Phosphinothricin (PPT) is one of the most prevalently using herbicides. The commercial phosphinothricin products are generally in the form of a racemic mixture, of which only the l-phosphinothricin (L-PPT) gives herbicidal function. Synthesis of optically pure L-PPT by deracemization of D/L-PPT is a promising way to cut down the environmental burden and manufacturing cost. To convert D/L-PPT to L-PPT, we expressed the catalytic enzymes by genomic integration in E. coli. The whole production was implemented in two steps in one pot using four catalytic enzymes, namely d-amino acid oxidase, catalase, glutamate dehydrogenase, and glucose dehydrogenase. Finally, after a series of process optimization, the results showed that with our system the overall L-PPT yield reached 86%. Our study demonstrated a new strategy for L-PPT synthesis, based on enzymes from chromosomal integrated expression, which does not depend on antibiotic selection, and shows a high potential for future industrial application.