Adaptive Laboratory Evolution Guided Engineering of Embden–Meyerhof–Parnas Pathway Disrupted Escherichia coli mutant and Its Application to Production of 3-Hydroxypropionic Acid

Key factors for high production performance in biosynthetic process include continuous supply of redox cofactors for catalyzing reactions, which becomes critical in case of a pathway with redox imbalance. There are natural strains with high [NADPH]/[NADP+] reported. However, the lack of whole genome sequence or an inefficient genomic engineering tool makes it difficult to engineer such strains in contrast to a well-studied model organism Escherichia coli. The most distinguishing feature of their sugar metabolisms is the lack of functional Embden–Meyerhof–Parnas (EMP) pathway, which is a major glycolytic pathway in other organisms. E. coli mutants with no functional EMP pathway were reported to have higher [NADPH]/[NADP] than their wild type strains while showing growth defect on glucose as a sole carbon source. One of the strategies for acquiring mutants with desired phenotype is adaptive laboratory evolution (ALE). One of biosynthetic pathways producing platform chemical 3-hydroxypropionic acid (3-HP) from glucose requires two NADPH per a 3-HP produced. We applied ALE approach to obtain an E. coli mutant growing well without the functional EMP pathway. Genetic modifications in isolated mutants were identified to insight into the molecular mechanisms linking 3-HP biosynthesis