Production Of 3-Hydroxy Propionic Acid By Recombinant Klebsiella Pneumoniae And Optimization Of Fermentation

3-hydroxypropionic acid(3-HP)is an important platform chemicals which can be used to synthesize a variety of important compounds,that has a very broad application prospect.Glycerol is accompanied by the massive production of biodiesel industry and the price is decreased in recent years.If a strain can be selected to convert glycerol into 3-HP efficiently,it will produce a great environmental and economic benefits.In order to realize synthesis 3-HP using recombinant Klebsiella pneumoniae strain,The major researches were summarized as follows.In this experiment,Cupriavidus necator Gab D4 and Azospirillum brasilense KGSADH aldehyde dehydrogenase gene were cloned by polymerase chain reaction(PCR),and ligated to pUC19 plasmid whose resistance was modified to Kan(p UC19Kan).Then,the recombinant plasmid was transformed into the host bacteria K.pneumoniae DSM2026.The recombinant strains were named KpKan/KGSADH and KpKan/Gab D4,respectively.Through shake-flask fermentation,their maximum 3-HP yield reached 3.66 and 2.56 g/L,KpKan/KGSADH was determined eventually as the starting strain which was used in fermentation optimization.Five factors were tested through single factor shake flask test including substrate concentration,yeast extract concentration,initial pH,loading fluid volume and inoculum size.Finally,the optimal conditions were: 30 g/L glycerol,4 g/L yeast extract,initial p H 7.0,2% inoculum size,125 mL medium in 250 mL bottle.The optimized yield of 3-HP was 3.81 g/L.Based on that,through controlling fermentation pH to 7.0 in the fermentation process,the maximum yield of 3-HP was 11.2 g/L after 64 h of fermentation.In 5 L fermenter,the 3-HP yield was obtained with maximum of 11.9 g/L using stepwise pH controlling during fermentation;The 3-HP yield continued to rise to 13.4 g/L using continuous controlling pH strategy.Finally,the maximum 3-HP yield of 15.1 g/L was obtained using continuous controlling pH and adding glycerol in batch strategy.This study first introduced C.necator Gab D4 aldehyde dehydrogenase into K.pneumoniae DSM2026 and used it for synthesis of 3-HP.Meanwhile,their performance differences were further investigated.Finally,the maximum 3-HP yield in this study reached 15.1 g/L,which was 4 times that of the initial level.This study provided the basis for further enhancing 3-HP yield using recombinant K.pneumoniae system.