Rare sugars are monosaccharides and derivatives that only occur in very small amounts in nature.Despite their limited natural abundance,rare sugars are potentially useful in a wide range of applications in pharmaceutical,medicinal,food,and synthetic chemistry and thus hold tremendous economic value.Most L-form sugars are rare in nature.Due to their improved biological activity and lower toxicity,L-form rare sugars are important precursors for certain pharmaceutical drugs.Some L-form rare sugars have been widely used for the production of antiviral and anticancer agents in medical fields.L-Xylulose and L-xylose are rare in nature and are isomers of each other.These aldo-and ketopentose are expensive but offer a wide range of potential applications in medical,pharmaceutical,and synthetic chemistry.L-Xylulose inhibits a-glucosidase and can be used to decrease blood glucose.It is also an indicator of hepatitis or liver cirrhosis for medical or clinical diagnosis and pentosuria,which is a rare autosomal recessive trait.In addition,L-xylulose is used to produce other rare sugars such as L-xylose,L-ribose,L-ribulose,or L-lyxose.L-Xylose is an important pharmaceutical intermediate that is used in the synthesis of numerous antiviral and anticancer drugs.L-xylose derivatives have also shown an inhibitory effect on urinary glucose reabsorption in vivo,suggesting their potential use for the treatment of diabetes.The chemical production of L-xylulose and L-xylose is complex with very low yields,as a result,both sugars remain very expensive.Higher yields and cost-effective processes are feasible using microbial or enzymatic methods.To date,there are reports on microbial production of both L-xylulose and L-xylose using very low substrate concentrations.However,none of those reports have been applicable to industrial-level production due to lower yields or the use of expensive starting materials.The economic feasibility of the biological production of the two sugars remains unsolved,hence the supply of L-xylulose and L-xylose is insufficient and at a high price in the market.In this study,the biosynthesis of L-xylulose and L-xylose was reported,using engineered whole E.coli cells.This dissertation includes the following part1.Enhanced L-xylulose biotransformation from xylitol.Recombinant E.coli expressing the xylitol-4-dehydrogenase（XDH）gene of Pantoea ananatis was constructed.A cost-effective culture media containing 27.3 g/L molasses,6.3 g/L corn steep liquor,and 9.5 g/L yeast extract was developed and media components were optimized using response surface methods to cultivate the recombinant E.coli strain constructed and used for L-xylulose production.The reaction conditions for the biotransformation of xylitol to L-xylulose such as temperature,pH,cell biomass,and substrate concentrations were also optimized in order to maximize the Lxylulose product.A high conversion rate of 96.5%was achieved under an optimized pH and temperature using 20 g/L xylitol,which is the highest among the reports available.The recombinant E.coli cells expressing the xdh gene were immobilized in calcium alginate to improve the recycling of cells.Effective immobilization was achieved with 2%（w/v）sodium alginate and 3%（w/v）calcium chloride.The immobilized E.coli cells retained were used for nine batches with a conversion between 53 and 92%which would be beneficial for the economical production of L-xylulose.2.Efficient L-xylulose biosynthesis using whole-cell biocatalyst with NAD+regeneration system.The XDH of P.ananatis is NAD+dependant dehydrogenase enzyme.Because the oxidation of xylitol using XDH reduces NAD+into NADH,the supply of NAD+is necessary to continue the process at higher xylitol concentration.Synthesis of L-xylulose by microbial oxidation of xylitol on an industrial scale demands efficient cofactor regeneration methods.Hence,cofactor engineering was applied to improve the efficiency of L-xylulose production from xylitol.A water-forming NADH oxidase from Streptococcus mutans ATCC 25175 was introduced into E.coli with XDH of P.ananatis resulting in recombinant cells harboring the vector pETDuet-xdh-SmNox.The co-expression system exhibited optimal activity at a temperature of 37℃ and pH 8.5,and the addition of Mg2+ enhanced the catalytic activity by 1.19 fold.The co-expression of NADH oxidase with the XDH enzyme increased the catalytic activity of XDH to 16.34 U/mL compared to E.coli BL21-pETDuet-l-xdh（11.63 U/mL）.After 20 h,the intracellular NAD+concentration of E,coli BL21-pETDuet-1-xdh-SmNox was increased to 18.9 μM which was higher than E.coli BL21 pETDuetl-xdh.To further harness the potential of the co-expression biocatalyst system developed,the initial cell biomass and substrate concentration were optimized,and maximum L-xylulose product was achieved using 10 gDCW/L of cell biomass and 50 g/L of xylitol.Using a 1 L bioconversion system the final concentration and productivity of L-xylulose from 50 g/L of xylitol reached 48.45 g/L,and 2.42 g/L.h respectively under the optimized reaction conditions.Overall,the study is a suitable approach for the large-scale production of L-xylulose from xylitol.3.Biosynthesis of L-xylose from xylitol using a dual enzyme cascade in Escherichia coli.A new strategy was developed for the production of L-xylose from the inexpensive starting material xylitol through L-xylulose intermediate using a dual enzyme cascade system coexpressed in E.coli.The L-fucose isomerase（L-FucI）gene from E.coli W and the xdh gene from P.ananatis ATCC 43072 were cloned and co-expressed in E.coli,resulting in recombinant cells harboring the vector pET28a-xdh/LfucI.A ribosomal binding site was inserted before the start codon of the L-FucI gene.The dual enzyme cascade system exhibited optimal activity at a temperature of 40℃ and pH 10.0,and the addition of 7.5 mM Zn2+ enhanced the catalytic activity by 1.5-fold.The effect of the amount of whole-cell catalyst harboring the dual enzyme cascade system and xylitol concentration on L-xylose production was also evaluated.The maximum L-xylose yield was obtained at OD600 of 80 cell biomass and 80 g/L of xylitol concentration.Using the dual enzyme cascade co-expression system,52.2 g/L of L-xylose was produced from 80 g/L of initial xylitol concentrations,corresponding to a conversion rate of 65%and productivity of 1.86 g/L.h.The study provides an economically feasible approach for the production of L-xylose from xylitol using a dual enzyme co-expression system harboring L-fucI and XDH in a one-pot reaction.4.Using deep eutectic solvents（DESs）increases the biosynthesis of rare sugar.The synthesis of L-xylulose and L-xylose from xylitol using whole-cell biocatalysts was improved by deep eutectic solvents（DESs）based on choline chloride（ChCl）formulation.The promotion effects on the product yield were observed in reactions with the presence of DESs ChCl/ethylene glycol and ChCl/urea,but ChCl/glycerol suppressed the reaction.DES compositions in the ratio of 2:1 displayed an improved yield than 1:1.The improvement effects appeared in the reactions with low DES contents（2.5%）.The enhancement of the efflux of intracellular molecules was proved by an analysis of the nucleic acid and protein contents in the medium.DES assisted in the increase in cell membrane permeability,accelerating quick contacts between the enzyme and substrate and improving the conversion rate.