Abstract
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.