Heterologous production of 3-hydroxypropionic acid in Methylorubrum extorquens by introducing the mcr gene via a multi-round chromosomal integration system based on cre-lox71 / lox66 and transposon

Background and aim Reprogramming microorganisms to enhance the production of metabolites is a part of contemporary synthetic biology, which relies on the availability of genetic tools to successfully manipulate the bacteria. Methylorubrum extorquens AM1 is a platform microorganism used to convert C1 compounds into various value-added products. However, the repertoire of available plasmids to conveniently and quickly fine-tune the expression of multiple genes in this strain is extremely limited compared with other model microorganisms such as Escherichia coli . Thus, this study aimed to integrate existing technologies, such as transposon-mediated chromosomal integration and cre-lox- mediated recombination, to achieve the diversified expression of target genes through multiple chromosomal insertions in M. extorquens AM1. Results A single plasmid toolkit, pSL-TP-cre-km, containing a miniHimar1 transposon and an inducible cre - lox71 / lox66 system, was constructed and characterized for its multiple chromosomal integration capacity. A co-transcribed mcr-egfp cassette [for the production of 3-hydroxypropionic acid (3-HP) and a reporting green fluorescent protein] was added to construct pTP-cre-mcr-egfp for evaluating its utility in mediating the expression of heterologous genes, resulting in the production of 3-HP with a titer of 34.7–55.2 mg/L by two chromosomal integration copies. Furthermore, in association with the expression of plasmid-based mcr , 3-HP production increased to 65.5–92.4 mg/L. Conclusions 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.