Engineering of a bi-enzymatic reaction for efficient production of the ascorbic acid precursor 2-keto-l-gulonic acid

•Enzymatic production of the vitamin C precursor 2-keto-l-gulonate.•NADP(H) and glucono-1,5-lactone inhibit the reaction.•The bi-enzymatic process was optimized by mathematical modeling. The enzymatic production of the vitamin C precursor 2-keto-l-gulonate (2-KLG) from 2,5-diketo-d-gluconate (2,5-DKG) was catalyzed by coupling 2,5-diketo-d-gluconic acid reductase (2,5-DKG reductase) via its coenzyme to glucose dehydrogenase. This bi-enzymatic process shows complicated inhibition patterns caused by reaction products, NADP+ and NADPH. The bioconversion was optimized by modeling. Rate equations were derived for both enzymes and solved simultaneously by a numerical method to predict 2-KLG production. A series of batch conversions were used to verify the model and construct isocharts to determine optimal process parameters. The following key parameters for a fast and efficient conversion were found: (1) the NADP(H) concentration, (2) the volumetric activity of 2,5-DKG reductase, (3) the ratio of synthetic enzyme activity to regenerate enzyme activity and (4) the glucono-1,5-lactone concentration. By modeling the space-time yield of the process was nearly doubled and the coenzyme concentration reduced threefold.