Studies On Fermentative Production Of L-2-aminobutyric Acid By Recombinant Escherichia Coli

L-2-aminobutyric acid(L-ABA),a non-proteinogenic amino acid,has been used as a precursor for synthesis of many chiral drugs.With the increasing market demand for L-ABA in both pharmaceutical and chemical industries in recent years,the preparation of optically pure L-ABA with high efficacy has attracted much attention.In this study,we expand the nature metabolic network of Escherichia coli W3110 using metabolic engineering approach for the production of L-2-aminobutyric acid.L-threonine was taken as a precursor for L-ABA synthesis based on threonine deaminase and leucine dehydrogenase.In accordance with the previous reports,a threonine producing strain E.coli THR was firstly constructed,which could produce 12.45 g/L L-threonine in shake flask at 35 ?C for 48 h.Then the ilvA gene from E.coli W3110 was amplified and overexpressed in E.coli THR.An ilvA overexpression strain E.coli THR/ pTrc-ilvA was constructed,which could produce 4.38 g/L 2-KB.The feedback inhibition of ilvA could be removed by replacing the 1054 th T with G,1055 th T with C,1084 th C with T,1085 th G with T and 1086 th T with C(F352A,R362F)using site-directed mutagenesis to obtain pTrc-ilvA*.The 2-KB titer of E.coli THR/pTrc-ilvA* increased by 83.79% which was up to 8.05 g/L.Moreover,two different sources of dehydrogenase,including leuDH from Thermoactinomyces intermedius and BleuDH from Bacillus cereus were amplified and overexpressed,respectively.Results showed that the accumulation of L-ABA reached to 5.39 g/L and 3.16 g/L in E.coli THR/pTrc-leuDH and E.coli THR/pTrc-BleuDH respectively,which were cultivated in TPM medium with additional feeding of 10 g/L 2-KB.These results demonstrated that the dehydrogenase leuDH from T.intermedius displayed a higher specific activity in E.coli THR than that of BleuDH from Bacillus cereus.Finally,the co-overexpression strain E.coli THR/pTrc-ilvA*-leuDH was constructed,which could produce 3.09 g/L L-ABA from glucose in shake flask at 35 ?C for 48 h in TPM medium.With the purpose of decreasing the L-threonine export capacity,the gene rhtA and rhtC were deleted from the E.coli THR strain chromosome,resulting in three strains E.coli THR ?rhtA/pTrc-ilvA*-leuDH,E.coli THR ?rhtC/pTrc-ilvA*-leuDH and E.coli THR ?rhtA?rhtC/pTrc-ilvA*-leuDH.The result of strain E.coli THR ?rhtA/pTrc-ilvA*-leuDH displayed higher concentration of L-ABA than the other two strains.The deletion of rhtA in the chromosome led to the increase of L-ABA concentration from 3.09 g/L to 3.72 g/L and the remaining L-threonine decrease from 3.47 g/L to 0.22 g/L.Then,the gene ilvIH from the E.coli THR ?rhtA chromosome was knocked out to reduce the metabolic flux from 2-KB to L-isoleucine,the resulting strain E.coli THR ?rht A ?ilvIH/pTrc-ilvA*-leuDH was capable of producing 4.42 g/L L-ABA.Furthermore,in order to obtain a better expression of ilvA* and leuDH,different promoters were respectively inserted or replaced before the two genes to regulate their expression,resulting in L-ABA accumulation up to 4.86 g/L.Finally,L-ABA titer of the optimal strain E.coli THR ?rhtA? ilvIH/Gap-ilvA*-Pbs-leuDH reached to 9.33 g/L with a yield of 0.19 g/L/h in a 5 L bioreactor for a total 60 h of fed-batch fermentation.This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of L-ABA.