This study used a multi-round chromosomal integration system based on cre-lox71/lox66 and a transposon to construct a single constructed vector. A heterologous mcr gene was introduced through this vector, and high expression of 3-hydroxypropionic acid was achieved in M. extorquens. This study provided an efficient genetic tool for manipulating M. extorquens, which not only help increase the expression of heterologous genes in M. extorquens but also provide a reference for strains lacking genetic manipulation vectors.