| Phenylpyruvic acid（PPA）, a multi-functional organic acid, is widely used in the pharmaceutical, food, and chemical industries. The traditional chemical synthesis causes environmental pollution and microbial fermentation produces relatively small amounts of PPA. For biotransformation, D-amino acid oxidase（D-AAO） was currently used to produce PPA from D-phenylalanine. This reaction produces toxic hydrogen peroxide. On the contrary, L-amino acid deaminases（L-AADs） form Proteus. sp catalyze the stereospecific oxidative deamination of L-phenylalanine（PLA） to PPA and ammonia without the formation of hydrogen peroxide. Moreover, PLA is cheaper than D-phenylalanine. In this study, the membrane-bound L-AAD gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21（DE3）, and then the resulting recombinant E. coli whole-cell biocatalyst was used to produce PPA via biotransformation from PLA. Major results are listed below.（1） L-AAD gene from Proteus mirabilis KCTC 2566 was expressed in E. coli BL21（DE3） and then the induction conditions were optimized to improve the expression level. The purification steps（detergents, ultracentrifugation, and buffer） were optimized, and L-AAD was purified 52-fold with an overall yield of 13%, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA·min-1·mg-1. Then, the biotransformation conditions for the pure enzyme were optimized. The maximal production of enzymatic biotransformation was 2.6 ± 0.1 g·L-1(86.7 ± 5% mass conversion rate and 1.0 g·L-1·h-1 productivity) under the optimal conditions(0.2 mg·m L-1 L-AAD, 3.0 g·L-1 PLA, 5 mmol·L-1 FAD, pH 7.4, and 35°C).（2） A two-step whole-cell biotransformation, involving growing and resting cells, was established to produce PPA from PLA using the constructed E. coli. First, the biotransformation conditions for growing cells were optimized(PLA concentration 10.0 g·L-1, temperature 35°C) and a two-stage temperature control strategy（i.e., 20°C for 12 h and increased the temperature to 35°C until the end of biotransformation） was performed, resulting in 7.2 ± 0.4 g·L-1 of PPA production and 72.0% of conversion within 16 h. Then the biotransformation conditions for resting cells were optimized(4 g·L-1 PLA, 1.2 g·L-1 wholecell biocatalyst, pH 7.4, and 40°C), resulting in 3.3 ± 0.2 g·L-1 of PPA production and 82.5% of conversion within 6 h. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of production, conversion, productivity, stability, need of external cofactors, and recycling.（3） The biotransformation conditions for two-step bioconversion process were optimized in 3-L bioreactor. First, the optimal PLA concentration for growing cell biotransformation was 30.0 g·L-1 and the PPA production was 21.5 ± 1.3 g·L-1 within 6 h. The biotransformation conditions for resting cells were then optimized and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and PLA concentration 15 g·L-1, resulting in 100% of conversion withini 3 h. Effect of cell activity, PLA and PPA concentrations on whole-cell biotransformation in 3-L reactor was investigated, and then a kinetic model was established. The kinetic model was accurately predicted for the experimental results within this range of initial conditions.（4） The PPA titer was low due to the degradation of PPA and low substrate specificity of L-AAD. Metabolic engineering of the PLA degradation pathway in E. coli and protein engineering of L-AAD from P. mirabilis were performed to improve the PPA titer. First, three aminotransferase genes were knocked out to block PPA degradation, which increased the PPA titer of resting-cell biotransformation from 3.3 ± 0.2 g·L-1 to 3.9 ± 0.1 g·L-1 and the substrate conversion ratio to 97.5% in the flask. Next, L-AAD was engineered via error-prone polymerase chain reaction followed by site-saturation mutation to improve its catalytic performance. The triple mutant D165K/F263M/L336 M produced the highest PPA titer of 10.0 ± 0.4 g·L-1 of resting-cell biotransformation, with a substrate conversion ratio of 100%, which was 3.0 times that of wilD-type L-AAD. Comparative kinetics analysis showed that compared with wilD-type L-AAD, the triple mutant had higher substrate-binding affinity and catalytic efficiency. The PPA production of growing and resting cell biotransformation were 19.8 ± 2.3 g·L-1 and 10.0 ± 0.2 g·L-1, respectively, in the flask. The PPA production of growing and resting cell biotransformation were 45.1 ± 2.1 g·L-1 and 30.0 ± 1.2 g·L-1, respectively, in the 3-L bioreactor.（5） The synthesis and regeneration rate of cofactor FADH2/FAD in E. coli was strengthened by metabolic engineering to improve the PPA production. First, extra addition of FAD can significantly improve the PPA production, indicating that the supply of cofactor FAD is a limiting step for L-AAD catalysis. Then the key genes of FAD synthesis pathway（ribH, ribC, and ribF） were overexpressed and fine-tuned, leading to a 90% increase of PPA titer(19.4 ± 1.1 g·L-1) of resting-cell biotransformation. Next, a formate dehydrogenase and NADH oxidase were overexpressed to strengthen the regeneration rate of cofactors FADH2/FAD. The PPA production of growing and resting cell biotransformation were 57.1 ± 2.3 g·L-1 and 34.4 ± 1.1 g·L-1, respectively, in the flask. The PPA production of growing and resting cell biotransformation were 75.3 ± 4.3 g·L-1 and 58.4 ± 3.2 g·L-1, respectively, in the 3-L bioreactor. |
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