l-Phenyllactic acid (l-PLA) is a small molecular organic acid that exhibits a powerful capacity for inhibition against foodborne pathogens. In this work, we developed a new cost-effective and environmentally friendly process for the biosynthesis of l-PLA. This strategy designed a novel whole-cell biotransformation system employing two heterologous enzymes, namely, phenylalanine dehydrogenase (PheDH) and l-hydroxyisocaproate dehydrogenase (l-HicDH). The novelty of this strategy lies in the first-time utilization of these two enzymes, which not only enables cascade catalysis for the production of l-PLA but also facilitates the regeneration of the coenzymes (NAD + /NADH) using only two enzymes rather than introducing more heterologous enzymes to the system. Consequently, this strategy can effectively simplify the biosynthesis process of l-PLA and minimize production costs. The initial l-PLA yield using this process achieved 2.53 ± 0.07 g/L. Furthermore, through meticulous optimization of the parameters for inducible enzyme expression and l-PLA biosynthesis, the l-PLA yield was successfully increased to 4.68 ± 0.04 g/L with a yield rate of 64.54 ± 0.29%. Moreover, this novel strategy is versatile in the biosynthesis of other organic acids, which can be achieved by easily modulating the combinations of substrates and enzymes.
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