Qin Zhao, Wang Dan, Luo Ruoshi, Li Tinglan, Xiong Xiaochao, Chen Peng
School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, China.
Department of Biological Systems Engineering, Washington State University, Pullman, WA, United States.
Front Bioeng Biotechnol. 2021 Dec 17;9:795885. doi: 10.3389/fbioe.2021.795885. eCollection 2021.
The biosynthetic production of D-penyllactic acid (D-PLA) is often affected by insufficient supply and regeneration of cofactors, leading to high production cost, and difficulty in industrialization. In this study, a D-lactate dehydrogenase (D-LDH) and glycerol dehydrogenase (GlyDH) co-expression system was constructed to achieve coenzyme NADH self-sufficiency and sustainable production of D-PLA. Using glycerol and sodium phenylpyruvate (PPA) as co-substrate, the BL21 (DE3) harboring a plasmid to co-express LfD-LDH and BmGlyDH produced 3.95 g/L D-PLA with a yield of 0.78 g/g PPA, similar to previous studies. Then, flexible linkers were used to construct fusion proteins composing of D-LDH and GlyDH. Under the optimal conditions, 5.87 g/L D-PLA was produced by expressing LfD-LDH-l-BmGlyDH with a yield of 0.97 g/g PPA, which was 59.3% increased compared to expression of LfD-LDH. In a scaled-up reaction, a productivity of 5.83 g/L/h was reached. In this study, improving the bio-catalytic efficiency by artificial redox self-equilibrium system with a bifunctional fusion protein could reduce the bio-production cost of D-PLA, making this bio-production of D-PLA a more promising industrial technology.
D-苯乳酸（D-PLA）的生物合成生产常常受到辅因子供应不足和再生的影响，导致生产成本高昂且难以实现工业化。在本研究中，构建了一种D-乳酸脱氢酶（D-LDH）和甘油脱氢酶（GlyDH）共表达系统，以实现辅酶NADH的自给自足以及D-PLA的可持续生产。以甘油和苯丙酮酸钠（PPA）作为共底物，携带共表达LfD-LDH和BmGlyDH质粒的BL21（DE3）产生了3.95 g/L的D-PLA，产率为0.78 g/g PPA，与先前的研究相似。然后，使用柔性接头构建由D-LDH和GlyDH组成的融合蛋白。在最佳条件下，通过表达LfD-LDH-l-BmGlyDH产生了5.87 g/L的D-PLA，产率为0.97 g/g PPA，与LfD-LDH的表达相比提高了59.3%。在放大反应中，生产率达到了5.83 g/L/h。在本研究中，通过具有双功能融合蛋白的人工氧化还原自平衡系统提高生物催化效率，可以降低D-PLA的生物生产成本，使D-PLA的这种生物生产成为一种更有前景的工业技术。
