Construction And Immobilization Of Biosynthesis System For L-phenylglycine Production

L-phenylglycine,a chiral unnatural amino acid,an important raw chemical and pharmaceutical intermediate for the synthesis of?-lactam antibiotics,such as penicillin,vanillin,pristinamycin I,and the antitumor drugs like paclitaxel,has a huge market potential in medical field.Currently,it is produced mainly by chemical synthesis method,which requires severe reaction conditions,complicated process and usually produces many by-products.The products obtained by chemical synthesis method has low optical purity,which needs further desymmetrization.Moreover,a large number of organic solvents and toxic substances are needed for chemical synthesis of L-phenylglycine,which are unfriendly to the environment.Therefore,this study focused on the development of biosynthesis system for L-phenylglycine production.The research work mainly included:construction of L-phenylglycine biosynthesis system and process optimization;scaffold-mediated multi-enzyme molecular self-assembly and magnetic immobilization;optimization of intracellular enzyme ratio and coenzyme level and whole-cell magnetic immobilization.The main findings were as follows:?1?The multi-enzyme catalytic synthesis system of L-phenylglycine from benzoylformic acid was constructed by leucine dehydrogenase from Bacillus cereus?BcLeuDH?and formate dehydrogenase from Candida boidinii?CbFDH?,and the transformation conditions were optimized.The results showed that the multi-enzyme system could be effectively used for L-phenylglycine synthesis.The optimum conversion conditions were as follows:substrate:benzoylformic acid 60 g/L,cosubstrate:ammonium formate 50.4 g/L,BcLeuDH 4 U/mL,Cb FDH 2 U/mL,NAD⁺0.14 g/L,pH 8.0,30°C.Under the optimal conditions,the conversion rate of substrate reached to 99%after 5 h,at 1 L scale,the yield of L-phenylglycine was 60.2g/L,and the ee value was>99?4?.?2?The enzyme co-aggregates with BcLeuDH and CbFDH activities were prepared by using the small protein CipB as molecular scaffolds,and they were magnetically immobilized for multi-batch conversion synthesis of L-phenylglycine.The two types of small proteins CipA and CipB from Photorhabdus luminescens were fused with BcLeuDH and CbFDH,respectively.The types of small proteins and short peptide linkers,and the fusion types were optimized.Finally,the fusion enzymes AIB-L and AIB-F with optimal expression and enzyme activity were obtained,the residual enzymatic activity of them were 68.8?4?and 52.2?4?respectively,and most of them were protein aggregates,meanwhile,the stability of them were improved.Furthermore,co-expression strain of fusion enzymes AIB-L and AIB-F was constructed,the intracellular co-aggregation of fusion enzymes AIB-L and AIB-F was achieved by molecular scaffold CipB,and the active enzyme co-aggregate AIB-M was obtained.The activity and conversional ability of AIB-M were analyzed.The results showed that the conversion rate of AIB-M was 50?4?higher than that of free enzymes with same activity.Lastly,the magnetic nanoparticles Fe₃O₄ was used for immobilization of AIB-M,and the immobilized AIB-M was used for multi-batch systhesis of L-phenylglycine.Finally,the yield of L-phenylglycine reached to 97.5g/L after 13 batches of conversion,it was improved by 62.5?4?than that of single-batch conversion.?3?The efficiency of whole cell transformation for synthesis of L-phenylglycine was increased by adjusting the enzyme ratio of BcLeuDH and CbFDH_A10C in the co-expression system.The copy number of the CbFDH_A10C gene in the co-expressing strain was varied between 1 and 4.The enzyme activity of CbFDH_A10C?0.45 U/mL?was 87.5?4?higher than that in the single-copy strain when the copy number of CbFDH_A10C was 3 in the co-expression system.Furthermore,the expression of BcLeuDH in co-expressing strains was regulated by RBS with different strengths.The ratio of Bc LeuDH to CbFDH_A10C was close to the optimal one?2:1?in recombinant strain CHL-1,and the activities of them were 1.45 U/mL and 0.65 U/mL respectively in CHL-1,meanwile,the whole cell catalytic activity of CHL-1 was increased by 3.3times.Using strain CHL-1 as catalyst,the final yiel of L-phenylglycine reached to 60g/L with conversion rate 100%and ee value>99%after 5 h,under the conditions of cell concentration 10 g/L DCW,pH 8.0,30°C,and ammonium formate:benzoylformic acid 2:1.?4?Intracellular coenzyme levels have an important influence on the coenzyme-dependent catalytic system.The strain CHL-2 was obtained by co-expression of nicotinic acid phosphoribosyltransferase?NAPRTase?and NAD⁺synthetase?NAD⁺Syn?in strain CHL-1.They are rate limiting enzymes in the salvage synthesis of NAD?H?in E.coli.The intracellular coenzyme level and cell catalytic activity were improved by 2.35 times and 56.2?4?respectively in strain CHL-2.Subsequently,coenzyme level of CHL-2 was further incresed by 2.42 times after addition of 20 mg/L nicotinic acid in the medium.Finally,for strain CHL-2,the intracellular coenzyme level and the whole cell catalytic activity were increased by9.24 and 1.4 times respectively,with the effect of intracellular key enzymes and addtion of NA in the medium,and the whole cell production efficiency was increased by 70.4?4?.Under the same conditions as before,the yield of L-phenylglycine reached to 60 g/L only in 4 h with conversion rate 100%and ee value>99%when CHL-2 as whole cell catalyst.The whole cell conversion efficiency was further improved by increase in intracellular coenzyme level.?5?Rapid separation and recycling of cells in the L-phenylglycine whole cell transformation system was achieved by magnetic immobilization of cells.According to the analysis and characterization,the prepared magnetic nanomaterials Fe₃O₄@PEI and Fe₃O₄ have standard cubic phase crystal morphology with a particle size of50-200 nm.The average thickness of the PEI coating on the Fe₃O₄@PEI surface is about 2 nm.The surface charge of Fe₃O₄@PEI is significantly higher than that of Fe₃O₄,and the isoelectric point of Fe₃O₄ is around 6,while the isoelectric point of PEI-modified magnetic nanoparticles rises to around 10,indicating that the surface of Fe₃O₄ was successfully coated by PEI.After the material preparation and adsorption conditions optimization,the E.coli cell loading of Fe₃O₄@PEI reached 4.5g DCW/g.The optimal concentration of chitosan for magnetic cell flocculation is 0.1 g/L.SEM shows that chitosan successfully coated on the surface of magnetic cell complex.The resulting immobilized cells have superparamagnetism and can be quickly separated by magnetic field,and the stability of immobilized cells was improved too.Finally,the immobilized cells were applied to the L-phenylglycine synthesis.The yield of L-phenylglycine reached to 105.5 g/L after 7 batches of conversion,at 1 L scale,and the cell yield reached to 21.1 g/g DCW,which were higher than that of single-batch conversion of free cells by 75.8?4?and 2.5 times,respectively.The whole cell transformation efficiency of L-phenylglycine was significantly improved by magnetic immobilization of cells.