Production Of3-hydroxypropionic Acid Via Global Perturbation And Homologous Recombination

3-hydroxypropionic acid (3-HP) is an improtant three-carbonnon-chiral organic molecule from which several commodity and specialtychemicals can be generated. This study focuses on the production of3-HPby recombinant microorganisms, and several molecular biologytechniques were used to engineer Klebsiella pneumoniae, the host, toproduce3-HP.1. The sigma factor encoding gene, rpoD, in K.pneumoniae wascloned by PCR and its mutant library was constructed with an error-pronePCR method. Subsequent ligation to expression vector and thetransformation into K. pneumoniae DSM2026led to plentiful positiveclones. Among them, one recombinant was screened and fermented usingglycerol as the sole carbon source. Under preliminary optimizedcondition, this recombinant presented3-HP of4.49g/L. Compared withthe control strain, it has increased the yield by242.75%. In addition, ithas increased the conversion ratio of glycerol by832.50%, and theglycerol productivity by239.39%, respectively.2. The aldehyde dehydrogenase encoding gene, aldH, from Escherichia coli was cloned by PCR, subsequently ligated to expressionvector pET-PK and transformed into K. pneumoniae DSM2026. Therecombinant K.p(pET-PK-aldH) was fermented using glycerol as the solecarbon source. It was showed that the gene aldH was overexpressedsucessefully, and the recombinant presented3-HP of2.96g/L. Comparedwith the control strain, it showed1.5-fold of yield, and57.36%increaseof productivity, respectively.3. Relying on homologous recombination strategy, the DNA cassetteof homologous recombination, containing the gene aldH, wastransformed into K.pneumoniae DSM2026and delivered to the IS1siteon the chromosome, via the homologous recombination system, RecA ofK. pneumoniae. The recombinant KpAD was fermented using glycerol asthe sole carbon source. It was showed that the gene aldH wasoverexpressed sucessefully. Under preliminary optimized condition,KpAD/Kan+presented3-HP of0.84g/L, while KpAD/Kan-presented3-HP of0.90g/L. Compared with the control strain, KpAD/Kan+showed1.5-fold of yield, while KpAD/Kan-showed1.4-fold of yield. In addition,KpAD/Kan+showed1.7and1.5-fold of glycerol conversion ratio andproductivity, while KpAD/Kan-showed1.4-fold of yield, and2.0and1.4-fold of glycerol conversion ratio and productivity, respectively.