Development of Biochar-Based Whole-Cell Biocatalysts for the Production of l-Tryptophan and l-Phenylalanine

Abstract

Microbial cell immobilization by adsorption is a simple and effective method to obtain sustainable whole-cell biocatalysts that provide unique advantages and are widely used for the biosynthesis of chemicals. Adsorption between cells and biochar allows direct diffusion of substrates and products but does not have high immobilization efficiency. In this study, a certain amount of negatively charged amino acids were displayed on the surface of engineered Escherichia coli cells using truncated ice-nucleation proteins (INPs) as anchoring motifs. An optimized FeCl3 solution modification was performed to obtain modified biochar MBC-Fe with a positively charged surface, facilitating enhanced adsorption between the cells and MBC-Fe. The strategy to enhance adsorption was applied to the previously developed E. coli strains for producing l-tryptophan and l-phenylalanine. Results showed that charged amino acids can be anchored on the cell surface by INaA, and the fusion proteins successfully provided corresponding charges to the cells. The constructed whole-cell biocatalysts Mp-trpAN45@MBC-Fe and Cc-pheAN45@MBC-Fe carrying the most negative charges yielded good results in terms of production of L-Trp and L-Phe and reusability in all five repeated batch fermentations. The novel strategy developed in this study was effective in reusability of whole-cell immobilization for improving the production of target compounds.