Screening Of Xylitol Producing Strains And The Biosynthesis Of Xylitol

Xylitol is a naturally occurring five-carbon sugar alcohol and widely exists in fruits, vegetables and algae. Xylitol has a sweetening power similar to sucrose but a lower caloric value. In addition, xylitol has anti-cariogenic property and its metabolism in human body does not require insulin. Due to these properties, xylitol has important applications in food, odontological and pharmaceutical industries. Although xylitol is industrially produced through the chemical reduction of D-xylose, this method is uneconomical and caused pollution due to the requirements of pure D-xylose as well as high temperature and pressure. Alternatively, the biosynthesis of xylitol has attracted large research attention as an economical and environmentally-friendly method because the conditions of the reaction are mild and high pure xylose is not needed in the process.In this work, a wild strain SK36.001 was isolated from soil and it showed an effective ability of producing xylitol. Based on the results of morphological observation, physiological characteristics and 26 S rDNA D1/D2 area sequence analysis, strain SK36.001 was identified as Candida tropicalis and named as C. tropicalis SK36.001, which was deposited at China Center for Type Culture Collection(CCTCC, M 2014428). The 26 S rDNA D1/D2 area gene sequence included 570 bases and was submitted to GeneBank with the accession number of KT 945155.In order to improve the production capacity of C. tropicalis SK36.001, strain mutagenesis was performed through ARTP(atmospheric and room temperature plasma) method and the mutant T31 was screened among more than 200 mutants with the highest xylitol yield of 61%, which increased by 22% compared to the wild-type. To study the mutant T31 genectic stability, 7 generations were cultured, and the results showed that the strain still maintained high xylitol yield. For further explaining the higher xylitol yield in mutant strains, the xylose reductase(XR) activities in several positive mutants and wild strain were evaluated and compared. Meanwhile, the relative expression level of XYL1(which encode xylose reductase) in both wild strain and high-yield mutants was quantified. The enzyme assay results revealed that the mutants with higher xylitol yield had sigificantly higher XR activities than that in wild strain. The real-time results indicated that the XYL1 expression level in the mutants was obviously higher than that in the original strain.To further improve the performance of the mutant T31, the medium components and growth conditions were optimized on a flask scale through single factor experiments and the orthogonal test. The optimium fermentation medium included: 100 g/L xylose, 2 g/L yeast extract, 4 g/L(NH4)2SO4, 0.5 g/L MgSO4·7H2O. The optimium fermentation conditions were: initial medium pH 5.0, temperature 28oC, shaking speed 200 r/min, inoculation 4%. The maximum xylitol yield of T31 reached 79% under the optimum conditions after cultivated for 30 h, and the improvement of the xylitol yield was markedly.Based on the data obtained from fermentation process on flask scale, the fermentation scale-up experiment was performed with mutant C. tropicalis T31 in 7-liter fermenter. A preliminary investigation was made into the effect of different agitator speeds on xylitol production, xylose consumption and biomass in batch fermentation mode. The fermenter conditions were controlled as following: work volume 3 L, medium initial pH 5.0, temperature 28oC, and the aeration rate was 1.0 vvm. Based on the results of the fermentation process, a two-stage agitator speed controlling strategy, in which the agitator speed was controlled at 500 r/min in the first 18 hours and then shift to 300 r/min, was suggested and experimentally verified. Following this strategy, the maximum xylitol production reached 77.5 g/L. Meanwhile, the mathematic models of cell growth, product accumulation and substrate consumption in batch fermentation mode under strategy described above were built according to the Logistic equation and the Luedeking-Piret equation. The parameters of kinetic models were estimated by none-linear fitting with OriginPro 8.0 software and the parameters of the models were obtained. The results turned out that the models could fit the batch fermentation process of C. tropicalis T31 well, which would benefit the optimizing control of xylitol fermentation in industrial producing.