Enhancing the Glucose Flux of an Engineered EP-Bifido Pathway for High Poly(Hydroxybutyrate) Yield Production.

BACKGROUND

As the greenhouse effect becomes more serious and carbon dioxide emissions continue rise, the application prospects of carbon sequestration or carbon-saving pathways increase. Previously, we constructed an EP-bifido pathway in by combining Embden-Meyerhof-Parnas pathway, pentose phosphate pathway and "bifid shunt" for high acetyl-CoA production. There is much room for improvement in the EP-bifido pathway, including in production of target compounds such as poly(hydroxybutyrate) (PHB).

RESULT

To optimize the EP-bifido pathway and obtain higher PHB yields, we knocked out the specific phosphoenolpyruvate phosphate transferase system (PTS) component II Cglc, encoded by . This severely inhibited the growth and sugar consumption of the bacterial cells. Subsequently, we used multiple automated genome engineering (MAGE) to optimize the ribosome binding site (RBS) sequences of (galactose: H (+) symporter) and (glucokinase gene bank: NC_017262.1), encoding galactose permease and glucokinase, respectively. Growth and glucose uptake were partially restored in the bacteria. Finally, we introduced the glf (UDP-galactopyranose) from mutase sugar transport vector into the host strain genome.

CONCLUSION

After optimizing RBS of , the resulting strain L-6 obtained a PHB yield of 71.9% (mol/mol) and a 76 wt% PHB content using glucose as the carbon source. Then when was integrated into the genome strain L-6, the resulting strain M-6 reached a 5.81 g/L PHB titer and 85.1 wt% PHB content.