Production Enhancement of Surface-Expressed D-hydantoinase and D-carbamoylase in E. Coli by Developing a Novel Coculture Process for the Efficient Biotransformation of D-p-hydroxyphenylglycine.

D-p-hydroxyphenyl glycine (D-PHPG) is a D-amino acid used as an intermediate in the synthesis of semi-synthetic antibiotics. It is synthesized from hydantoin derivatives through two sequential enzymatic reactions involving D-hydantoinase (D-hase) and D-carbamoylase (D-case). Although whole-cell biocatalysis of D-PHPG is cost-effective, its efficiency suffers from transport obstacles, intracellular degradation, and limited substrate solubility. This study utilized a bacterial surface display system to express D-hase and D-case in Escherichia coli for D-PHPG production. Enzyme production optimization was carried out in two stages. Initially, key factors influencing cell density during co-culture were identified through culture media and fermentation parameters screening using the Plackett-Burman design, followed by optimization with the D-optimal method. Next, induction parameters were fine-tuned using response surface methodology. The optimal culture medium was found to contain glycerol (12 g/L) and yeast extract (15 g/L) under optimal induction conditions (0.17 mM IPTG, OD600 of 1.3, and 21 °C). These conditions achieved an OD600 of 15.6, with expression levels of 20.18% for D-case and 20.82% for D-hase. Scaling up in a stirred tank bioreactor resulted in an OD600 of 32.15, with D-hase and D-case expression levels increasing to 25.8 and 24.2%, respectively, and enzymatic activities improving by 2.83 times for D-case and 3.42 times for D-hase. The optimized co-culture approach under optimized induction conditions achieved a conversion yield of 95% and a D-PHPG production yield of 90%. The study results showed that the suggested fermentation conditions will contribute to future scale-up studies aimed at improving enzyme activities for other surface protein production.