Construction And Optimization Of Non-PTS Glucose Transport Pathway In Escherichia Coli

Glucose is the most widely used substrate in the fermentation industry, and high glucose utilization rate is very important for cost effective production. In Escherichia coli, glucose is firstly transported into cytoplasm through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase systems (PTS). One PEP is required for transport and phosphorylation of one glucose. PEP is an important precursor for synthesis of many industrially important chemicals, such as succinate, malate, and aromatic compounds. For improving synthesis of succinate or other compounds which use PEP as an essential precursor, PTS was usually inactivated to increase PEP supply to improve metabolic fluxes towards desired products.Although inactivating PTS system could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization of those PTS" strains, two glucose transport proteins and two glucokinases (Glk) from Escherichia coli and Zymomonas mobilis were recruited in this work and combinatorial modulation of galP/glk E.coli, glf/glk E.coli, glf/glk zmobilis gene expression by artificial regulatory parts was carried out in chromosome. Their impacts on glucose utilization and succinate production were investigated. All PTS-strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk e. coli (JT014, Glfl2,Glk E.coli,12) was 2.13 g/L·h, which was 81% higher than the wild-type E. coli and 30% higher than the highest rate obtained by combinatorial modulation of galP and glk E. coli (JL043,GalP93,Glk E.coli 37). On the other hand, although glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L·h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells.For succinate production, using GalP led to a 20% increase of succinate productivity, while recruiting Glf led to a 41% increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.