Enhancement of 3-hydroxypropionic acid production in engineered Escherichia coli by modulating endogenous aldehyde dehydrogenase expression system

Abstract

Replacement of conventional petro-based chemicals with biomass-based substances is a central paradigm in the chemical industry. 3-Hydroxypropionic acid (3-HP) was selected as a target product in this thesis, which is a precursor for various chemicals including acrylic acid, methyl acrylate and acrylamide. In previous research, accumulation of glycerol was observed during 3-HP producing culture from a mixture of glucose and xylose, which is known to inhibit glycerol dehydratase, one of the key enzymes in biosynthesis of 3-HP. It was confirmed that the cause of glycerol accumulation is that the aldehyde dehydrogenase (ALDH) catalyzing the oxidation (dehydrogenation) of 3-hydroxypropionaldehyde acts as a rate-limiting step. Therefore, it is necessary to alleviate glycerol accumulation for improving 3-HP production. For this purpose, a novel system that endogenously overexpresses PuuC in the chromosome of Escherichia coli together with an existing vector system that overexpresses ALDH (Pseudomonas aeruginosa) was introduced. The first strategy was to replace the monocistronic promoter of puuC with a strong inducible promoter. As a result, the expression of the puuC transcript increased by a 42.4-fold and the specific activity of aldehyde dehydrogenase in the crude extract increased by a 2.2-fold. Batch culture of this strain (E. coli BL21 star (DE3) ∆gyp-PT7 / pELDRR / pCPaGGRmGalP) in R/5 medium containing 14 g/L of glucose and 7 g/L of xylose reduced glycerol accumulation by 36% and enhanced 3-HP production by 60% compared to the control strain (E. coli BL21 star (DE3) ∆gyp / pELDRR / pCPaGGRmGalP). As the second strategy, a method of relieving the negative inhibition mechanism acting on the polycistronic promoter of the puu operon was used. As a result, the expression of the puuC transcript increased by a 8.3-fold and the specific activity of aldehyde dehydrogenase in the crude extract increased by a 2.3-fold. Batch culture of this strain (E. coli BL21 star (DE3) ∆gypr / pELDRR / pCPaGGRmGalP) in R/5 medium containing 14 g/L of glucose and 7 g/L of xylose reduced glycerol accumulation by 29% and enhanced 3-HP production by 79%. Finally, a combination of the two strategies was performed. As a result, the expression of the puuC transcript increased by a 91-fold and the specific activity of aldehyde dehydrogenase in the crude extract increased by a 2.9-fold. Batch culture of the strain (E. coli BL21 star (DE3) ∆gypr-PT7 / pELDRR / pCPaGGRmGalP) in R/5 medium containing 14 g/L of glucose and 7 g/L of xylose reduced glycerol accumulation by 61% and enhanced 3-HP production by 127%. Furthermore, fed-batch fermentation using the combinational strain was carried out to produce high concentration of 3-HP. As a result, 53.7 g/L of 3-HP could be produced from a mixture of glucose and xylose. In addition, when xylose was supplied as a sole carbon source using the same strain, 62.2 g/L of 3-HP could be produced. These results suggest that an endogenous ALDH (puuC) overexpression system based on the strong promoter replacement in combination with the elimination of transcriptional repression is expected to be universally applicable to the production of biochemical materials using metabolically engineered microorganisms.