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Abstract
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 E. coli 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-l3-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.
Highlights
Phenyllactic acid (PLA) widely exists in honey and fermented food and includes two enantiomers as D-phenyllactic acid (D-PLA) and L-phenyllactic acid (L-PLA) (Sorrentino et al, 2018; Luo et al, 2020a)
D-lactate dehydrogenase (D-lactate dehydrogenase (LDH)) was overexpressed to produce D-PLA from PPA in Leuconostoc mesenteroides ATCC 8293, and the results showed that growing cells produced 35 mM D-PLA with a yield of 75.2–83.3% (Li et al, 2014)
Fusion protein engineering of D-LDH and glycerol dehydrogenase (GlyDH) is conducted to aim for effective regeneration of coenzyme NADH for D-PLA biosynthesis
Summary
Phenyllactic acid (PLA) widely exists in honey and fermented food and includes two enantiomers as D-phenyllactic acid (D-PLA) and L-phenyllactic acid (L-PLA) (Sorrentino et al, 2018; Luo et al, 2020a). The whole-cell cascade catalysis using recombinant E. coli co-expression of glucose dehydrogenase (GDH)/formate dehydrogenase (FDH) and LDH has been widely used in the synthesis of PLA (Zhao et al, 2018; Rajanikar et al, 2021). Recombinant E. coli co-expressing LDH and GDH produced 17.25 g/L PLA from PPA with a productivity of 0.86 g/L/h (Zhu et al, 2017). Fusion protein engineering of D-LDH and GlyDH is conducted to aim for effective regeneration of coenzyme NADH for D-PLA biosynthesis.
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