Abstract
BackgroundYarrowia 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.ResultsFor 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.ConclusionsAlkane-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.
Highlights
Cytochrome P450s (CYPs) are a large, ubiquitous family of heme-containing monooxygenases that are responsible for the oxidative metabolism of a wide variety of drugs, environmental chemicals and endogenous compounds, such as steroids, prostaglandins and fatty acids [1]
Several important CYPs such as the vitamin D3 25-hydroxylase CYP27B1 are associated to mitochondrial membrane [4]
Expression takes place in different tissues, but the highest levels are found in the liver, where CYPs have the principal function to introduce an oxygen atom into hydrophobic substrates
Summary
Cytochrome P450s (CYPs) are a large, ubiquitous family of heme-containing monooxygenases that are responsible for the oxidative metabolism of a wide variety of drugs, environmental chemicals and endogenous compounds, such as steroids, prostaglandins and fatty acids [1]. Most cytochrome P450 systems are composed of a monooxygenase and one or two additional proteins, constituting an electron transfer chain. Genes encoding these components are either expressed individually or linked resulting in self sufficient CYPs. To some extent, the natural electron transport chain from NAD(P)H to the heme containing cytochrome P450 can be replaced by either homologues or different proteins with similar function e.g. flavodoxin and flavodoxin reductase to support catalytic activity [2]. 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
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