The Construction And Optimization Of The Synthesis Pathway For 3-Hydroxypropionic Acid Production

3-Hydroxypropionic acid(3-HP), as for its good properties and wide application prospect, secures the third position in a list of the top 12 platform chemicals accordinging to the United States Department of Energy. The major problem for biosynthesis of 3-HP is the low production, therefore, develop an excellent strain by genetic engineering has becoming more important. In this study, the biosynthesis pathway for 3-HP production was constructed by Escherichia coli W3110, as well as the regeneration pathway of coenzyme NAD+. Beside, the byproduct metabolic pathway was knocked out for enhancing the 3-HP production. The main results is as follows:?The gene dha B and gdr A from K. pneumoniae DSM2026 encoding glycerol dehydratase and glycerol dehydratase activity factor were cloned by polymerase chain reaction and ligated to vector p ACYCDuet-tac and p Trc99 a.The glycerol dehydratase(GDHt) activity of the resulting expression vector p ACYCDuet-tac-dha B1-4 was 2.00 U/m L, the specific enzyme activities of GDHt was 8.94 U/mg. The GDHt enzyme activity of the resulting expression vector p Trc99a-dha B1-4 was 2.14 U/m L and the specific enzyme activities of GDHt was 9.59 U/mg. The gene TUkgsadh from A.brasilense encoding ?-ketoglutaric semialdehyde dehydrogenade(KGSADH) were cloned by polymerase chain reaction and ligated to vector p CDFDuet-tac. The enzyme activity of KGSADH with the resulting expression vector p CDFDuet-tac-TUkgsadh was 0.68 U/m L, and the specific enzyme activitie was 3.11 U/mg. The 3-HP production of the recombinant strain E.coli BL21(DE3)(p CDFDuet-tac-TUkgsadh/p ACYCDuet-tac-dha B1-4) and E.coli BL21(DE3)(p CDFDuet-tac-TUkgsadh/p Trc99a-dha B1-4)under aerobic condition was 1.09 g/L and0.41 g/L, respectively.?The gene gpd1 from Saccharomyces cerevisiae encoding glycerol-3-phosphate dehydrogenase and the gene sth A from Escherichia coli encoding pyridine nucleotide transhydrogenase were cloned by polymerase chain reaction and ligated to the vector p CDFDuet-tacTUkgsadh. The enzyme activity of p CDFDuet-tac-gpd1-TUkgsadh and p CDFDuet-tac-sth A-TUkgsadh were 1.04 times and 2.03 times higher than that of p CDFDuet-tac-TUkgsadh.? In the end, the gene of endogenous propanediol oxidoreductase for byproduct production and the gene of glycerol pathway repressor were knocked out by genetic method. The 3-HP production of E.coli W3110_?yqh D(p CDFDuet-tac-gpd1-TUkgsadh/p ACYCDuet-tac-dha B1-4) was 4.46 g/L, 9.42 times higher than that the orginal strain. Meanwhile, the 3-HP production of E.coli W3110_?glp R(p CDFDuet-tac-gpd1-TUkgsadh/p ACYCDuet-tac-dha B1-4) was 4.02 g/L, 8.40 times higher than that of the original one.During the 3-HP fermentative process, the main problems were the imbalance enzyme activities between Dha B and Ald H, redox imbalance and by-products formation. Based on those, the biosynthesis pathway for 3-HP production was constructed and optimized, which would provid the foundation for improving production of 3-hydroxypropionic acid.