Combinatorial protein engineering and transporter engineering for efficient synthesis of L-Carnosine in Escherichia coli

•A cell factory of L-Car was built with transporter engineering and protein engineering.•G148D/T168S mutant was first found to significantly increase L-Car yield by 41.6%.•Transporter protein YeaS was identified as involved in L-Car production.•By biocatalysis, the yield of L-Car was 133 mM, which was the highest reported. L-Carnosine has various physiological functions and is widely used in cosmetics, medicine, food additives, and other fields. However, the yield of L-Carnosine obtained by biological methods is far from the level of industrial production. Herein, a cell factory for efficient synthesis of L-Carnosine was constructed based on transporter engineering and protein engineering. Firstly, a dipeptidase (SmpepD) was screened from Serratia marcescens through genome mining to construct a cell factory for synthesizing L-Carnosine. Subsequently, through rationally designed SmPepD, a double mutant T168S/G148D increased the L-Carnosine yield by 41.6% was obtained. Then, yeaS, a gene encoding the exporter of L-histidine, was deleted to further increase the production of L-Carnosine. Finally, L-Carnosine was produced by one-pot biotransformation in a 5 L bioreactor under optimized conditions with a yield of 133.2 mM. This study represented the highest yield of L-Carnosine synthesized in microorganisms and provided a biosynthetic pathway for the industrial production of L-Carnosine.