Heterologous Lignan Production in Stirred-Tank Reactors-Metabolomics-Assisted Bioprocess Development for an In Vivo Enzyme Cascade

Towards establishing a prospective industrial microbial lignan production process, we set up and investigated the biotransformation of coniferyl alcohol to secoisolariciresinol with recombinant Escherichia coli in a stirred-tank reactor (STR). Initially, we tested different cofactor concentrations and antifoam additions in shake flasks. Next, we designed an STR batch bioprocess and tested aeration rates, pH regulation, and substrate-feeding strategies. Targeted metabolomics of phenylpropanoids and lignans assisted the bioprocess development by monitoring the lignan pathway activity. We found that the copper concentration and the substrate-feeding strategy had considerable impact on lignan production. Furthermore, time-resolved monitoring of pathway metabolites revealed two maximal intracellular lignan concentrations, the first shortly after induction of gene expression and the second after the cells entered the stationary growth phase. During STR cultivation, a maximal intracellular titer of 130.4 mg L-1 secoisolariciresinol was achieved, corresponding to a yield coefficient of 26.4 mg g(-1) and a space-time yield of 2.6 mg L-1 h(-1). We report for the first time the in-depth evaluation of microbially produced lignans in a well-controlled STR bioprocess. Monitoring of the lignan pathway activity showed that lignan accumulation is highly dynamic during the cultivation and points towards the need for a more efficient coniferyl alcohol dimerization system for optimal microbial production conditions.