Exploration of an Efficient Electroporation System for Heterologous Gene Expression in the Genome of Methanotroph

Abstract

One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of <i>Methylomicrobium buryatense</i> 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered <i>M. buryatense</i> 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous <i>fadE</i> gene in the genome with a heterologous <i>kivd</i> gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs.