Molecular Transformation,Construction And Expression Of The Key Enzyme In 3-hydroxypropionic Acid Producing Bacteria

3-Hydroxypropionic acid（3-HP）is a valuable platform chemical,which can be produced biologically by engineered microorganisms from glycerol.Currently,the reported highest yield of 3-HP was 71.9 g/L.However,according to the paper,the commercial production of organic acids needed to reach 100 g/L or more.Therefore,in order to achieve industrial production of 3-HP as soon as possible,the limiting factor from glycerol to 3-HP to increase the activity of the key enzyme and substrate conversion rate need to be deeply studied.So,in this paper,the strain E.coli BL21（pET-28b-kgsadh）is used for the study of the molecular transformation and characterization of aldehyde dehydrogenase.At last,the strain E.coli BL21（pCDFDuet-dhaB1-4,pET-28b-kgsadh）was successfully constructed.The main results are as follows:（1）According to KGSADH gene sequence on NCBI database,the3JZ4 of 40%similarity to homology modeling was found.Using the reported enzyme active center information and energy analysis software,the KGSADH amino acid point mutation of 120E,163K,177K,219P,225K,252E,278K were determined.The enzyme specific activities of KGSADH using acetaldehyde as substrate with point mutation of E120D,K177R,and P219A improved greatly,which was 1.84,1.61 and 1.54 fold than original enzyme activity.When KGSADH combined with two mutations,the highest specific activity was E120D/P219A mutant,which was 4.22 times more than the original enzyme.In the end,the homologous model of the mutant E120D/P219A enzyme was constructed,and the results of molecular docking indicated that the protein structure was slightly changed by mutation,which caused the specific activity increased.（2）The purified aldehyde dehydrogenase was obtained by Ni-NTA column.Then the enzymatic properties of the pure enzyme,including the optimum temperature,thermal stability,pH,pH stability,and the influence of metal ions on the enzyme activity,reaction kinetic parameters and so on,were studied.The results showed that the optimum temperature of the enzyme was 30°C,The optimum pH was 8.0 and its stability was improved slightly under pH 6.0.When adding metal ions and surfactant into the reaction system,Co²⁺,Fe³⁺and Fe²⁺showed the promoting effect on enzyme activity,but Zn²⁺had a great inhibition on enzyme activity.Using acetaldehyde as substrate,the K_m and V_maxax of the original enzyme was 7.58 mM and 10.6 U/mg,and values of the mutant enzyme could reach to 6.28 mM and 12.3 U/mg,respectively.（3）The glycerol dehydratase DhaB1-4 was cloned into the vector pCDFDuet-1 and constructed expression vectors,which transformed into E.coli BL21（DE3）together with pET-28b-kgsadh.And the co-expression recombinant E.coli BL21/（pCDFDuet-dhaB1-4,pET-28b-kgsadh）was successfully constructed.The results indicated that the optimum inducing time was 5 h,and the single enzyme activity of DhaB1-4（109.3 U/mg）was much higher than KGSADH activity（6.3 U/mg）.When the two enzymes were co-expressed,DhaB1-4 and KGSADH activity were reduced to 18.6 U/mg and 2.8 U/mg,respectively,which was due to the instability of DhaB and the imbalanced activity between DhaB and KGSADH.The fermentation results in the flask showed that biomass was3.98 g/L,residual glycerol was 10.6 g/L and the 3-HP production was66.8 mM（6.0 g/L）,which was 2.1 times more than the original bacteria.This study aimed at the molecular transformation of the limiting factor of aldehyde dehydrogenase during the 3-HP fermentative process.Besides,a 3-hydroxypropionic acid producing bacteria was constructed,which provided the foundation for solving the issues on the synthesis pathway and improving production of 3-hydroxypropionic acid.