang, Jie Zhou, Fengxue Xin, Xinhai Zhou, Hao Wu Int J Biol Macromol . 2018 Nov;119:1171-1178. doi: 10.1016/j.ijbiomac.2018.08.036.
d‑Phenyllactic acid (d‑PLA) shows great potential for biopreservative production owning to its anti-microbial activity. In this study, strain ZX1, which could inhibit the growth of other microbes was isolated and identified as Lactobacillus genus. Strain ZX1 could produce d‑PLA with 0.16 g·L-1. Furthermore, a novel d‑lactate dehydrogenase gene was identified and expressed in Escherichia coli BL21 (DE3) with the specific activity of 71.64 U·mg-1protein under the optimal temperature and pH of 35 °C and 6.2. The kinetic constants for Kcat, Km, Vmaxwere 10.71 s-1, 2.356 mM and 11.27 μM·mg-1·min-1for phenylpyruvic acid (PPA), respectively. 18.21 g·L-1d‑PLA from PPA with yield of 90.49% and productivity of 2.49 g·L-1·h-1was obtained by the recombinant E. coli BL21 harboring d‑LDH, indicating that engineered E. coli BL21 (d‑LDH) has excellent potential in commercial d‑PLA production.
