Engineering And Fermentation Of A High-yield D-arabitol Strain Of Hansenula Polymorpha

D-arabitol can be used widely in food, chemical and pharmaceutical field. However, the disadvantages of the direct extraction and chemical synthesis limit the large-scale production of D-arabitol. Nowadays, the production of D-arabitol by fermentation is recognized as a green and promising production alternative. Unfortunately, the strains for D-arabitol production used have many defects, such as low yield and much more by-products. Therefore, it is important to generate an industrial strain of Hansenula polymorpha(H.polymorpha) capable of yielding greater levels of D-arabitol.Here wild-type H.polymorpha DL-1 strains were engineered using a nitrogen ion beam. Firstly, the optimum parameters of low energy ion implantation were investigated. According to the high-yield D-arabitol strains with the ability of hypertonic-resistant, a high-throughput screening method based on the combination of high-glucose medium, paper chromatography and high performance liquid chromatography (HPLC) was determined. Secondly, the genomic DNA of H.polymorpha was transformed into wild-type H.polymorpha DL-1 to breed high-yield recombinant strains. Then, high-yield D-arabitol recombinant strains were constructed using the high-throughput screening method. Finally, the fermentation process of high-yield D-arabitol recombinant strains was optimized. The results are shown as follows:(1) H.polymorpha yeast was implanted by the nitrogen ion beam with different doses under the condition of low-energy ions energy, the "saddle" curve was made based on the survival of test strains. The implantation conditions of H.polymorpha DL-1 are shown as follows:10 sec of the implanted time and 10 sec of the interval time at an energy level of 25 keV and dose of 2.5×1016 ions/cm2 under the vacuum pressure of 1.0× 10-3Pa.(2) A high-throughput screening method was establelished. After high-glucose screening medium was used for prescreening, the combination of paper chromatography and HPLC was used for re-screening.(3) A high-yield strain with the yield of 113.76g/L designated as H.polymorpha DLg4-14 was obtained using low ion implantation technology and high-throughput screening method.(4) The optimum fermentation conditions were determined using single factor experiments, orthogonal experiment, range analysis and variance analysis. The fermentation condition is the seed culture time of 21h, the inoculums of 5% dextrose with 250g/L, and the yeast extract:ammonium sulfate (mol/mol) of 10: 1. The results showed that the glucose content has a significant influence on the yield of D-arabitol. Under the optimal fermentation condition, the yield of D-arabitol reached 119.03g/L(5) The best variable temperature was determined using variable temperature regulation experiments. After H.polymorpha DLg4-14 were cultivated for 24 hours at the optimum growth temperature of 37℃, H.polymorpha DLg4-14 was further cultured at the un-optimum growth temperature of 48℃ for 24 hours, and then H.polymorpha DLg4-14 was cultured at the optimum fermentation temperature of 34℃for 80 hours. The yield of D-arabitol reached 123.92/L, which has an increase of 3.73% compared with the yield of 119.46g/L under the condition of constant temperature fermentation.