Effect of cultivation conditions on heterologous expression of oxidoreductases by the yeast Blastobotrys adeninivorans

[摘要] English: Different oxidoreductases are being investigated for hydroxylation reactions to satisfy the global need for environmentally friendly industrial processes. These enzymes are found in microorganisms, animals and plants. Comparative study of different yeasts has shown the best heterologous expression of the fatty-acid hydroxylase (CYP505A1) and vanillyl-alcohol oxidase (VAO) in B. adeninivorans UOFS Y-1220. In an attempt to improve this, we studied the effect of cultivation conditions on biomass production and the expression of both genes. Medium constituents and bioreactor conditions that play significant roles and the nature of those roles on CYP505A1 and VAO expressions were investigated. Plackett-Burman shake-flask studies revealed that vitamins, substrate concentration, inoculum size, buffer concentration, initial pH and culture volume had significant effects on biomass production, while factors significant for VAO expression were buffer concentration and initial pH. Higher culture volume and vitamins had a negative effect on biomass production while substrate concentration, inoculum size, buffer concentration and initial pH showed a positive effect. The yeast preferred potassium phosphate buffer, glycerol as carbon source and longer cultivation times but not δ-aminolevulinic acid although these were statistically insignificant. CYP505A1 expression was better when using MES buffer, glycerol and shorter cultivation times but not δ-aminolevulinic acid. In addition, we observed that substrate concentration and culture volume had negative and positive effect, respectively. Higher buffer concentration and initial pH had a positive effect on VAO expression. Moreover, though not statistically significant, substrate concentration and culture volume had negative effect while vitamins, metals, basal salts and inoculum size showed positive effect. Lastly, high DOT, in the bioreactors, had positive impact on biomass production while it decreased the level of VAO expression. In conclusion, the importance of cultivation conditions for gene expression has been highlighted and thus, based on this study, further research on medium composition and bioreactor cultivation can be pursued for optimization purposes.

[发布机构] University of the Free State