Heterologous Expression And Molecular Modification Of Proline-4-hydroxylase

Trans-4-hydroxy-L-proline is an important unnatural amino acid,widely used in food,cosmetics,medicine,chemical industry and other fields.Now the market demand is increasing constantly.The price is more than 600,000 yuan per ton.Biocatalysis has mild reaction condition,process and high optical purity product,and is environmentally friendly,thus has gradually attracted the attention of researchers.The development of technology for trans-4-hydroxy-L-proline production with high efficiency and low cost is currently a hot issue in biocatalysis field.In order to improve the production efficiency of trans-4-hydroxy-L-proline via biocatalysis,in this work we studied the key factors affecting trans-4-hydroxy-L-proline biosynthesis,including the activity of the key enzyme proline-4-hydroxylase,biotransformation system and conditions.Five recombinant Escherichia coli strains harboring genes encoding proline-4-hydroxylase,constructed and deposited in our laboratory were used for the study.The main research contents and results are as follows:（1）Based on fermentation cultivation,induced expression and SDS-PAGE analysis of five proline-4-hydroxylase recombinant E.coli strains constructed by our laboratory previously,Three strains E.coli BL21（pET28b-p4hP）,E.coli BL21（pET28b-p4hBr）,and E.coli BL21（pET28b-p4hBa）withsuccesfulexpressionof proline-4-hydroxylase were screened.They were named 17908,17909,and 17911 respectively for short.Then the whole-cell catalytic synthesis conditions and systems for trans-4-hydroxy-L-proline production with the three strains were optimized respectively.The optimized reaction conditions were determined:temperature 35℃,the MES buffer,pH 6.5,30℃overnight incubation;the optimized reaction system were as follows:8 mM 2-oxoglutarate,6 mM L-ascorbic acid,Fe²⁺concentration was 0.8 mM for 17908 and 17909,and 1.2 mM Fe²⁺for 17911.After optimization,whole-cell catalytic synthesis of trans-4-hydroxy-L-proline with three strains 17908,17909 and 17911 were all improved,trans-4-hydroxy-L-proline titer reached 34.86 mg/L,30.66 mg/L,31.43mg/L,respectively;increased by 32.25%,26.85%and 26.85%compared with that of pre-optimization.（2）In order to increase the soluble expression level of proline-4-hydroxylase,expression vector pET-32 a（+）with soluble label was used for cloning and expression of proline-4-hydroxylase;through molecular operation,plasmid（pET32a-p4hP）and recombinant E.coli BL21（pET32a-p4hP）were succesfully constructed;In order to enhance the expression level of proline-4-hydroxylase,the original T7 promoter of pET28b-p4hP and pET32a-p4hP was replaced with a trp tandem promoter,respectively;and recombinant E.coli BL21（pET28b-ptrp2-p4hP）and E.coli BL21（pET32a-ptrp2-p4hP）were constructed;through the analysis of proline-4-hydroxylase expression and the enzyme activity,we found that proline-4-hydroxylase of E.coli BL21（pET28b-ptrp2-p4hP）and E.coli BL21（pET32a-ptrp2-p4hP）was almostly not expressed;but soluble protein expression level of E.coli BL21（pET32a-p4hP）was improved substantially compared with that of E.coli BL21（pET28b-p4hP）,indicading that the problem of low soluble expression level of proline-4-hydroxylase could be resolved;However the ability of trans-4-hydroxy-L-proline biosynthesis of whole-cell ability was not significantly improved.（3）In order to improve the trans-4-hydroxy-L-proline synthesis ability of the proline-4-hydroxylase recombinant E.coli strains 17908,17909,and 17911,site-saturation mutagenesis was conducted for the molecular modification of proline-4-hydroxylase.With the software BioXM2.6,we designed primers for PCR on site-saturation mutagenesis forproline-4-hydroxylase,theconcentrationof trans-4-hydroxy-L-proline in the catalytic reaction supernatant was determined by chloramine T method and amino acid analyzer.The results showed that proline-4-hydroxylase of two strains E.coli BL21（pET28b-p4hBr）and E.coli BL21（pET28b-p4hBa）were not screened out forward-mutant after mutation;In the seven mutations library of proline-4-hydroxylase P4HP,the enzyme activity of mutant proline-4-hydroxylase F137R and Y205K were obviously improved.With0.35 g/L L-proline,55.09 mg/L and 52.29 mg/L trans-4-hydroxy-L-proline were synthetized,which was increased by58%and 50%than the original strain respectively.The results of molecular docking with software AutoDock showed that the active centers of the mutant enzyme F137R and Y205K was not apparently changed.The reason why enzymatic activity was improved might be that the protein crystal structure were slightly changed after site mutation,which increased the affinity of proline-4-hydroxylase with substrate or co-substrate.