Development of a semi-endogenous PRT-based expression system for Methylotuvimicrobium alcaliphilum and its application to enhance 3-hydroxybutyric acid production from methanol

Endogenous expression systems, such as those incorporating promoters, ribosome-binding sites (RBS), and terminators (i.e., PRT systems), have been developed for various bacterial host strains. However, intact native promoters are typically long sequences that include regulated regions, which hinder the production of recombinant proteins. In this study, a semi-endogenous PRT system based on 20-40 nucleotide motif sequences derived from the genome of the target host strains was developed using machine learning-based prediction tools. This system was subsequently fine-tuned and optimized based on transcription and translation rates as well as the Gibbs free energy of the terminator structure. This system was validated, and its effectiveness was demonstrated by using it to induce the expression of dTomato, ZeocinR, and phaCAB cluster genes in E. coli. The semi-endogenous PRT was also used for enhanced production of 303 ± 6.39 mg/L 3-hydroxybutyric acid (3-HB) from methanol as the sole carbon source in a 50-mL nitrate mineral salt (NMS) flask after 234 h with overexpression of exogenous phaA and phaB genes in the Methylotuvimicrobium alcaliphilum 20ZX strain. The semi-endogenous PRT system was efficiently designed and served as an endogenous expression system in both E. coli and methanotroph. This system will offer more choices for selecting an endogenous expression platform tailored to the target host strain.