Construction And Optimization Of The Synthetic Pathway Of 3-Hydroxypropionic Acid With Glucose As Carbon Source In Klebsiella Pneumoniae

3-Hydroxypropionic acid(3-HP)bears both hydroxyl and carboxyl groups and therefore can be used to synthesize various economically valuable chemicals.3-HP can also be polymerized into degradable materials.Previous studies reported the feasibility of 3-HP production by coenzyme Aindependent pathway using glycerol as substrate and Klebsiella pneumoniae as the host bacterium.Although glycerol is a common carbon source to produce3-HP,the recent decline of international biodiesel industry has led to decreased quantity but increased price of by-product glycerol.That is,glycerol has no price advantage over glucose.In this study,a glucose-based 3-HP synthetic pathway was engineered in K.pneumoniae.Specifically,the genes encoding glycerol-3-phosphate dehydrogenase(GPD1,EC 1.1.1.8)and glycerol-3-phosphate(GPP2,EC 3.1.3.21)were cloned from Saccharomyces cerevisiae,and a vector was constructed to co-express gpd1 and gpp2 to convert glucose into glycerol which would then be converted to 3-HP through the glycerol metabolic pathway of K.pneumoniae.Next,to reduce the glucose consumption by EMP pathway and by-product formation,CRISPRi system was developed in above recombinant strain to inhibit the expression of glyceraldehyde-3-phosphate dehydrogenase gene(gap A)and 2,3-butanediol production gene(bud A).To determine the performance of CRISPRi system,the recombinant K.pneumoniae was subjected to shake-flask and fed-batch cultivation.In shake flask cultivation,this bi-functional strain produced 2.8g/L glycerol and 0.78 g/L 3-HP using 20 g/L glucose as carbon source.In fedbatch cultivation,this bi-functional strain produced 1.77 g/L 3-HP.The above experiments revealed that heterologous expression of 3-HP synthesis genes substantially halted the growth of K.pneumoniae.To address this issue,we attempted to engineer an orthogonal ribosome system to decouple exogenous gene expression and host metabolism.In principle,this orthogonal system holds promise to redistribute metabolic flux by dividing ribosomes into two distinct pools: One is responsible for expression of heterologous genes,the other sustains host inherent metabolisms.The above study provides insights for efficient bioproduction of 3-HP.