Molecular Transformation Of Aldehyde Dehydrogenase And Its Application In The Synthesis Process Of 3-hydroxypropionic Acid

Amount of glycerol was producted as the by-product in large-scale production of biodiesel.Therefore,large numbers of scientists dedicate to research the new way to use glycerol.In recent years,production of 3-hydroxypropionic acid(3-HP)by genetically engineering bacteria with glycerol has become a hot topic.3-hydroxypropionic acid has excellent physical and chemical properties and extensive application prospect.In order to increase the production of 3-hydroxypropionic acid,many works has been done,including the screening of aldehyde dehydrogenase and expression vectors,the choice of the host,and the optimization of fermentation conditions.The topic aims at increasing the production of 3-hydroxypropionic acid.In this study,the strain E.coli DH5α(pET-28b-kgsadh,pCDFDuet-DhaB)was built and preserved in our laboratory.Aldehyde dehydrogenase was transformed by EP-PCR,the reaction system of 3-hydroxypropionic acid biotransformation by resting cell was investigated,and the fed-batch fermentation process of 3-hydroxypropionic acid production was optimized.The main results are as follows:(1)Molecular modification of aldehyde dehydrogenase:EP-PCR mutagenic primer was designed and the primer annealing temperature was identified,then the kgsadh gene was cloned and random mutated by changing the concentration of Mn2+ in EP-PCR reaction system.And the the mutant library was constructed by the above operation.(2)The reaction system for 3-hydroxypropionic acid production with the resting cell was optimized by single factor experiments.The optimal sampling time was 6～7 h based on the reaction progress curves;the optimum concentration of cell and glycerol were all 20 g/L;the optimal concentration of VB12 and NAD+were 10 mg/L and 0.15 mM,respectively;and the temperature was 35℃,the buffer solution was 0.05 mol/L Tris-HCl(pH=7.0).Under the above conditions,the enzyme activity of Kgsadh and DhaB could reach to 16.77 U/gDCW and 57.91 U/gDCW,respectively.The production of 3-hydroxypropionic acid could reach to 3.17 g/L,which was improved by 33%.(3)On the basis of batch fermentation process,VB12 was added the same amount twice.After 36 h cultivation,the production of 3-hydroxypropionic acid could reach to 6.81 g/L and the dry cell weight was 3.98 g/L,which increased more than 12%and 17%compared to add VB12 once.In the fed-batch fermentation,glucose was added when its concentration was lower than 3 g/L and the dry cell weight and 3-hydroxypropionic acid production were 3.97 g/L and 6.34 g/L,respectively.The fed-batch process adding glycerol and glucose mixed medium was determined as follows:the mixed medium was fed with a constant speed at 10 h,and the production of 3-hydroxypropionic acid and dry cell weight could reached to a maximum value of 7.17 g/L and 4.62 g/L,respectively,which were increased 12%and 13%than that of adding glucose.This study lay the foundation for improving and solving the issues on the synthesis pathway of 3-hydroxypropionic acid.