LAUSR

Abstract Biofilms are promising candidates for sustainable bioprocessing applications. This work presents a rational design of biofilm catalysts by integrating extra‐ and intracellular catalysis systems with optimized substrate channeling to realize efficient multistep biosynthesis. An assembly of four enzymes in a “three‐in‐one” structure was achieved by rationally placing the enzymes on curli nanofibers, the cell surface, and inside cells. The catalytic efficiency of the biofilm catalysts was over 2.8 folds higher than that of the control whole‐cell catalysis when the substrate benzaldehyde was fed at 100 mm. The highest yield of d‐phenyllactic acid catalyzed by biofilm catalysts under optimized conditions was 102.19 mm, also much higher than that of the control catalysis test (52.29 mm). The results demonstrate that engineered biofilms are greatly promising in integrating extra‐ and intracellular catalysis, illustrating great potentials of rational design in constructing biofilm catalysts as sustainable supports for whole‐cell catalysis.