Abstract: An efficient biosynthetic pathway in Escherichia coli MG1655 ΔlacI for the production of sarcosine was constructed in this study. First, the β-piperidone-2-carboxylic acid reductase gene (dpkA) from Pseudomonas putida was overexpressed via a plasmid. This enzyme is capable of catalyzing the synthesis of sarcosine from glyoxylate and methylamine. The optimal catalytic temperature, pH, and cell concentration were determined through single-factor experiments. Subsequently, to enable the engineered strain to accumulate glyoxylate through endogenous metabolism, the repressor gene iclR and the malate synthase gene aceB were knocked out, while the isocitrate lyase gene aceA was genomically overexpressed. Furthermore, to enhance the supply of nicotinamide adenine dinucleotide phosphate (reduced form), the glucose-6-phosphate dehydrogenase gene (zwf) was overexpressed by both genomic integration and plasmid-based expression, thereby improving the fermentation yield of sarcosine. Ultimately, the engineered strain E. coli S6 successfully synthesized 55.6 mmol/L of sarcosine using glucose and methylamine as substrates in a 5-L bioreactor. This study constructed an innovative microbial cell factory for the efficient biosynthesis of sarcosine, achieving directional substrate conversion through metabolic engineering, thereby providing a scalable technological pathway and theoretical framework for the green production of industrial chemicals.

Key words: sarcosine; metabolic engineering; β-piperidone-2-carboxylic acid reductase; Escherichia coli