Analysis of two-liquid-phase multistep biooxidation based on a process model: Indications for biological energy shortage

A process model for whole-cell biocatalysis in a two-liquid-phase system including cell growth and bioconversion kinetics was developed. The reaction considered is the kinetically controlled multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde catalyzed by recombinant Escherichia coli expressing the Pseudomonas putida genes encoding xylene monooxygenase (XMO). XMO catalyzes the successive oxygenation of one methyl group of xylenes to corresponding alcohols, aldehydes, and acids. The biocatalytic process includes cells growing in fed-batch mode and a two-liquid-phase system consisting of bis( 2-ethylhexyl) phthalate as organic carrier solvent and an aqueous minimal medium in a phase ratio of 1: 1. The process model comprises a description of the bioconversion kinetics, mass transfer kinetics, cell growth, and mass balances for both the aqueous and the organic phase. Bioconversion kinetics and consistent process simulation indicated the occurrence of direct substrate uptake from the organic phase and provided evidence for a pH-influenced competition for NADH between XMO and the respiratory chain with its consequential impact on bioconversion and cell growth. For the simulation of such differential NADH limitation, a pH-dependent feedback inhibition of the NADH consuming bioconversions was introduced as a modeling tool, which allowed good simulations of biotransformation experiments performed at varying pH, scale, and initial substrate concentration. Moreover, modeling indicated a product inhibition in the second oxidation step, which could be confirmed experimentally. A sensitivity analysis for the aqueous-organic mass transfer coefficient showed that this transfer is not critical for the process performance and emphasized the efficient substrate-cell transfer in the investigated two-liquid-phase process. Based on a process model, this study provides an in-depth analysis of a biooxidation process based on growing cells with indications for biological energy shortage as a limiting factor.