Enhancing production of surface-displayed D-Hydantoinase and D-carbamoylase using lactose-induced co-culture for efficient biotransformation D, L-hydroxyphenylhydantoin into of D-p-hydroxyphenylglycine

D-p-hydroxyphenyl glycine (D-PHPG) is a D-amino acid that serves as an intermediate in the synthesis of semi-synthetic antibiotics such as amoxicillin. D-PHPG is synthesized from hydantoin derivatives through two sequential enzymatic reactions involving D-Hydantoinase (D-Hase) and D-Carbamoylase (D-Case). While whole-cell biocatalysis is a cost-effective method for producing D-PHPG, its efficiency is limited by transport issues, intracellular degradation, and poor substrate solubility. This study employed a bacterial surface display system to express D-Hase and D-Case in Escherichia coli for D-PHPG production. Enzyme expression was optimized in two stages: (1) optimizing co-culture media components using the Taguchi method, and (2) fine-tuning induction conditions with lactose as the inducer via response surface methodology. Results showed that adding glycerol (10 g/L) and yeast extract (20 g/L) under optimal induction conditions (8 mM lactose, induction at an optical density OD600 of 2.3, and 20 °C) achieved a maximum expression level of 19.75%, along with suitable cell growth (OD600 8.8). When scaled up to a stirred-tank bioreactor, cell densities reached OD600 of 25.2 before optimization and 28.4 after, corresponding to total enzyme expression levels of 32.42% and 28.62%, respectively. Enzymatic activities increased by 2.6-fold for D-Case and 2.9-fold for D-Hase. Using optimized co-culture fermentation and lactose induction, a conversion yield of 96% and a production yield of 91% for D-PHPG were achieved, making it one of the highest yields reported to date.