Rational Engineering Of The Key Enzymes For Efficient Biosynthesis Of (S)-2-hydroxybutyric Acid

Poly-hydroxy acids synthesized from?-hydroxy acids are important degradable plastics.Compared with the polylactic acid,poly(2-hydroxybutyric acid,2-HBA)showed improved tensile characteristics and better biocompatibility.2-HBA has been widely used for the synthesis of biomedical materials,and is also an important intermediate for chiral drug and cosmetic chemicals.Synthesis of 2-HBA by traditional chemical methods usually get mixed chiral products(S-2-HBA/R-2-HBA),which needs combine with chiral resolution to obtain the chiral 2-HBA,while it would be easier to synthesize homochiral 2-HBA using microbial synthesis.Construction of multi-enzyme cascade reaction system would realize the biosynthesis of(S)-2-HBA from the bulk chemical L-threonine.However,some major problems exist in these reactions:key enzyme L-lactate dehydrogenase(LDH)showed a relatively low activities towards 2-OBA and other?-keto acids with long side chain;the unbalance conversion rate in the multi-enzyme cascade reaction system;the intermediate accumulation results to the enzymatic activity inhibition and the followed reaction termination;poor enzymatic stability and the low efficiency for the cofactor-NADH regeneration.Therefore,aimed at the efficient preparation of(S)-2-HBA by multi-enzyme cascade reaction system,and the thesis content was divided into four modules to achieve the final efficient preparation of(S)-2-HBA,including:1)rational engineering of the key enzyme L-LDH to improve the conversion efficiency of(S)-2-HBA;2)construction of a tunable multi-enzyme co-expression system in recombinant Escherichia coli for efficient production of(S)-2-HBA;3)optimized enzymatic conversion efficiency of(S)-2-HBA production by using enzymatic aggregates with fused self-assembled hydrophobic short peptide to the enzymes;4)preparation and purification of(S)-2-HBA from the enzymatic conversion solution.(1)For the low activities of P.falciparum L-lactate dehydrogenase(PfLDH)towards2-OBA and other?-keto acids with long side chain,the loop(G193-V200)with catalytic active residue H195 on was studied.Rational engineering of this loop was carried out based on the analysis of LDHs amino acid sequence alignment,and the variant N197D_ldh was obtained with enzyme activity and catalytic efficiency k_cat/K_m increased 1.15 times and 2.73times,respectively,compared with wild type PfLDH.The t_1/2 under 40°C increased to 77.9 h,much higher than 50.4 h for the wild type PfLDH.The change of the enzymatic activity and stability might be due to the change of loop charge characteristics-increased electronegativity based on the study of protein secondary structure,molecular docking and molecular dynamics simulation,which improved enzymatic catalytic efficiency and stability towards 2-OBA substrate.The results showed that the rational engineering of amino acid residues in the active site loop not only played a key role in the catalytic efficiency,but also played an important role in the stability of the enzyme.In addition,by coupling with Candida boidinii fomate dehydrogenase(Cb FDH)for NADH regeneration,N197D_ldh showed the enzymatic conversion of 2-OBA to(S)-2-HBA with 95.8 g/L production titer within 12 h,which was1.05times higher than that of wild type PfLDH,and the conversion rate was 98.8%.These results showed that rational engineering of amino acid residues on catalytic active loop played an important role in improving enzyme activity,and the variant N197D_ldh would better serve the efficient production of(S)-2-HBA.At the same time,by coupling Geobacillus stearothermophilus alcohol dehydrogenase(Gs ADH)or Cb FDH of cofactor NADH regeneration with co-expressing E.coli threonine deaminase(Ec TD)and PfLDH or its mutant N197D_ldh,a multi-enzyme cascade catalytic system was constructed in E.coli,and we achieved the multi-enzyme cascade catalytic system for the asymmetric conversion of chiral pure(S)-2-HBA from bulk chemical L-threonine.(2)To solve the problem of kinetic imbalance in the multi-enzyme cascade reactions,a tunable multi-enzyme co-expression system was constructed by regulating the expression of Ec TD and Gs ADH of cofactor NADH in recombinant E.coli,the kinetic balance in the multi-enzyme reaction steps was achieved for the efficient preparation of(S)-2-HBA.In vitro enzymatic transformation experiments confirmed that the optimal enzyme ratio of Ec TD,N197D_ldh and Gs ADH in the multi-enzyme cascade reaction system was 1.0:1.0:0.3.The expression of Ec TD and Gs ADH were optimized by changing RBS sequence and T7promoter variants,respectively.A strong promoter P21285 was screened,which could increase the expression level and get 2.6 times higher enzyme activity for Gs ADH.Twenty-one recombinant E.coli strains harboring Ecivl A with different RBS intensities(T.I.R.94670 to 924197)or different promoter intensities were designed and constructed,with conversion rates ranging from 3.8 to 23.1 g/(L·h).Finally,the recombinant strain P21285ADH-T7V7827 with better transformation ability was obtained.The intracellular enzyme activity ratio of the strain was Ec TD:N197D_ldh:Gs ADH=1.1:1.0:0.3.The maximum titer for(S)-2-HBA production was 144.2 g/L with the molar conversion rate at 97.2%,and the space-time yield of(S)-2-HBA was 6 g/(L·h),which was 1.85 times of the maximum titer reported so far,and this strategy showed great potential for industrial application.(3)In order to further improve the enzymatic stabilities and mass transfer efficiency during the cascade reaction transformation process,an optimized enzymatic method was designed to produce(S)-2-HBA based on the construction of active enzyme aggregations induced by fusion of self-assembled hydrophobic short peptide to enzymatic C-terminal.The results showed that compared with using GFIL8 and GFIL16 short peptide,the enzyme fused with GFIL12 short peptide showed a relative higher enzyme activity in the precipitation and a higher aggreration rate,while the aggreration rate using different length peptide all reached over 90%.Moreover,the fusion of hydrophobic short peptide GFIL12 to the enzyme did not affect the enzymatic catalytic efficiency,and the k_cat/K_m value was similar to that of the wild type.The half-life(t_1/2)values of Ec TD-GFIL12,N197D_ldh-GFIL12 and Gs ADH-GFIL12were 74.6 h(50?),103 h(45?)and 70.2 h(40?),respectively,which were 6.6 times,10times and 5.4 times of the t_1/2 values of the soluble enzymes Ec TD,N197D_ldh and Gs ADH with unfused hydrophophilic short peptides,respectively.The p H stability and solvent tolerance of all the enzymes were also enhanced,the enzyme activity could be maintained in a wide p H range and under a higher concentration of solvent.These results showed that the conversion efficiency of(S)-2-HBA using GFIL12 fused enzyme aggregates was higher than unfused multi-enzyme cascade system.After the 14 h transformation,the titer of(S)-2-HBA reached 155.2 g/L,2.05 times of the highest(S)-2-HBA titer reported so far,the molar yield and space-time yield were 98.7%and 11.1 g/(L·h),respectively.When the transformation was repeated for 5 times,the relative catalytic activities was 82.1%.(4)Further purification and preparation of(S)-2-HBA in the enzyme aggregation transformation solution.The characteristics of pretreatment process including ethanol precipitation method and ethyl acetate(EA)extraction method were compared.After pretreatment procedures including centrifugation,heating,filtration and ultrafiltration,the treating solution was basically transparent and clear.The recovery of(S)-2-HBA for ethanol precipitation and EA extraction method was 98.8%and 96.3%,respectively.In the final step using ethanol for(S)-2-HBA precipitation,when the ratio of ethanol to concentrated solution is 3:1,the yield and purity of the product were relatively high with 92.2%yield and 97.0%purity.The 87.3%yield and 96.8%purity of(S)-2-HBA were obtained using EA extraction method.These results showed that the high catalytic activity of enzyme aggregates could effectively reduce the separation cost and would better serve for industrial application.