Metabolic engineering of Halomonas bluephagenesis for high-level mevalonate production from glucose and acetate mixture

Mevalonate (MVA) plays a crucial role as a building block for the biosynthesis of isoprenoids. In this study, we engineered Halomonas bluephagenesis to efficiently produce MVA. Firstly, by screening MVA synthetases from eight different species, the two efficient candidate modules, specifically NADPH-dependent mvaESEfa from Enterococcus faecalis and NADH-dependent mvaESLca from Lactobacillus casei, were integrated into the chromosome, leading to the construction of the H. bluephagenesis MVA11. Through the synergetic utilization of glucose and acetate as mixed carbon sources, MVA11 produced 11.2 g/L MVA with a yield of 0.45 g/g (glucose + acetic acid) in the shake flask. Subsequently, 10 beneficial genes out of 50 targets that could promote MVA production were identified using CRISPR interference. The simultaneous repression of rpoN (encoding RNA polymerase sigma-54 factor) and IldD (encoding L-lactate dehydrogenase) increased MVA titer (13.3 g/L) by 19.23% and yield (0.53 g/g (glucose + acetic acid)) by 17.78%, respectively. Furthermore, introducing the non-oxidative glycolysis (NOG) pathway into MVA11 enhanced MVA yield by 12.20%. Ultimately, by combining these strategies, the resultant H. bluephagenesis MVA13/pli-63 produced 13.9 g/L MVA in the shake flask, and the yield increased to 0.56 g/g (glucose + acetic acid), which was the highest reported so far. Under open fed-batch fermentation conditions, H. bluephagenesis MVA13/pli-63 produced 121 g/L of MVA with a yield of 0.42 g/g (glucose + acetic acid), representing the highest reported titer and yield in the bioreactor to date. This study demonstrates that H. bluephagenesis is one of the most favorable chassis for MVA production.