Production Of 2,5-furandicarboxylic Acid Using Whole-cell Biotransformation By Recombinant Raoultella Ornithinolytica BF60

2,5-Furandicarboxylic acid?FDCA?is an important bio-based building block and has been widely used in the polymers,medicines,and other industrial areas.Currently,numerous methods have been reported for oxidation of 5-hydroxymethylfurfural?HMF?to FDCA.However,some limitations for these methods,such as high pressure/temperature and noble or non-noble additives for chemical reactions,low HMF concentration used for enzymatic reactions and low production was obtained from fermentation.Compared with these methods,whole-cell biotransformation was considered as a promising approach.In this work,we engineered a newly isolated Raoultella ornithinolytica BF60 strain as a whole-cell biocatalyst for FDCA production from HMF.Furthermore,the conversion conditions and metabolic mechanism of HMF were studied.The main results achieved in this study are highlighted as follows.?1?The strain R.ornithinolytica BF60 which could produce FDCA from HMF was isolated from soil.Subsequent studies on HMF tolerance and FDCA production by R.ornithinolytica BF60 showed that cell growth was severely inhibited when HMF concentration was higher than 40 mM,and the higher FDCA production?5.4 mM?could be obtained when HMF concentration was 10 mM.To relieve the effect of HMF on cell growth,whole-cell biotransformation was used for FDCA production,and higher catalytic efficiency was achieved when the later logarithmic phase cells was used.The optimal conditions for FDCA production were as follows:OD₆₀₀=100 whole-cell biocatalyst,100 mM HMF,30^oC,and 50 mM sodium phosphate buffer?pH 8.0?.Under this optimal conditions,51.0 mM FDCA with 51.0%yield was obtained.?2?The synthesis efficiency of FDCA was further improved by reducing the degradation of FDCA,optimizing the expression of the key genes in FDCA synthesis pathway,and HMF pulse-feeding strategy.The degradation of FDCA was reduced by 66.7%and 58.3 mM of FDCA was achieved through dcaD gene deletion.Subsequently,various expression cassettes of hmfH and hmfO genes were constructed for tuning FDCA synthesis from HMF with different strength promoters and different copy number plasmids,and 108.9 mM FDCA was obtained when hmfH was expressed under the control of trc promoter.Additionally,different RBS mutants were designed and assembled into hmfH expression cassettes,and 139.6 mM FDCA was achieved.Finally,FDCA production was further improved to 157.0 mM with a yield of 78.5%by the HMF pulse-feeding strategy.?3?The metabolic mechanism of HMF in R.ornithinolytica BF60 was analyzed based on transcriptomics and proteomics data.Firstly,the transcriptomics data under HMF stress at 12h and 36 h were analyzed,and it was found that 2502 and 2675 genes were significantly differentially expressed,respectively.These significantly differentially expressed genes mainly involved in intracellular redox process,transport process and central metabolic process,and maybe helpful in cells to resist and repair the injury caused by HMF.Subsequently,proteomics data under HMF stress was analyzed and 34 proteins showed significant differentially expressed and were identified as participating in intracellular translation,redox process,nucleotide metabolism and central metabolic pathways.Finally,based on the comprehensive analysis of transcriptomics and proteomics data,a total of 37genes which possibly participated in FDCA synthesis were predicted.?4?The production and yield of FDCA were further improved by reducing the formation of byproduct 2,5-dihydroxymethylfuran?DHMF?and eliminating the accumulation of intermediate 5-formyl-2-furoic acid?FFA?.To reduce the formation of DHMF,aldR,dkgA,akR,adhP1 and adhP2 genes were identified and combinatorially deleted based on the transcriptomics and proteomics data,89.3%reduction in DHMF formation and 175.6 mM of FDCA was achieved.In addition,after adhP3 and alkR genes deletion,DHMF formation was further reduced and FDCA production was increased to 242.0 mM.Subsequently,the newly identified aldehyde dehydrogenase?AldH?was overexpressed to eliminating the accumulation of FFA,and 264.7 mM(41.3 g·L^-1)FDCA with a yield of 96.2%was obtained.