Engineering Biosynthetic Pathways Of Poly(3-hydroxypropionate) In Klebsiella Pneumoniae

Due to remarkable biocompatibility and biodegradation,poly(3-hy droxypropionate)(P3HP)has emerged as an alternative to conventional feedstock of plastics.However,P3HP cannot be synthesized by any wild type microbes identified so far.Clearly,it is a grand challenge to achieve large-scale production of P3HP.The aim of this study is to engineer P3HP biosynthesis pathways in Klebsiella pneumoniae.To this end,heterologous genes were expressed and inexpensive carbon sources were used to grow K.pneumoniae.K.pneumoniae can utilize glycerol for rapid growth and synthesize a series of chemical intermediates including 3-hydroxypropionic acid(3-HP)and 3-hydroxypropionaldehyde(3-HPA)in P3HP metabolic pathways.K.pneumoniae has genetic background similar to Escherichia coli.These advantages make K.pneumoniae an excellent host for production of P3HP.This study includes the following contents:1.The propionaldehyde dehydrogenase gene pduP from Salmonella entericaserovar serovar Typhimurium LT2 and PHA synthase gene phaC from Ralstonia eutropha H16 were cloned by PCR.The promoter in plasmid pET-PK was replaced by tac promoter,resulting in a novel vector named pET-tac.This vector was employed for simultaneous expression of pduP and phaC in K.pneumoniae,leading to a recombinant strain named K.p(pET-tac-pduP-tac-phaC).This recombinant strain was cultivated in shake flasks for 24 h with glycerol as the sole carbon source.Gas chromatography(GC)analysis showed that K.p(pET-tac-pduP-tac-phaC)could generate P3HP,indicating P3HP biosynthetic pathway was successfully constructed in K.pneumoniae.2.To improve P3HP production,two strategies were recruited to optimize the expression of phaC which encodes the key PHA synthase in metabolic pathway.Consequently,two recombinants were engineered.For strain K.p(pET-tac-pduP-tac-phaC),the gene sequence of tac promotor was inserted betweenpduP and phaC.For strain K.p(pET-tac-phaC-pduP),the order of pduP and phaC in vector was rearranged to preferentially express PHA synthase gene.Consequently,the strain K.p(pET-tac-pduP-tac-phaC)produced 0.091 g/L P3HP in 24-hour shake flask fermentation,while another strain only produced 0.03 g/L P3HP.Notably,the initial strain K.p(pET-tac-pduP-phaC)generated 0.054 g/L P3HP.Fed-batch cultivation of the double-promotor strain was performed in a 5 L bioreactor containing 3 L medium.After 48 h cultivation,0.44 g/L P3HP was generated.SDS-PAGE analysis demonstrated the expression of heterologous proteins.Reverse transcription PCR(RT-PCR)results showed that the phaC transcription levels in two optimized recombinants were higher than that in reference strain.3.Propionyl-CoA synthetase gene prpE from Escherichia coli was cloned and co-expressed with phaC,resulting in a new vector pET-tac-phaC-prpE.To develop a double-plasmid system in 3-HP high-yield strain,the Kan resistance gene was replaced by Cm resistance gene.As a result,the recombinant strain K.p(pET-tac-puuC)(pET-tac-phaC-prpE)was engineered.After fermentation with glycerol as carbon source,a small amount of P3HP was detected by GC.SDS-PAGE analysis showed that all heterologous proteins were expressed.