Synthesis Of 2,5-Furandicarboxylic Acid By Recombinant E.coli_VDH1_HmfH Cells

2,5-Furandicarboxylic acid(FDCA)is an important biobased building block to produce biobased polymers,pharmaceuticals,and metal-organic frameworks.FDCA can be synthesized via the oxidation of the biobased platform chemicals 5-hydroxymethylfurfural(HMF).Currently,the chemical methods remain predominant in the synthesis of FDCA,because of short reaction periods and high substrate conversions.However,they suffer from many issues such as using metal catalysts and requiring harsh reaction conditions.In terms of the selectivity and conditional mildness as well as environmental friendliness,biocatalysis appears to be more promising compared to chemical catalysis.However,biocatalytic oxidation ofHMF to FDCA remains challenging,since HMF is highly toxic and inhibitory toward biocatalysts,thus resulting in poor substrate tolerance and low catalytic efficiencies of biocatalysts.Recently,high HMF-tolerant aldehyde dehydrogenases(ALDHs)with high activities were mined from Comamonas testosteroni SC1588 by our group,but these enzymes are only capable of oxidizing the formyl group present in HMF.It was previously reported that HMF/furfural oxidoreductase(HmfH)enabled efficient oxidation of the primary hydroxyl in HMF;unfortunately,its functional expression was hard to be achieved in Escherichia coli.Therefore,the coexpression of ALDHs with HmfH in E.coli was attempted for constructing an efficient and selective biocatalytic approach toward FDCA in this thesis.Then,the mechanistic insights into the toxicity ofHMF toward the recombinant cells were provided.The major results were summarized as follows.1.Constructing an efficient biocatalytic approach toward FDCA.It was found that the functional expression ofHmfH was accomplished under the control of a Trc promoter in E.coli when a low-copy-number plasmid vector p ACYCDuet1 harboring an optimized ribosome binding site sequence(RBS11)was used.The optimal p H and temperature were 7.0 and 30 °C,respectively,for the oxidation ofHMF catalyzed by E.coli?VDH1?HmfH.Under the optimal reaction conditions,the tolerance level of E.coli?VDH1?HmfH cells toward HMF was approximately 150 m M.When the substrate concentrations were lower than 150 m M,the yields of FDCA could reach 94-98%.The toxicity and inhibition of FDCA toward microbial cells were mainly attributed to its acidity.When the reaction system was scaled up to 50 m L,approximately 101 m M of FDCA was formed after 36 h.FDCA was isolated with a yield 92% via acid precipitation combined with tetrahydrofuran(THF)extraction.FDCA of 0.7 g was obtained with the purity of 99% and a space-time yield up to 0.4 g/L h.2.Mechanistic insights into the toxicity ofHMF toward the cells.It was found that HMF had a strong toxic effect on the E.coli?VDH1?HmfH during the catalytic process.The cell viability decreased with the elongation of the incubation time.Besides,the presence ofHMF caused the increased intracellular reactive oxygen species(ROS)levels,damage the cell membranes,and reduce the activities of the intracellular vanillin dehydrogenase 1(VDH1)and HmfH as well as the intracellular NAD(H)and ATP levels.In this study,a novel approach toward FDCA catalyzed by E.coli?VDH1?HmfH was preliminarily constructed,and the mechanistic insights into the toxicity ofHMF toward a whole-cell biocatalyst were provided.This study may not only enrich the theoretical knowledge in biocatalytic upgrading of biobased platform chemicals,but also lay a technical foundation for the industrial production of FDCA.