Research On Enzymatic Synthesis Of 4-hydroxyisoleucine Using Cell Surface Display System In Escherichia Coli

4-Hydroxyisoleucine(4-HIL)is a non-protein amino acid with biological activities such as inducing insulin secretion,so it has attracted much attention as a potential alternative drug for treating diabetes.The existing methods for producing 4-HIL have some disadvantages such as complex process,consuming high energy and difficult extraction.In this study,we have proposed the strategy that the enzymatic synthesis of 4-HIL by cell surface.The main research and results are as follows:We had screened the transmembrane structures(LAA,LPP'OmpA)which are suitable for constructing fusion proteins with L-isoleucine dioxygenase(IDO).And we had screened the expression vectors and host strains.The strain E.coli BL21(DE3)/pTrc99a-laa-ido was constructed.The experiments of extracting membrane proteins and whole-cell catalysis experiments showed that the fusion protein LAA-IDO expressed in recombinant strains successfully and it has catalytic activity.The production of 4-HIL was 1.49 g/L.In the process of fusion protein expression,there are too many inclusion bodies.We had optimized the linker of fusion protein.Ten kinds of linkers with different lengths and types selected to reconstruct the fusion proteins.The results of enzyme catalytic showed that the productions of 4-HIL were 2.36 g/L and 2.61 g/L,which increased 53.2%and 69.5%compared with the original strain respectively when(GGGGS)4 and(Gly)20 used as linker.In order to increase the number of L-isoleucine dioxygenase on the outer membrane of host strain,the surface display strains which have two and three copies of L-isoleucine dioxygenase fusion proteins were constructed using(GGGGS)4 and(Gly)20 as linkers.When(Gly)20 used as a linker,the yields of 4-HIL that catalyzed by L-isoleucine dioxygenase double-copy and triple-copy strains were 3.13 g/L and 3.35 g/L,respectively,which were 4.68%and 12.04%higher than that of the original strain.In order to increase the number of fusion proteins,the outer membrane of the host strain modified.The genes which are encoding OmpA and OmpW were knocked out by CRISPR/Cas9.The results of enzyme catalysis showed that the yields of 4-HIL were 3.73 g/L and 3.83 g/L respectively which were 8.21%and 11.10%higher than that of the original strain.In this study,we had explored the production method of 4-HIL by surface display.After optimization and modification,the maximum production of 4-HIL can reach 3.83 g/L.This method used whole-cell as enzyme source to catalyze,which can eliminate the operation of breaking up cell and can provide reference for producing 4-HIL cleanly with low energy consumption.