Metabolic Engineering Of Corynebacterium Glutamicum For Efficient Production Of 3-hydroxypropionic Acid From Acetate

3-Hydroxypropionic acid(3-HP)is an important chemical raw material.Due to its two active terminals,it can be applied to produce various chemicals and has been widely used in the agriculture,food,pharmaceutical industries and materials.On account of its significant market value and wide applications,3-hydroxypropionic acid was listed as one of the twelve priority compounds for development both in 2004 and2010 by the US Department of Energy.Corynebacterium glutamicum was used as the host for further metabolic engineering in this project.This is the first report about 3-hydroxypropionic acid production by acetic acid through the malonyl-CoA pathway in Corynebacterium glutamicum.Firstly,malonyl-CoA reductase(MCR)was introduced into Corynebacterium glutamicum using a plasmid to construct 3-HP synthesis pathway.To increase the MCR enzyme activity,three point mutations was introduced to MCR-C;the two functional domains of MCR N-terminal and C-terminal were disassembled and the order was exchanged;promoter of gene mcr was replaced to stronger one.Subsequently,to reduce the flux of the TCA cycle and increase the intracellular content of acetyl-CoA,gradient weakening of the citrate synthase,which was the entrance enzyme of the TCA cycle,was performed.Three promoters with different promote strength were used to weaken citrate synthase in genome,and then the start codon was replaced to TTG on the basis of the weakest promoter.The final engineered strain Cgs13 produced 3.73 g/L 3-HP in48 h with 9.91 g/L acetic acid consumed.In order to increase the flux of malonyl-CoA,we increased the enzymatic activity of acetyl-CoA carboxylase by removing the negative feedback regulation of fas R.The3-HP titer of the obtained engineered strain Cgs16 did not increase but decreased slightly.It was assumed that the imbalance of enzyme activities of MCR and ACC caused the decrease of 3-HP titer.To prove our hypothesis,the same genetic manipulation was performed on the basis of the strain Cgs8,and found that the titer of3-HP was significantly improved.In order to explore the potential of the subsequent gene modification of the fatty acid synthesis pathway of strain Cgs13,15 μmol/L cerulenin was added at 12 h of fermentation,and 3-HP titer was increased to 4.67 g/L.Finally,a molecule of MCR was integrated on the genome of strain Cgs13,which was scaled up in a 5 L fermenter,at which 14.90 g/L 3-HP was finally produced.In this study,several strategies were taken to engineer Corynebacterium glutamicum as the host to produce 3-HP,which included introducing malonyl-CoA reductase and then increase its enzyme activity,weakening citrate synthase by gradient,adding cerulenin to inhibit fatty acid synthesis pathway,and finally scaling up in the 5L fermenter.This is the first report about 3-HP production from acetic acid via the malonyl-CoA pathway in Corynebacterium glutamicum.