Construction Of Engineering Bacteria For Efficient Conversion Of Glycerol To 1,3-Propanediol

1,3-propanediol is a very important chemical raw material.In addition to being used as a solvent for the preparation of a variety of chemical substances,1,3-propanediol plays an important role in the synthesis of polyesters as monomers.Poly(trimethylene terephthalate)is a new polyester material,which is synthesized from terephthalic acid and 1,3-propanediol.PTT not only has the advantages of high elasticity,easy dyeing and no static electricity,but also easy to degrade and will not cause pollution to the environment.It has attracted wide attention because of its excellent properties.As a result,the market for 1,3-propanediol has also expanded.In recent years,with the increasing shortage of fossil fuel resources,the chemical synthesis method based on oil resources has been seriously restricted.The production of 1,3-propanediol by biotransformation has become a research hotspot in today’s society.This paper provides a genetic engineering strain for efficient transformation of glycerol to 1,3-propanediol with K.pneumoniae 2-1 and E.coli BL21 as host and its construction method and application.The following research work has been carried out from the aspects of metabolic pathway transformation and fermentation condition optimization.(1)In this study,using Klebsiella pneumoniae 2-1 as host,the isozyme of formate dehydrogenase(FDH)from Shewanella onedensis MR-1and isozyme of 1,3-propanediol oxidoreductase from E.coli JM109 were heterogeneously expressed in the same host,and the recombinant strain K.pneumoniae/pET28a-dhat-yqhD-FdhD was constructed.In this study,using Klebsiella pneumoniae 2-1 as host,formate dehydrogenase(coding gene FdhD)from Shewanella onedensis MR-1,1,3-propanediol oxidoreductase from Klebsiella pneumoniae 2-1(coding gene dhaT)and isozyme of 1,3-propanediol oxidoreductase from E.coli JM109(coding gene yqhD)were heterologous expressed respectively.The recombinant bacteria K.pneumoniae/pET28a-dhat-yqhD、K.pneumoniae/pET28a-dhat-dhaT、K.pneumoniae/pET28a-dhat-FdhD、K.pneumoniae/ pET28adhat-yqhD-FdhD and K.pneumoniae/pET28a-dhat-dhaT-FdhD were constructed.The ability of recombinant bacteria to transform glycerol to 1,3-propanediol was studied.The performance of its conversion of glycerol to 1,3-propanediol was investigated.After fed-batch fermentation for 29 h in a 5 L fermentor,the highest yield of K.pneumoniae/pET28a-dhat-yqhD-FdhD strain was 77.18 g/L,which was 97.93% higher than that of wild type strain(38.99 g/L).The fermentation intensity and glycerol conversion rate were 2.34 g/(L·h)and 55.79%,respectively.The results showed that the co-expression of yqhD and FdhD genes further promoted the transformation of 3-hydroxypropionaldehyde to 1,3-propanediol,thus increasing the yield of 13-propanediol.(2)The glycerol dehydrase and its activator genes(dhaB,dhaC,dhaE,dhaF and dhaG)of 4.9kb were amplified from K.pneumoniae 2-1 genome to construct the recombinant strain E.coli BL21/p ETDuet-dhaB-dhaC-dhaE-dhaF-dhaG.At the same time,1.16 kb PDOR isozyme coding gene yqhD was cloned from E.coli JM109 genome to construct recombinant bacteria E.coli BL21/p ETDuet-dhaB-dhaC-dhaE-dhaF-dhaG-yqhD.The production of 1,3-propanediol by recombinant bacteria fermented with glycerol for 24 h was investigated.The results showed that the recombinant strain obtained by introducing only glycerol dehydrase and its activators(dhaB,dhaC,dhaE,dhaF and dhaG)genes into E.coli BL21 was not conducive to the production of 1,3-propanediol from glycerol.Only by increasing the copy number of yqhD gene and strengthening the expression of 1,3-propanediol oxidoreductase isozyme can we achieve the purpose of efficient transformation of glycerol fermentation to produce 1,3-propanediol.After fed-batch fermentation for 30 h in a 5 L fermentor,the yield,conversion rate and production intensity of 1,3-propanediol of recombinant strain E.coli BL21/p ETDuet-dhaB-dhaC-dhaE-dhaF-dhaG-yqhD reached 50.76 g/L,47% and 1.69g/(L·h).