Metabolic and plasmid engineering to produce D-phenyllactic acid from glucose–xylose co-substrates in <i>Escherichia coli</i>

Plasmid DNA comprises various modules, including promoter and terminator regions, replication origins, and antibiotic resistance genes as selection markers. The 5'UTRs of the promoter and ribosome binding site (RBS) have been extensively studied, and multiple synthetic promoters and RBSs have been developed. In this study, we focused primarily on the components of plasmid vectors in <i>Escherichia coli</i>, excluding the 5'UTRs. Six replication origins and four selection markers were shuffled, resulting in the construction of 24 different plasmids. The gene encoding D-lactate dehydrogenase from <i>Cupriavidus necator</i> was cloned into each plasmid in this plasmid library, and the resulting 24 plasmids were evaluated based on D-phenyllactic acid production. The maximum concentration and yield of PheL from glucose were 5.45 g/L and 0.34 g/g, respectively. To the best of our knowledge, this yield represents the highest reported for PheL production. Furthermore, to examine the relationship between culture profiles and plasmid selection, clustering analysis was conducted, revealing the correlation between PheL production, and replication origins and selection markers. To demonstrate PheL production from biomass-derived resources, we also performed fermentation using mixed sugars of glucose and xylose with a metabolic pathway-compartmentalized <i>Escherichia coli</i> host. The gene cluster, including the Dahms pathway that converts xylose into acetyl-coenzyme A, was optimized, and the resulting transformant produced 3.5 g/L PheL with a glucose yield of 0.24 g/g.IMPORTANCEAs genetic and metabolic engineering has evolved, those technologies have enabled us to create synthetic microbes to produce chemicals of interest with high productivity. Although research on promoter regions and 5' UTRs in plasmid DNA for the effective expression of heterologous genes has been widely conducted, there are few reports about the effect of replication origins and antibiotic resistance markers on production. In this study, we focused on the combination of those two factors and the effect on culture profiles. Using D-phenyllactic acid as the model chemical, the production and selection of replication origins and antibiotic resistance were evaluated. Additionally, we applied the metabolic pathway compartmentalized technology to the transformant showing the highest production, and D-phenyllactic acid production from the mixed sugars with glucose and xylose was demonstrated. This study provides new insights of the heterologous gene expression in microbial biosynthesis.