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

Loading...
Thumbnail Image
Date
2004-05
Authors
Shiningavamwe, Andreas Ndafudifwa
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
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.
Afrikaans: Sitochroom P450 is heem bevattend mono-oksigenases wat algemeen in die lewende organismes voorkom. Hierdie ensieme speel ‘n belangrike rol in prosesse soos koolstof assimilasie, biosintese van hormone en detoksifisering van xenobiotiese verbindings. As die aktiwiteit van P450’s beter verstaan kan, word kan hierdie ensieme gebruik word in die ontwikkeling van terapeutiese middels, die bestudering van plant patogeen interaksie asook bioremediëring. Giste wat aan die basidiomisete genus Rhodotorula behoort kan groei op nie-koolhidraat koolstofbronne soos alkane, aromatiese verbindings en selfs monoterpene. Slegs een sitochroom P450 koderende geen, die van ‘n bensoaat para-hydroksilase (CYP53B1), is al uit ‘n basidiomisete gis, Rhodotorula minuta, geïsoleer. Yarrowia lipolytica is ‘n askomisete gis wat hidrofobiese substrate soos n-alkane en vetsure kan benut. ‘n Wye reeks uitdrukking-sisteme is reeds vir Y. lipolytica ontwikkel. Dit was die doel van hierdie studie om ‘n tweede P450 koderende geen (een wat kodeer vir ‘n alkaan of vetsuur hidroksilase) uit ‘n Rhodotorula sp, wat limoneen benut, te isoleer en hierdie geen sowel as die CYP53B1 geen van R. minuta in Y. lipolytica uit te druk. PKR (wat gebruik gemaak het van priemstukke gebaseer op die basispaaropeenvolging van die heliks I en die heem bindings domein van 15 alkaan- en vetsuur-hidroksilases van askomisete giste) en OPKR (omgekeerde PKR) was gebruik om ‘n sitochroom P450 te isoleer uit ‘n limineen benuttende stam Rhodotorula sp. CBS 8446. Basispaaropeenvolging- analise van die totale geen het gewys dat hierdie geen introne bevat en Southern-klad-hibridisasie het gewys dat daar slegs een kopie van die geen teenwoordig is in die genoom. Deur geen spesifieke priemstukke te gebruik is die volledige cDNA insluitend die begin en eind-kodons geïsoleer. Vergelyking met ander P450 proteïene het getoon dat die afgeleide aminosuur opeenvolging van die nuwe P450 minder as 40% ooreenstemming met enige bekende P450 het, dit was dus toegeken aan ‘n nuwe P450 familie, CYP557 en die nommer CYP557A1 gegee. BLAST soektogte van die NCBI databank het gewys dat CY557A1 die beste ooreenkoms het met plant vetsuur-hidroksilases naamlik CYP86 en CYP94 families. Die bensoaat para-hidroksilase koderende geen, CYP53B1 van R. minuta, is eerstens gekloneer in Y. lipolytica deur gebruik te maak van dieselfde uitdrukkings sisteem wat voorheen gebruik is vir die uitdrukking van ‘n menslike sitochroom P450, CYP1A1. Om die hoogste vlakke van bensoaat-hidroksilase aktiwiteit met heel selle van Y. lipolytica te verkry, moes verskeie kopieë (10-13 kopieë) van die CYP53B1 geen sowel as ‘n addisionele kopie van die Y. lipolytica sitochroom P450 reduktase geen in Y. lipolytica ingevoeg word. Uitdrukking van CYP557A1 in Y. lipolytica was meer ingewikkeld omdat hierdie gis sy eie vetsuur- en alkaan hidroksilase bevat. Biotransformasie van heksielbenseen na fenielasynsuur kan egter gebruik word om verhoogde alkaan-hidroksilase aktiwiteit waar te neem. Die klonering van verskeie kopieë van CYP557A1 in ‘n Y. lipolytica transformant wat alreeds ‘n addisionele kopie van die CPR bevat het, het geen transformante gelewer wat verhoogde feniel-asynsuur produkise vanaf heksielbenseen vertoon het nie. Dit is moontlik dat CYP557A1 geen alkaanhidroksilase aktiwiteit het nie, maar slegs vetsuur-hidroksilase aktiwiteit. Dit was moeilik om vetsuur-hidroksilase met heel selle van Y. lipolytica wat nog ‘n intakte β-oksidasie sisteem het te toets aangesien dit die toets substraat, oleïnsuur, as beide koolstof en energiebron benut en dit in lipiede inkorporeer. Transformante met ‘n addisionele kopie van die CPR geen en verskeie kopieë van die CYP557A1 geen, het oleïnsuur egter vinniger opgeneem as die kontrole stam wat slegs ‘n addisionele CPR geen bevat het en transformante wat die CPR geen en ‘n enkel kopie van die CYP557A1 bevat het. In hierdie studie het is bewys dat Y. lipolytica gebruik kan word vir die heteroloë uitdrukking van P450 gene uit fungi. Heel selle in plaas van mikrosomale fraksies kon in hierdie geval gebruik word vir die bepaling van P450 aktiwiteit. Hierdie is die eerste studie van hetereloë uitdrukking van P450 gene vanuit fungi in Y. lipolytica en het die weg gebaan vir die uitdrukking van ander soortgelyke P450 gene in Y. lipolytica.
Description
Keywords
Cytochrome P450, CYP52 family, Helix I, Heme-binding, Rhodotorula, Yarrowia lipolytica, Genetic regulation, Molecular cloning, Thesis (Ph.D. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2004
Citation