Molecular cloning and expression of cytochrome P-450 monooxygenases from Rhodotorula spp. in Yarrowia lipolytica

English: Cytochrome P450s are heme-containing monooxygenases that are widely distributed in nature and found in all kingdoms. These monooxygenases play a vital role in cell processes such as carbon assimilation, biosynthesis of hormones and detoxification of drugs and xenobiotics. Understanding the mechanism of action of the cytochrome P450s can help to develop effective therapeutic drugs, understand plant-pathogen interactions and eliminate pollutants in nature. Yeasts belonging to the basidiomycetous genus Rhodotorula can grow on non-carbohydrate carbon sources such as alkanes, aromatic compounds and even monoterpenes. Only one cytochrome P450 encoding gene, that of a benzoate-para-hydroxylase (CYP53B1) has been isolated from a basidiomycetous yeast, Rhodotorula minuta. Yarrowia lipolytica is an ascomycetous yeast that can metabolise hydrophobic substrates such as n-alkanes and fatty acids. A wide range of expression tools have been developed for Y. lipolytica. It was the goal of this study to isolate a second cytochrome P450 encoding gene, coding for an alkane or fatty acid hydroxylase, from a limonene utilizing Rhodotorula sp. and to express this gene as well as the CYP53B1 gene from R. minuta in Y. lipolytica. PCR (using primers based on the sequences of the helix I and heme binding domains of 15 alkane and fatty acid hydroxylases from the ascomycetous yeasts) and IPCR were used to clone a cytochrome P450 encoding gene from the limonene utilizing strain Rhodotorula sp. CBS 8446. The sequence analysis of the full-length gene sequence showed that the amplified gene is interrupted by introns and southern hybridization showed that only one copy of the gene was present in the genome. By using gene specific primers the full-length cDNA sequence that included the start and stop codons was isolated. Comparison with the protein sequences of other P450s showed that the deduced protein sequence had less than 40% amino acid identity with any classified P450 and therefore it was assigned to a new P450 family CYP557 and given the number CYP557A1. BLAST searches of the NCBI databases showed that CYP557A1 has the highest amino acid identity with fatty acid omega hydroxylases belonging to the CYP86 and CYP94 families from plants. The benzoate-para-hydroxylase encoding gene CYP53B1 from R. minuta was first expressed into Y. lipolytica using the same expression system previously used for the expression of a human cytochrome P450, CYP1A1. In order to obtain maximum benzoate para-hydroxylase activity with whole cells of Y. lipolytica multiple copies of the CYP53B1 gene as well as an additional copy of the Y. lipolytica cytochrome P450 reductase (CPR) gene had to be inserted into Y. lipolytica. Expression of CYP557A1 in Y. lipolytica was more complicated, because Y. lipolytica has its own fatty acid and alkane hydroxylases. Biotransformation of hexylbenzene to phenyl acetic acid can, however, be used to detect increased levels of alkane hydroxylase activity. When multiple copies of CYP557A1 were cloned into a Y. lipolytica strain already carrying an additional copy of the Y. lipolytica CPR, none of the transformants displayed an increased rate of phenyl acetic acid production from hexylbenzene. It is possible that CYP557A1 has no alkane hydroxylase activity, but only fatty acid hydroxylase activity. It was very difficult to test fatty acid hydroxylase activity with whole cells of a Y. lipolytica strain, which still has an intact β-oxidation system, because it utilizes the test substrate, oleic acid, as carbon and energy source and incorporates it into lipids. However transformants that contained an additional copy of the CPR and multiple copies of the CYP557A1 gene showed the fastest consumption of oleic acid in comparison with the control strain carrying only the additional CPR gene and with strains carrying the CPR gene and a single copy of the CYP557A1 gene. In this study we have demonstrated the use of Y. lipolytica for the heterologous expression of other fungal P450s. Whole cells instead of microsomal fractions could in this case be used for the detection of P450 activity. This is the first study on heterologous expression of fungal P450 genes in Y. lipolytica, and has paved the way for the expression of other fungal cytochrome P450s in Y. lipolytica.