Construction And Optimization Of The Key Pathway For 3-hydroxypropionic Acid Production

3-Hydroxypropionic acid(3-HP)is a 3-hydroxy isomer of lactic acid and is an important building block in the synthesis of many industrially valuable chemicals.At present,the main problem is that the biological conversion of glycerol to 3-HP can not reach the level for commercial applications.A potential strategy to solve the problems is to construct excellent recombinant strains.In this study,Escherichia Coli BL21(DE3)was used to construct the metabolic pathway for synthesis of 3-HP.The glycerol dehydratase regeneration factor GDR and NAD+ coenzyme regeneration genes were introduced into the pathway,and then the strong promoter trc was used to replace the acetyl-CoA natural promoter for enhancing 3-HP.The main results were as follows:1.The plasmid pETDuet,pET28 a and pCDFDuet with different copy numbers were used to express dha B and gdrA genes from Klebsiella pneumoniae DSM 2026.The result showed the glycerol dehydratase enzyme activity of pETDuet-dhaB1-4,pET28a-dha B1-4 and pCDFDuet-dha B1-4 were 11.56 U/mg,7.94 U/mg and 6.94 U/mg,respectively.Then two different aldehyde dehydrogenase of GabD4 and TUKGSADH were compared,and the aldehyde dehydrogenase activity of the pCDFDuet-tacTUkgsadh and pCDFDuet-tac-GabD4 were 3.11 U/mg and 1.71 U/mg,respectively.Finally,the recombinant strains of E.coli BL21(DE3)(pCDFDuet-tac-TUkgsadh/p ET-28a-dhaB1-4),E.coli BL21(DE3)(pCDFDuet-tac-TUkgsadh/p ETDuet-dhaB1-4),E.coli BL21(DE3)(pCDFDue-tac-GabD4/pET28a-dhaB1-4)and E.coli BL21(DE3)(pCDFDuet-tac-GabD4/pETDuet-dhaB1-4)were cultured for 3-HP production.The 3-HP production were 0.37 g/L,1.14 g/L,0.31 g/L and 0.86 g/L,respectively,which indicated that the balance of the two enzymes activity was important for enhancing 3-HP production.2.To further improve the 3-HP production,the glycerol dehydratase regeneration factor gene gdrB,NAD+ coenzyme regeneration factor gene were introduced,and the promoter for acetyl-CoA synthetase was exchanged.The gene gdrB from K.pneumoniae DSM 2026 was cloned and ligated to recombinant plasmid pETDuet-dhaB1-4.The resulting recombinant strain was E.coli BL21(DE3)(pCDFDuet-tac-TUkgsadh/ pETDuet-dhaB-gdrAB)produced 5.12 g/L of 3-HP,which was 4.5 times higher than before.But the strains containing NAD+ coenzyme regeneration factor of gpd1 and sthA showed lower 3-HP production,which was mainly resulted from the lower activity of aldehyde dehydrogenase for the leak of NAD+.The recombinant strain that exchange the acetyl-CoA natural promoter to trc promoter using the CRISPR-Cas9 system gained 5.5 g/L 3-HP,and the cells grew to a higher density and reach the stationary phase ahead of time.3.The recombinant strain was cultured in a 5L bioreactor.The 3-HP production reached up to 8.79 g/L under the condition of pH7.0,which improved 68% compare with uncontrolled pH.And the output improved greatly to 12.11 g/L by feeding glycerol at 15-20 g/L and 3-HP production increased to 14.10 g/L with VB12 supplementation.