Institute of Molecular Biotechnology, Graz University of Technology, ACIB GmbH, Petersgasse 14, Graz, Austria.
Microb Cell Fact. 2012 Aug 9;11:106. doi: 10.1186/1475-2859-11-106.
Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase.
For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems.Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains.
Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.
解脂耶氏酵母能够有效地代谢和吸收疏水性化合物，如正烷烃和脂肪酸。这种酵母天然分泌的乳化剂以及经过修饰的细胞表面疏水性和突起，使有效底物吸收成为可能。我们正在研究重组解脂耶氏酵母作为生物催化剂用于氧化难溶性疏水性甾体的潜力。此外，还评估了两相双液相培养系统以增加底物的可用性。在这种培养系统中，细胞与水溶性营养素一起保留在水相，而底物和大部分产物则包含在第二相水不混溶的有机溶剂相中。
我们首次在解脂耶氏酵母中共表达了人细胞色素 P450 2D6 和 3A4 基因以及人细胞色素 P450 还原酶（hCPR）或解脂耶氏酵母细胞色素 P450 还原酶（YlCPR）。这些全细胞生物催化剂用于在两相系统中转化疏水性差的甾体。在使用有机溶剂和解脂耶氏酵母碳源油酸乙酯的两相系统中进行孕酮的全细胞生物转化时，在 1.5 L 搅拌釜生物反应器中的初始特异性羟化速率进一步提高了 2 倍。此外，与水相相比，产物形成显著延长。与共表达天然解脂耶氏酵母 CPR 基因相比，共表达人 CPR 基因可使特异性活性提高 4-10 倍。与单拷贝菌株相比，多拷贝转化体的活性提高了 50-70 倍。
烷烃同化酵母解脂耶氏酵母与所描述的表达策略相结合，证明了其在两相双液相系统中转化疏水性底物的巨大潜力。特别是细胞能够有效吸收和/或代谢的有机溶剂可能比水相更有效地进行生物转化，甚至可以实现简单、连续或至少长时间高收率的过程。
