Construction Of An Efficient Trichosporonoides Oedocephalis Mutant With Gene Deletion Of HOG1 And Research Of The Fermentation Regulation

Trichosporonoides oedocephalis produces large amounts of polyols(e.g. erythritol, ribitol and glycerol) in culture. Erythritol is internationally recognised as food additives due to its low calorific value, and ribitol is one type of important pharmaceutical intermediates for antiviral medicines. However, glycerol is the by-product of erythritol production. It not only makes it difficult to separate erythritol from the culture but also limits the increasing of erythritol yield.HOG1 genes can activate genes which are related to glycerol synthesis enzymes. The center signal cascade system of HOG1 signaling pathways that consists of the MAP kinase(mitogen- activated protein kinases, MAPK) can adjust production of polyol under high osmotic pressure.In this paper, we controlled production of polyols from the level of molecule and fermentation. It mainly included constructing an efficient T. oedocephalis mutant with gene deletion of HOG1 and researching the fermentation regulation. Firstly, in order to get an excellent method for T.oedocephalis transformation. The study investigated an achievable method combining with Zymolyase-100T(an enzyme to digest yeast cell wall).Transformed cells was selected on G-418 sulfate(G418) media. Concentration of zymolyase- 100, digestion time, lithium acetate, calcium chloride and lithium chloride were the five single factors that we set for the influence study on the T. oedocephalis transformation efficiency. Through single factor experiments, we further optimized the method for T. oedocephalis transformation by response surface methodology. The optimal method for T. oedocephalis transformation was as follows: 30.17 μ g/m L Zymolyase-100 T, 40.04 min incubation, and 99.05 μ L/m L Ca Cl2. The predicted transformation efficiency of T. oedocephalis reached 44.30 %. Secondly, TO-HOG1 null mutation was constructed in T. oedocephalis by loxp-kan-loxp/cre system as target gene replacement to control production of polyols via molecular level. The engineering strains should be cultured and sub-cultured for 6 generations to keep genetically stable. Finally, research the difference between TO-HOG1 mutant and original strains by fermentation culture. In order to study the effect of citric acid on the production of polyols by TO-HOG1 mutant and original strains, citric acid was added to the media. Yield of erythritol was up to 69.12 g/L, when the concentration of citric acid was 0.3 %. Single factor experiment was performed to improve the yield of T. oedocephalis. The result of single factors are: glucose concentration is 20 %, sodium chloride concentration is 0.75 g/L, speed is set at 200 r/min, yeast powder concentration is set in 1.0 %, magnesium sulfate concentration is 0.7 g/L. This paper provides theoretical and technological support for the industrialized production of erythritol.