Biosynthesis Of D-arabinitol And Targeted Modification Of Key Enzyme

D-arabitol,a high-value-added novel sugar substitute,has garnered widespread applications in fields such as food,pharmaceuticals,and chemicals.It is considered one of the twelve high-value-added bio-based chemicals（C3-C6）.Biological production of D-arabitol boasts advantages,including cost-effective substrates,mild conditions,and eco-friendliness,aligning with the contemporary development concepts of carbon neu-trality and carbon recycling.As such,it has emerged as a research hotspot in recent years.Hitherto,researchers have mainly focused on increasing the production of D-arabitol by selecting high-yield strains,fermenting glucose and glycerol as substrates,and optimizing fermentation using traditional methods.There has been relatively less research on using xylose as a substrate.Xylose is the second most abundant monosac-charide in nature,and xylitol,an industrial product derived from xylose,has a mature production technology,making it a low-cost industrial raw material.By constructing genetically engineered strains for the production of D-arabitol using molecular biology techniques and expanding substrate selectivity,combined with targeted enzyme engi-neering and metabolic engineering research using modern biotechnology,it is possible to provide support for more efficient D-arabitol production.In this study,the xylitol dehydrogenase（XDH）and D-arabitol dehydrogenase（Ard H）genes involved in the D-arabitol synthesis pathway from Gluconobacter thailandicus were cloned,thereafter,genetically engineered bacteria,was constructed.and then the production of D-arabitol using xylitol as substrate was explored.Furthermore,a semi-rational protein design strategy was employed to directionally modify and enhance the catalytic activity of XDH.And then,a genetically engineered strain capable of high-efficiency production of D-arabitol was established accordingly.The yield of D-arabitol was improved by fermentation optimization.The main research results are shown as follows:（1）The xylitol dehydrogenase gene（xdh）and D-arabitol dehydrogenase gene（ardh）were cloned from Gluconobacter thailandicus,and the co-expression recombi-nant vector p ETduet-xdh-ardh was constructed.This vector was introduced into E.coli BL21（DE3）host cells,resulting in the recombinant strain EC-0.Flask fermentation was carried out using EC-0 was conducted with an initial substrate concentration of 60 g/L xylitol at 37°C and 220 rpm for 60 hours.This process generated 11.99 g/L of D-arabitol with a conversion ratio of 19.98%.（2）A semi-rational protein design strategy was employed on the modification of the key enzyme XDH in the D-arabitol synthesis pathway to enhance activity.Initially,based on bioinformatics analysis and using 1ZEM.1.A as a template,a three-dimen-sional structural model of XDH was constructed via SWISS-MODEL.Subsequently,molecular docking of the substrate xylitol with XDH was performed to analyze the critical amino acid residues within a 5（?）range of the pocket space which may influence the catalytic performance of XDH.In conjunction with multiple sequence alignment analysis,non-conserved amino acid residues were identified for site-directed mutagen-esis,and then a mutant library was constucted.The single mutants XDH^G161A and XDH^S163A,and the double mutant XDH^G161A/S163A with improved enzymatic activity were selected.These three mutants successfully expressed a 28 k Da protein in E.coli BL21（DE3）.Compared with the wild-type XDH,the enzymatic activity of XDH^G161A,XDH^S163A and XDH^G161A/S163A increased to 39.82 U/mg,45.83 U/mg and 50.58 U/mg,respectively.The activity of XDH^G161A/S163A was 1.60 times higher than that of the XDH.（3）Based on the results of XDH’s site-directed mutagenesis,the genetically engi-neered strains E.coli BL21/p ETduet-xdh^G161A-ardh（referred to as EC-1）,E.coli BL21/p ETduet-xdh^S163A-ardh（referred to as EC-2）,and E.coli BL21/p ETduet-xdh^G161A/S163A-ardh（referred to as EC-3）were constructed to investigate their D-arabi-tol fermentation production capabilities.Results showed that the ability of these engi-neered strains to produce D-arabitol followed the order of EC-3>EC-2>EC-1>EC-0.（4）Through single-factor experiments,the fermentation conditions of the genet-ically engineered strain EC-3 were optimized,including medium composition and cul-ture conditions.The optimal medium composition was:initial xylitol 80 g/L,peptone20 g/L,sodium chloride 5 g/L,yeast extract 12 g/L,（NH₄）₂SO₄ 2 g/L,K₂HPO₄ 14 g/L,KH₂PO₄ 5 g/L,and Mg SO₄ 1.8 g/L.The optimal culture conditions were:inoculation age of 12 h,inoculation volume of 12%,fermentation temperature of 31°C,shaking speed of 210 rpm,and fermentation time of 36 h.Under optimal conditions,the EC-3strain can produce D-arabitol at 18.44 g/L,with residual xylitol concentration at 31.82g/L and a conversion ratio of 23.05%.Compared to EC-0,the yield and conversion ratio increased by 4.77 g/L and 5.97%,respectively.