DNA AND CELL BIOLOGY Volume 11, Number 10, 1992 Mary Ann Lieber!, Inc., Publishers Pp. 767-780

Identification and Characterization of Additional Members of the Cytochrome P450 Multigene Family CYP52 of Candida tropicalis WOLFGANG SEGHEZZI, CHRISTOPH MEILI, ROLF RUFFINER, RALF KUENZI, DOMINIQUE SANGLARD, and ARMIN FIECHTER

ABSTRACT

Using different DNA probes from the first two previously described alkane-inducible cytochrome P450 genes of the Candida tropicalis CYP52 gene family, we isolated five independent additional members by screening a genomic library under low-stringency conditions. These genes are not allelic variants and, when taken gogether, constitute the largest gene family known in this organism. The seven members of this gene family are located on four different chromosomes and four of them are tandemly arranged on the C. tropicalis genome. The products of the seven genes, alkl to alk7, were compared to each other and revealed a high degree of divergence: the two most diverged proteins exhibit a sequence identity of only 32%. Six of the seven genes were shown to be induced by a variety of different aliphatic carbon sources but repressed when the organism was grown on glucose. Three of the five additional CYP52 genes could be successfully expressed in Saccharomyces cerevisiae and display different substrate specificities in in vitro assays with model substrates: alk2 and alk3 exhibit a strong preference for hexadecane, while alk4 and alk5 preferentially hydroxylate lauric acid.

INTRODUCTION P450

form a superfamily of monooxygenases catalyzoxygénation reactions on a variety of different endogenous and exogenous substrates. This superfamily consists of more than 25 gene families with a total of over 150 members described so far (Nebert et al, 1991). In higher eukaryotes, the presence of P450 gene families containing multiple members is very common, in contrast to the situation found with lower eukaryotes such as yeasts. The yeast Candida tropicalis needs an alkane-inducible P450 monooxygenase system, consisting of the P450 and the corresponding NADPH-P450 oxidoreductase, for the terminal hydroxylation of rc-alkanes and other aliphatic carbon sources (Sutter et al, 1990). This reaction constitutes the first and rate-limiting step in the assimilation of alkanes (Gmünder et al, 1981; Sanglard et al, 1984). In addition, this enzymatic function is required in the synthesis pathway of fatty acid alcohols and dicarboxylic acids, thus rendering this yeast interesting for industrial applications (Bühler and Schindler, 1984).

(P450) Cytochromes ubiquitous heme-containing ing diverse

Institute of

Biotechnology,

The study of this P450 monoxygenase system at a molecular level was made possible by the isolation of the corresponding genes: a first alkane-inducible P450 gene (P450alk) was cloned from a Xgtll C. tropicalis expression library and its gene product characterized by heterologous production in Saccharomyces cerevisiae (Sanglard et al, 1987; Sanglard and Loper, 1989). P450alk was assigned to a new P450 gene family called CYP52 (Nebert et al, 1989), and its product was shown to be functional by in vitro assays for the terminal hydroxylation of lauric acid. Nevertheless, several observations soon led to the hypothesis that P450alk-related sequences must be present in the yeast genome. For example, a mass difference was observed between P450alk produced in S. cerevisiae and the main P450 form detected in C. tropicalis wild-type cells. Furthermore, a conserved P450alk probe cross-hybridized with multiple bands of restricted C. tropicalis genomic DNA under low-stringency conditions. Upon reexamination of the Xgtll C. tropicalis expression library, a partial DNA sequence of such a related P450 gene was isolated and sequenced. Using this sequence as a probe, the existence of additional P450alk-related sequences was pre-

Swiss Federal Institute of Technology, ETH

767

Hönggerberg,

CH-8093

Zürich, Switzerland.

SEGHEZZI ET AL.

768

dieted (Sanglard and Fiechter, 1989). The sequence of this second gene named CYP52A2 was completed and its product, alk2, was synthesized heterologously in S. cerevisiae. In vitro assays showed that alk2 hydroxylates hexadecane at the terminal position but not lauric acid (Seghezzi et al,

1991).

Here we present the isolation and characterization of five additional CYP52 genes by nucleotide sequence analysis, determination of their chromosomal localization, and heterologous expression in S. cerevisiae. Together with CYP52A1 and CYP52A2, these five CYP52 genes enlarge the CYP52 gene family of C. tropicalis to a total of seven members.

MATERIALS AND METHODS

Strains and media A wild-type strain C. tropicalis ATCC 750 was used in these studies and grown on synthetic medium (Hug et al., 1974) supplemented with glucose or different aliphatic substrates as sole carbon source at a final concentration of 1 % (wt/vol). S. cerevisiae YS18 [(a, his3-U, his3A5, leu2-3, leu2-\\2, ura3A, canR); Sengstag and Hinnen, 1987], a derivative of GRF18, was transformed by the Li-acetate procedure (Rothstein, 1985) and was grown on synthetic medium (Hug et al, 1974). Cultivations were carried out in batch or continuous mode in a 2.5 liter bioreactor (Bioengineering, Switzerland) or a 7 liter bioreactor (Chemap, Switzerland). E. coli DH5a (Hanahan, 1985) was used as a recipient of all plasmid constructions and was grown on LB medium supplemented with the required antibiotic. E. coli LE (P2) 392 (Borck et al, 1976; Murray et al, 1977) was used for the preparation of phage DNA.

Construction of a C. and screening

tropicalis genomic library

The constructions of the Xgtll expression library used for the isolation of CYP52A1 and the XDASH II genomic library used for the isolation of the additional CYP52 genes have been described previously (Sanglard et al, 1987; Seghezzi et al, 1991). The screening procedure involved the use of 5' and 3' segments of the first two described genes as probes: a 1.5-kb Eco RI restriction fragment containing the complete CYP52A1 gene (Sanglard and Loper, 1989) and a 0.4-kb Eco RI restriction fragment containing the 3'-terminal nucleotide sequence of CYP52A2 including the HR2 region (Sanglard and Fiechter, 1989). Their genomic positions are diagrammed in Fig. 1. The DNA probes were prepared as described for Southern blots. The XDASH II plaques were immobilized on nylon membranes (Gene Screen Plus, Dupont) and hybridized overnight in a buffer containing 20% formamide, 1% NaDodSO«, 10% dextran sulfate, and 5x SSC at 42°C (low-stringency hybridization for heterologous signals). The filters were exposed to X-ray film after successive washes in 2x SSC, 1% NaDodS04 at increasing temperatures ranging from 50°C (low-stringency wash) to 65 °C (high-stringency wash) as described earlier (Sanglard

and Fiechter, 1989). Phage DNA was prepared from X-sensitive E. coli cells after preamplification at 37 °C overnight and phage particles were precipitated by addition of 10% PEG 6000 on ice and centrifugation at 7,000 x g for 15 min. After chloroform extraction, the phages were purified in a CsCl gradient at 288,000 x g and 4°C for 20 hr. The phage DNA was recovered by digestion of the dialyzed and purified phage particles with proteinase K (250 fig ml"1) in 0.5% NaDodS04 at 65°C for 1 hr. After a phenol/chloroform extraction step, the DNA was ethanol

precipitated (Sambrook et al, 1989). Southern blots

Digested genomic DNA was separated on a 1 % agarose gel, transferred to a Genescreen Plus membrane according to the supplier's instructions (New England Nuclear). DNA probes were labeled with [a-"P]dCTP (Amersham) by random primer elongation (Feinberg and Vogelstein, 1983). The hybridization buffer consisted of 50% formamide, 10% dextran sulfate, 1% NaDodS04, 1 M NaCl, 100 fig ml"1 heat-denatured salmon sperm DNA, and 106 cpm ml"1 probe. The hybridization temperature was kept constant overnight at 42°C. The hybridization membranes were washed at 60°C in 2x SSC, 1% NaDodS04 (low-stringency wash) or 65°C in 0.1 x SSC, 0.1% NaDodS04 (high-stringency wash) and exposed to X-ray films (Fuji) at -70°C.

Subcloning and DNA sequencing Restriction fragments of phage DNA adequate in size subcloned into the plasmid pBluescript (Stratagene) and used for determining the nucleotide sequence. Restriction enzymes and T4 DNA ligase were from Boehringer Mannheim. Plasmids were isolated from E. coli DH5a as minipreparations without further purifications and the resulting DNA was directly used for sequencing. One microgram of each size-selected plasmid was alkali denatured and annealing of the reverse primer, M13 primer (New England Biolabs), or individual primers (Microsynth, Switzerland) was performed. Sequencing was done with [35S]dATP (Amersham) using the T7 DNA polymerase (Pharmacia) in combination with the Sequenase kit (USB) following the supplier's instructions. were

Northern blots Total RNA of C. tropicalis cells was extracted following the method of Chirgwin et al (1979) and electrophoresed after formaldehyde denaturation in 1 % agarose containing 20 mM MOPS buffer pH 7.0 and 0.6 M formaldehyde. EtBr was added at a concentration of 10 fig ml"1 to allow direct UV-visualization of ribosomal RNA bands (Davis et al, 1986). Northern transfer was performed on Genescreen Plus according to the supplier's recommendations.

Gene-specific probes were prepared by kinasing oligonucleotides with [7-"P]ATP (Amersham) using T4 polynucleotide kinase (Boehringer Mannheim). The probes were purified from nonincorporated [7-32P]ATP by liquid chro-

IDENTIFICATION OF P450 GENES

769

matography (Sephadex G50, Pharmacia). The composition Isolation and analysis of microsomal fractions of the hybridization buffer is identical to the one described containing P450 products for Southern blots. Washing conditions were stringent (6 x The isolation of microsomal fractions from yeast transSSC, 1% NaDodS04, 52°C). formants and the subsequent analysis of the products by SDS-PAGE and immunoblotting was done as described Chromosome separations earlier (Seghezzi et al, 1991). CO-difference spectra for C. tropicalis cells were harvested from an overnight cul- the detection of P450 products in microsomal fractions were recorded according to Omura and Sato (1962). ture and DNA samples were prepared as described by Monod et al (1990) with the exception of using Zymolyase-100T (1.5 mg/sample; Seikagaku Kogyo Co. Ltd., In vitro assays with model substrates using P450 Japan). Electrophoresis was done in 0.5% agarose (chro- products mosomal grade, BioRad) using the clamped homogenous Lauric acid and hexadecane hydroxylation activities in electric field technique (CHEF-DRII, BioRad) with the microsomes of yeast transformants were determined as defollowing settings: 75 V, 500 sec switching time, 76 hr, and scribed et al (1991). by Seghezzi 0.5X TBE buffer (Sambrook et al, 1989) at 14°C. After with and to UVEtBr, electrophoresis, staining exposure light for 2 min, the chromosomal DNA was transferred RESULTS with 0.5 M NaOH, 1.5 M NaCl by capillarity to Genescreen Plus membranes, which was cut into stripes for inEvidence for a multigene family and isolation dividual hybridization as described for Southern blots. of individual CYP52-related genes Construction

of yeast expression

vectors

The coding region of the individual CYP52 genes was amplified by the polymerase chain reaction (Saiki et al, 1988). The chosen primers were designed to contain the respective start (primer A) and stop (primer B) codons with the incorporated restriction sites Sal I and Bam HI for oriented cloning of CYP52A6, A7, A8 and Cl into the yeast expression vector YEp51 (Broach et al, 1983). As CYP52B1 contains an internal Bam HI site, both primers were designed to contain the Sal I restriction site. The oligonucleotide primers used were as follows: CYP52A6: A : ( 5

'

gcg ' tcg ' aca cta tgg ' cca cac ' aag ' aaa tt ) •

•

B : (5'GCG'GAT'CCA'ACC'AGT'ATT'ACA'TTT'TAA'CGA'A)

CYP52A7: A:

(5 'GCG'tcg'ACA'taa'tga'ttg'aacagg'ttt'ta)

B : (5'GCG'GAT'CCT•AGC'TAA'TTA'ATC'CAT'CTT'GAC)

CYP52A8: A : ( 5

•

•

gcg tcg ' aca caa ' tgt ' acg ' aac ' aag ' ttg ' tt) •

B : (5'GCG'GAT'CCG'TCT'CGT'CAC'GAT'CTC'TAG'GT)

CYP52B1: A:

(5 gcg'tcg'ACA'cca'tgt'cat'taa'cag1 aaa'ca)

B : (5'GCG'TCG'ACT'AAA'AAC'TTA'TAA'TCT'ATG'GAA)

CYP52C1: A: (S'gcg'tcg1 aca'taa'tgt•atcraat tat ttt•gtt'tc) B : (5'GCG'GAT'CCA'ATC'CTA'TTA'TAT'TCA'TAT'AAA'C)

.

Ten nanograms of the phage DNA containing the desired gene, 2 units of Taq DNA polymerase (Boehringer), and 10 pmoles of each primer were used in a 100-/d reaction buffer (Biofinex, Switzerland). Thirty cycles of denaturation at 95°C for 1 min, annealing at 52°C for 2 min, and elongation at 72°C for 4 min were performed and followed by a last elongation step at 72°C for 10 min. The amplification products were cut with the required restriction enzymes and ligated into the expression vector where the genes were under control of the inducible GALIO promoter.

The possible existence of a CYP52 multigene family in the yeast C. tropicalis was first hypothesized due to differences observed between low- and high-stringency hybridizations of restricted genomic DNA with CYP52A1 and CYP52A2 gene-specific probes. Whereas under high-stringency conditions only the homologous signals were detected, low-stringency conditions led to the appearance of a whole pattern of cross-hybridizing bands (Sanglard and Fiechter, 1989). To determine whether these additional signals could be attributed to additional P450 genes, a XDASH II genomic library was screened using probes derived from CYP52A1 and CYP52A2 (see Materials and Methods) under low-stringency hybridization conditions. Only the signals appearing at low stringency were purified because those still apparent at high stringency probably represented signals arising from homologs of CYP52A1 and CYP52A2. The DNA extracted from the isolated clones was used for restriction mapping analysis with Eco RI, and the fragments hybridizing with the above-mentioned probes were identified. Comparative analysis of the Eco RI restriction sites and their alignments allowed distinction of overlapping and non-overlapping clones, and thus suggested the presence of at least five additional putative CYP52 genes (Fig. 1). Their transcriptional orientations were determined in a first attempt by hybridizations of Eco Rl-digested phage clone DNA with different probes containing either the complete CYP52A1 gene or the 3' fragment of CYP52A2, and were later confirmed by nucleotide sequence analysis. Apart from CYP52A1 and CYP52A2, a second set of tandemly repeated CYP52 genes could be detected on clone X44, while the other genes were located on independent clones. In the case of phage clone X13, a restriction site heteromorphism for Eco RI was detected when comparing different clones containing the putative CYP52A6 gene, as indicated by a black arrow above the site in Fig. 1. This restriction site heteromorphism can be explained by the diploid nature of C. tropicalis and was also confirmed in genomic digests (not

shown).

SEGHEZZI ET AL.

770

C-terminal CYP52A2 probe CYP52AI probe

lkb

hT7,

T3™

CYP52A1 CYP52A2

—i_

-1

17s Q

T7W

tu

w w

-I T3

J_U_

k CYP52A6 tu

T3

w

m

J_l_

_l_LL_

T7

clone 13

201s 4_L_

T7 L-

J T3

clone 201

CW52A7

44s o

J_l_

T3

-

sr

-

CW52/1« CYP52B1

_lT7

801

T3"v

J_l_

T7

clone 801

CYP52C1

FIG. 1. Eco RI restriction maps of XDASH II clones containing DNA fragments hybridizing to CYP52A1 and CYP52A2. CYP52AI and CYP52A2 have been mapped and sequenced previously (Seghezzi et al, 1991). Grey bars and black bars denote the Eco RI DNA fragments identified by a CYP52A1 probe and a carboxy-terminal CYP52A2 probe, respectively (see Materials and Methods). The arrows represent the position and orientation of the individual open reading frames. The transcriptional direction of CYP52C1 on X801 has not been determined (double arrow). The Eco RI fragments called 17s, 201s, 44s, and 801 were used as gene specific probes for the determination of their chormosomal location in Fig. 3. A black triangle on clone 13 indicates the only Eco RI restriction heteromorphism detected. T3 and T7 mark the insertion sites of the genomic DNA into the XDASH II vector. For all P450 genes, overlapping clones have been identified, but only one map per gene is shown. E(Dash), Eco RI restriction sites of the XDASH II cloning vector; E, genomic Eco RI restriction sites. Dashed lines on the maps of clones 16, 17, and 801 represent the distance to the adjacent genomic Eco RI site.

The nucleotide and deduced amino acid sequence the five additional CYP52 genes

of

After restriction mapping of the five additional putative P450 genes, their complete nucleotide sequence was determined by subcloning of adequate-sized restriction fragments, wherever possible, or by individual primers designed after each round of sequencing (see Materials and Methods; the nucleotide sequence data reported here for CYP52A6, A7, A8, Bl, and Cl appears in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession numbers Z13010, 11, 12, 13, and 14, respectively). In each case, an open reading frame was detected with a significant similarity to the previously de-

scribed P450 genes, CYP52A1 and CYP52A2. Thus, the additional genes isolated could actually encode P450 proteins and are not due to artifactual isolation. In addition, extensive restriction mapping and nucleotide sequence data revealed the nonallelic nature of all seven CYP52 genes isolated from C. tropicalis (see Discussion). Their nomenclature and the terminology of their corresponding gene products is given in Table 1. With the exception of CYP52C1, where only 69 bp of the 5' noncoding region were sequenced, a possible consensus TATA box of all genes could be located between 71 and 205 bp upstream of the ATG codon. In all cases apart from CYP52C1, a wellconserved purine residue is situated at nucleotide position -3, but in contrast to CYP52A1 and CYP52A2, no con-

IDENTIFICATION OF P450 GENES Table 1. Nomenclature

\-DASHII clone*

Xalk 16 Xalk 17 Xalk 201 Xalk 44 Xalk 801

771 All Seven CYP52 Gene Family Members Their Corresponding Gene Products

of

P450 gene*

P450alk protein (amino acid residues)0

CYP52A1 CYP52A2 CYP52A6 CYP52A7 CYP52A8 CYP52B1 CYP52C1

alkl (543) alk2 (522) alk3 (524) alk4 (507) alk5 (517) alk6 (506) alk7 (505)

sequence for highly expressed S. cerevisiae genes, as proposed by Hamilton et al (1987), was found to be present. No repeated element common to all CYP52 genes that could serve as a putative regulatory sequence could be detected within the sequenced 5' noncoding region, as had been observed in CYP52A1 and CYP52A2 (Seghezzi et al, 1991). In CYP52A6, the sequence TAGCTAT was repeated three times, whereas the other 5' noncoding regions contained different short sequences repeated only twice. In no case could repeats of the pentanucleotide ATGTG be found, as has been reported to be present in the 5' region

of three distinct C. maltosa alkane-inducible P450 genes

(Ohkuma eí al, 1991b). To examine the degree of divergence among all members of the C. tropicalis CYP52 gene family, an alignment of the deduced amino acid sequences was prepared and is shown in Fig. 2. Looking at the heme-binding region HR2, it becomes obvious that even such a region of high conservation has undergone remarkable changes when comparing alkl or alk2 to the last member, alk7. In addition, the amino-terminal hydrohobic domains (18-22 amino acid residues; Fig. 2, underlined) found in alkl through alk5, which are thought to be responsible for anchoring the P450 protein into the endoplasmic reticulum membrane, was less prominent in alk6 as judged by hydrophobicity profiles (data not shown). In the case of alk7, it was present but reduced in length (only 12 amino acid residues) and even contained a lysine residue. This contrasts with the distribution of positive charges observed in the amino-terminal hydrophobic domains of alkl, alk2, and alk3. In these cases, positively charged residues (Lys, Arg) are flanking the hydrophobic domains and may serve as halt-transfer signals during protein translocation across the endoplasmic reticulum membrane, as suggested by Kuroiwa et al (1991). Table 2 gives the identity and similarity values of the comparisons between all seven P450 proteins obtained by applying the method of Needleman and Wunsch (1970). The identity values of alk6 and alk7 with the other members of the CYP52 gene family were as low as 39% and

C. tropicalis

and

Reference Sanglard and Loper (1989) Seghezzi et al (1991)

This This This This This

aFor X-clone numbering and Eco RI restriction maps see Fig. 1. bThe nomenclature recommended by Nebert et al. (1991) for P450 genes. cThe number of amino acid residues in the individual P450 proteins with trivial the nucleotide sequences.

sensus

of

work work work work work

names was

deduced from

32%, respectively. Therefore, their corresponding genes were

assigned

to two

additional subfamilies, CYP52B and

CYP52C, following the recommended nomenclature (Nebert et al, 1991). The CYP52 genes isolated so far from C. maltosa all belong to the first subfamily (Ohkuma et al,

1991b). The degree of divergence between the C. maltosa proteins around the HR2 region (Ohkuma et al, 1991b) is lower than in C. tropicalis. We conclude that either the sequences in C. maltosa are less divergent or that the isolation procedure used by Ohkuma et al (1991b) excluded the signals arising from genes with the lowest homology. The highest degree of similarity between the C. tropicalis and C. maltosa P450 proteins is found between alk3 and Cml, the product of CYP52A3. This is reflected in their immunological crossreactivity with a polyclonal antibody directed against Cml (W. Seghezzi, unpublished results). P450

Chromosomal assignment of the CYP52 genes The nonallelic nature of the seven CYP52 genes was further demonstrated by their assignment to distinct C. tropicalis chromosomes. Allelic genes are expected to be located on chromosomal homologues. Eight chromosomes could be separated by pulse-field electrophoresis, as shown in Fig. 3. A similar resolution was also achieved by Fukuda et al (1991) and Kamiryo et al (1991) but using a different C. tropicalis strain. Using gene-specific probes, the assignment of the seven CYP52 genes to the eight chromosomes was undertaken. All gene probes were first tested by Southern blots of genomic DNA to determine their specificity and no cross-hybridization was detected under the chosen conditions (data not shown). Figure 3 shows that CYP52C1, CYP52A6, and the repeat CYP52A1/CYP52A2 are located on chromosomes I, III, and IV, respectively. In the case of CYP52A 7 and the repeat CYP52A8/ CYP52B1, two signals of equal intensity appeared corresponding to the chromosomes VI and VIII. Because the two probes used were shown to be specific, different hypotheses could be formulated to explain this observation

(see Discussion).

SEGHEZZI ET AL.

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alkl

alkl

MSSSPSIAOEFLATITPYVEYCQENYTKWYYFIPLVI-LSLNLISMLHTKYLE-RKFKAK

alk2 alk3 alk4 alk5 alk6 alk7

MSI-QDIVETYS- -TKWYVWLVAL-IVYKVFDFFÏARYLM-YKLGAK MAT-QEIIDSALPYL- -TKWYTVITLAA-LVF-LISSNIKNYVKAKKLKCR M-IEQVLHY-- -WYYVLPAFI-IFHWIVSAIHTNSLR-RKLGAK M-YEQWEY-- -WYWLPIA'F-IL'HKVFDMWHTRRLM-KQLGAA -YNYWYIIFHLYFYTTSKIIKYHHTTYLMI-KFKAS Ms-LTETTATFIj-

"-""-FLAGIIWYKAAQ-YYKRRTLVTKFHCK

alk7

alkl alk2 alk3 alk4 alk5 alk6

--P-LAVYV0DYTFGLITPLVLIYYKSKGTVMOF-ACDLWDKKLIVSDPKAKTIGLKILG

alkl

M-YQLFC-

—

--PFTHTQLDGF-YGFKFGRDFLKAKRIG-RQVDLINSRFPDDIDTFSSYTFG-

alk4 alk5 alk6 alkV

DPPYFKG--AGWT-GISPLIEIIKVKGNGRLARF-WPIKTFDDYPN-HTFYMSIIG

--PVTNQLHDNF-FGIINGWKHLSSRKKVELKNIMIINLPIRKFQVWVHMLVP-SLEPP--LNYINKGF-FGIQATFTELKHLICHTSIDYAIDQFNNVPF-PHVHTFVTKVLG

alkl alk2 alk3 alk4 alk5 alk6 alk7

VL-IISTLEPDNIKAILA-TQFNDFSLGTRHSHFAPLLGDG IFTLDGAGWKHSRSMLTPQ ALKIVLTVIQENIKAVLA-TQFTDFSLGTRHAHFYPLLGDG IFTLDGEGWKHSRAMLRPQ -NHVIFTRDPENIKALLA-TQFNDFSLGGRIKFFKPLLGYG IFTLDGEGWKHSRAMLRPQ -QSSSSQKIRRNIKALLA-TQFSDFSLGKRHTLFKPLLGDG IFTLDGQGWKHSRAMLRPQ -NELIMTKDPENIKVLLRFPVFDKFDYGTRSSAVQPSLGMG IFTLEGENWKATRSVLRNM

alk3 alk4 alk5 alk6 alk7

VNKALNFTPEEIEEKSKSGYVFLYELVKQTRDPKVLQDQLLNIMVAGRDTTAGLLSFAMF

VQKALDATPEELEKQ--SGYVFLYELVKQTRDPNVLRDHHSISLLAGRDTTAGLLSFAVF VQRALDATPEELEKQ--SGYVFLYELVKQTRGPNVLRDQSLNILLAGRDTSAGLLSFAVF VQKELENTCNDK-FVFVHQLAKHTTNKTFIRDQALSLIMASRDTTAELMAFTIL IDRWGMSEEELNNHPKS-YVLLYQLARQTKNRDILQDELMSILLAGRDTTASLLTFLFF

ELSRNPEIFAKLREEI ENKFGLGQDARVEEISFETLKSCEYLKAVINETLRIYPSVPHNF ELARTPRVANKLREEI EDKFGLGQDARVEEISFESLKSCEYLKAVLNECLRLYPSVPQNF ELARHPEIWSKLREEI EVNFGVGEESRVEEITFESLKRCEYLKAILNETLRMYPSVPVNE ELARNPHIWAKLREDV ESQFGLGEESRIEEITFESLKRCEYLKAVMNETLRLHPSVPRNA ELARNPHIWAKLREDV ESQFGLGEQSRIEEITFESLKRCGYLKAFLNETLRVYPSVPRNF ELSRKSHHLGKLREEI DANFGLESP---DLLTFDSLRKFKYVQAILNETLRMYPGVPRNM ELSHHPEVFNKLKEEI ERHF-PDVESVTFGTIQRCDYLQWCINETMRLHPSVPFNF

RVATRNTTLPRGGGEGGLSPIAIKKGQWMYTILATHRDKDI-YGEDAYVFRPERWFEPE RVATRNTTLPRGGGKDGLSPVLVRKGQTVMYSVYAAHRNKQI-YGEDALEFRPERWFEPE

KTTYV-TKDIENIRHILSATEMNSWNLGARPIALRPFIGDG IFASEGQSWKHSRIMLRPV

alk7

RTAANDTVIPRGGGKSCTDPILVHKGEQVLFSFYSVNREEKY-FGTNTDKFAPERWSESL

FAREQVSHVKLLEPHMQVLFKHIRKHHGQTFDIQELFFRLTVDSATEFLLGESAESLRDE FAREQVSHVKLLEPHMQVFFKHIRKHHGQTFDIQELFFRLTVDSATEFLFGESVESLRDE FARDQIGHVKALEPHIQILAKQIKLNKGKTFDIQELFFRFTVDTATEFLFGESVHSLYDE

alkl alk2

TRKLGWAYV

FAREQLPMSPSLEPHFNV--KAYPQEQRWVFDIQELFFRFTVDSATEFLFGESVNSLKSA FAREQVAHVTSLEPHFQLLKKHMVKNKGGFFDIQELFFRFTVDSATEFLFGESVHSLKDE

FDRKSIDKVHDFEPHFKTLQKRI-DGKVGYFDIQQEFLKLGLELSIEFIFGQVVFAKEHVKQITSMEPYVQLLIKIIKNHEGEPLEFQTLAHLFTIDYSTDFLLGESCDSLKDF SVGLTPTTKDFDGRNEFADAFNYSQTNQAYRFLLQQMYWILNGSEFRKSIAIVHKFADHY

alk2

SIGMLNDALDFDGKAGFADAFNYSQNYLASRALMQQMYWILNGKKFKECNAKVHKFADYY KLGI-PTPNEIPGRDNFATAFNTSQHYLATRTYSQTFYFLTNPKEFRDCNAKVHYLAKYF

alk4 alk5 alk6 alk7

VQKALELTDEDLEKKE--GYVFLFELAKQTRDPKVLRDQLLNILVAGRDTTAGLLSFLFF VEKALELTPDQLEKQD--GYVFLYELVKQTRDRQVLRDQLLNILVAGRDTTAGLLSFVFF

alkl alk2 alk3 alk4 alk5 alk6

IP-LIETKDPENVKAILA-TQFNDFSLGTRHDFLYSLLGDG IFTLDGAGWKHSRTMLRPQ

alkl alk3

alk5 alk6

alk2 alk3

PARISPNKSWLEYLGIASWHADEMIRKGGL--YSEIDGRFKSL-DVSTFKSITLG

alk2

alk3 alk4

--PFLQSQTDGY-LGFRVPFELMGKKSEGTLIDF-TYQ-RTLELDNPDIPTFTFPIFS

alk7

alkl

alk2

SIGC-DEETELEERKKFAEAFNKAQEYISTRVALQQLYWFVNNSEFKECNEIVHKFTNYY TIGSYQDDIDFVGRKDFAESFNKAQEYLAIRTLVQDFYYLVNNQEFRDCNKLVHKFTNYY -SEDVPHYDDFTQAWDRCQDYMMLRLLLGDFYWMANDWRYKQSNQIVQAFCDYL

LGEESNSTLDTSLRLAFASQFNKTQQQMTIRFMLGKLAFLMYPKSFQYSIQMQKDFVDVY

alk3 alk4

RTATRDTTLPRGGGPNGTDPIFIPKGSTVAYIVYKTHRLEEY-YGKDADDFRPERWFEPS RFALKDTTLPRGGGPDGKDPILVRKMSCSIF-ISGTQIDPKH-YGKDAKLFRPERWFESS RIATKNTTLPRGGGSDGNSPVLVKKGEAVSYGINSTHLDPVY-YGDDAAEFRPERWNEPS

KTAKCTTTLPKGGGPDGQDPILVKKGQSVGFISIATHLDPVLNFGSDAHVFRPDRWFDSS

PFNGGPRICLGQQFALTEASYV TVRLLQEFGNLKQDPNTEYPPKLQNTL

TVRLLQEFSHLTMDPNTEYSPKKMSHL ITRLVQMFETVSSPPDVEYPPPKCIHL LVRLAQSFDTLELKPDTEYLTK-ISHL PFNGGPRICLGQQFALTEAGYV LVRLAQSFDTLELKPPWYPPKRLTNL PFNAGPRTCLGQQYTLIEASYL LVRLAQTYETVESHPDSVYPPRKKALI PFSAGPRACLGQQLPRVEASYV TIRLLQTFHGLH-NASKQYPPNRWAA

TKKLGWAFL PFNGGPRICLGQQFALTEASYV TKKLGWAYV PFNGGPRICLGQQFALTEASYV TRNLGWAYL PFNGGPRICLGQQFALTEAGYI

alk5 alk6 alk7

TRKLGWAYL MKNLGCKYL

alkl alk2 alk3 alk4 alk5

TLSLFEGAEVQMYLIL

alk6 alk7

NMCAADGVDVK-FHRL TMRLTDGCNV-CFI--

RRT-EFI

TMSLFDGANIQMYTMSHDDG-VFVKM TMCLF-GAFVKM-D

TMSLQDGTIVKI-D

Comparison of the deduced amino acid sequences of all seven CYP52 genes. The amino acid alignment was done using the program CLUSTAL implemented on the GCG software package (University of Wisconsin). The amino acids conserved in all genes are marked by an asterisk and a dot denotes one or more conserved substitutions at the indicated amino acid position. The HR2 region is boxed. Underlined sequences represent amino-terminal hydrophobic domains and were determined using the algorithm of Kyte and Doolittle (1982) at a window of 19 amino acid residues. The highest obtained hydrophobicity indices within the respective hydrophobic domains are as follows: alkl (first domain), 1.5; alkl (second domain), 1.7; alk2, 2.0; alk3, 1.6; alk4, 1.5; alk5, a.6; alk6, 0.6; alk7, 1.4. The dashed line denotes the weak hydrophobic segment of alk6. FIG. 2.

Table 2. Comparison

C.

Identity/similarity

alkl

alkl alk2 alk3 alk4 alk5 alk6 alk7

68.2 57.3 56.0 56.7 39.8 39.5

of the

Deduced Amino Acid

tropicalis CYP52 Genes*

Sequences of

alk2

alk3

alk4

alk5

alk6

alk7

81.6

70.9 75.0

70.8 72.5 70.2

72.1 74.8 72.9 77.8

60.8 61.4 61.3 63.6 64.3

61.3 62.4 59.2 59.7 60.0 56.2

58.7 57.1 59.1 38.4 37.4

55.9 58.5 40.6 37.0

65.5 40.2 36.3

40.7 37.4

32.1

«The sequence comparison was done using the algorithm of Needleman and Wunsch implemented in the GCG software package (University of Wisconsin). The values are given in % (amino acid simi-

larity/identity).

IDENTIFICATION OF P450 GENES

va

S C. tropicalis chromosome VIII VII VI V IV III

S. cerevisiae size standards

tv

00

~,

C

no.

VIII VI IV

^*

III

1.6 Mb —

II I

1.13 Mb-

C.

tropicalis

chromosome

Southern blot using CYP52 specific probes

separation

FIG. 3. Localization of the seven CYP52 genes on the C. tropicalis chromosomes. Eight C. tropicalis chromosomes (IVIII) could be resolved as described in Materials and Methods. Southern blots were hybridized with individual gene specific probes (right side). For the tandemly repeated CYP52A1 and CYP52A2 genes, the probe consisted of a central 1.2kb Hind III DNA fragment of CYP52A2. For the detection of the second tandem repeat (CYP52A8 and CYP52B1), a 2.1-kb Eco RI fragment called 44s (see Fig. 1) was chosen, which contained the entire CYP52B1 gene except for 250 bp at the amino terminus. The gene-specific probes for CYP52A6, CYP52A7, and CYP52C1 consisted of the Eco RI DNA fragments 17s, 201s, and 801, respectively, as shown in Fig. 1. The double signals generated by the CYP52A7, CYP52A8, and CYP52B1 probes remained both present under high-stringency conditions (0.1 x SSC, 65°C).

Induction of the CYP52 genes by carbon sources

different

mainly induced by substituted aliphatic subaldehyde and oleic acid, CYP52A2 To test the inducibility of the CYP52 genes, and thus the shows its strongest induction by n-alkanes. This is in good relevance of their gene products for hydrocarbon hydrox- agreement with the in vitro substrate specificities described ylation, and to identify possible pseudogenes, the role of for alkl and alk2 (Seghezzi et al, 1991). It should also be different carbon sources as inducers was investigated with mentioned that the induction detected on oleic acid conC. tropicalis grown in continuous culture in a bioreactor. trasts with the induction of the CYP52A genes of C. malTotal RNA was isolated from samples withdrawn from the tosa, where no signals could be found when cells were growing cultures. Northern blot analysis was performed grown on this carbon source (Ohkuma et al, 1991b). A direct comparison of the signals generated by the difusing gene-specific oligonucleotides for each CYP52 gene. As illustrated in Fig. 4A, transcripts of six genes could be ferent probes on the different substrates was achieved by readily detected at different levels when cells were grown normalizing the signal intensity with the help of an internal standard (i.e., the ADE2 gene; Sanglard and Fiechter, on the indicated carbon sources. In general there is a good correlation between the total P450 contents of C. tropicalis 1992) and measuring by scanning densitometry. The result grown on the different substrates (data not shown) and the shown in Fig. 4B indicates that the strongest absolute incorresponding accumulated signal intensities of the CYP52 duction value is obtained with CYP52A2 on dodecene as gene transcripts. In no case could transcripts of CYP52A7 the sole carbon source (left side). The normalized signals be seen, which means that either the conditions needed for generated by CYP52A8, CYP52B1, and CYP52C1 were induction were not met or that this gene could possibly plotted separately at four-fold increased resolution for represent a pseudogene. All genes are repressed by glucose better comparison of the small differences in signal intensiwith the exception of CYP52B1, where a weak but unam- ties (Fig. 4B, right side). Thus, it is obvious that the carbon biguous signal was found. In general the first three genes, source plays an important role as a regulator of gene inCYP52A1, A2, and A6, show a 10-fold stronger induction duction, but an additional experiment revealed that this is not the only regulation of the CYP52 genes. Replacing on all substrates than the others. While CYP52A1 and CYP52A6 aie

strates such as lauric

SEGHEZZI ET AL.

774

c

c

4J

y

.iS

c

S "^

"111 | 1 11 11 £ a .i J.

U "

a j.

x r j. j oo

CYPy2Al

CyP52Ai?

CYPS2A2

CYP52B1

CYP52A6

CYP52C1

fi

j.-

í

x s

-

(3

U

U

j o o

-m

ADE2

CYPS2A7

B

Decane Decene Dodecane Dodecene Tetradecane Hexadecane Dodecanol Laurie aldehyde Oleic acid Glucose

Decane Decene Dodecane Dodecene Tetradecane Hexadecane Dodecanol Lauric aldehyde Oleic acid Glucose

FIG. 4. A. Induction of the CYP52 genes by the growth of C. tropicalis on different C-sources. Blots of CYP52A1, A2, A6, A7, and ADE2 were exposed for 6 hr, and those specific for CYP52A8, Bl, and Cl for 24 hr. RNA was isolated

and separated as described in Materials and Methods from cells growing on the different carbon sources indicated above each lane. In the case of 1-dodecene and hexadecane, where RNA samples were loaded twice, the normalized signal intensities given in B were averaged. B. Normalized signal intensities for direct comparison of the induction levels of individual CYP52 genes. The signals generated by the gene-specific probes were scanned (Hoefer Scientific Instruments) and the absorbance values normalized with the help of the ADE2 gene, which served as an internal standard. The highest normalized induction value (CYP52A2 on dodecene) was assigned to 100 arbitrary units. For the direct comparison of signals generated after different exposure times (6 hr and 24 hr, respectively), a conversion factor was calculated using CYP52AÄ-specific signals of both exposure times. The scale of the graphic on the right panel is enlarged for better visualization of the relative induction levels.

IDENTD7ICATION OF P450 GENES continuous culture growth conditions by growth of cells in batch culture, where culture parameters such as the oxygen partial pressure or concentrations of medium components and metabolites are different, led to a higher induction level of CYP52A6 as compared to CYP52A2 (data not

shown).

Expression of the isolated CYP52 genes in S. cerevisiae

The products of the individual CYP52 genes were characterized by heterologous expression in S. cerevisiae. Their corresponding genes were cloned into the yeast expression vector YEp51 (Broach et al, 1983) to place them under control of the inducible GALIO promoter (Fig. 5). Efforts to clone CYP52B1 into YEp51 were unsuccessful, possibly due to a toxic effect of the gene product in E. coli cells. Transformants of S. cerevisiae YS18 cells were cultivated on glucose for 15 hr in a 2.5 liter bioreactor and then galactose was added to a final concentration of 3%. Maximum productivity was obtained for alk3, alk4, and alk5 after 8-9 hr on galactose as judged by CO-difference spectral analysis of whole cells. The highest P450-specific content in whole cells was obtained for alk3 (58 nmoles P450/ gram of dry weight), whereas alk4 yielded a specific P450 content of only 13 nmoles P450/gram of dry weight. The microsomal fractions were isolated and analyzed for their specific P450 content (see Table 2). All P450 proteins gave typical CO-difference spectra (Fig. 6) with the exception of alk7 (Fig. 6F), where no absorption at 450 nm but a strong peak at 420 nm, chracteristic for the denatured P450 form,

775

recorded (see Discussion). The microsomal fractions of alk2 and alk4 also contained a considerable amount of P420, which could indicate their lower stability as compared to alkl, alk3, and alk5. The microsomes of the S. cerevisiae transformants were also analyzed by comparative immunoblotting with a polyclonal antibody raised against the main P450 fraction of C. tropicalis grown on tetradecane (Loper er al, 1985). As can be readily seen in Fig. 7B, the cross-reacting signals for alk3 (51.8 kD) and alk5 (57.6 kD) are weaker than the signal generated by alk2 (53.0 kD), which is the putative main C. tropicalis P450 protein when this yeast is grown on tetradecane. The observed signal intensities are thus in good agreement with their respective degrees of identity. The very weak reactive specific band corresponding to alk4 (52.3 kD) can be explained by the low specific P450 content of its microsomal fraction. When expressed in S. cerevisiae alk5 shows some degradation products similar to alk2. In the case of alk7, spectral analysis suggests the presence of denatured P450 protein. When compared to the negative control, a corresponding band can indeed be detected in an enlarged reproduction (Fig. 7C) of the Coomassie-stained NaDodSO, gel (Fig. 7A). However, the sequence identity between alk7 and alk2 is only 37.4%, which could explain why no specific reactive band for alk7 is detectable by immunoblotting. was

In vitro

hydroxylation

activities

The terminal hydroxylation activities toward the two model substrates, lauric acid and hexadecane, were tested using microsomes from yeast transformed with the corresponding expression plasmid. As a positive control, microsomes isolated from C. tropicalis grown on tetradecane were used while microsomes from S. cerevisiae transformed with the parent expression vector YEp51 served as negative control. As summarized in Table 3, alk3 displays a preference to hexadecane hydroxylation, whereas both alk4 and alk5 preferentially hydroxylate lauric acid at high turnover rates. The four-fold higher NADPH-cytochrome P450 reducíase (CPR) activity determined for C. tropicalis microsomes as compared to the S. cerevisiae isolates was expected since CPR is induced by alkane and thus accounts for the high lauric acid conversion rate of C. tropicalis microsomes. This high activity cannot, however, be attributed to a particular P450 isoenzyme, but obviously represents the accumulated activities of all produced P450alk proteins. It is interesting to note that although under no conditions CYP52A7 transcripts could be detected in Northern blots (Fig. 4A), its gene product alk4 is still a fully active protein.

DISCUSSION FIG. 5. Yeast expression vector for the production of the different P450 proteins in 5. cerevisiae. The respective genes (CYP52A6, A7, A8, Cl) were cloned into the Sal 1/ Bam HI-restricted vector and thus placed under the control of the inducible GALIO promoter to yield the plasmids pRRl, pWS4, pWS5, and pWS7, respectively.

Applying low-stringency hybridization techniques to the screening of a C. tropicalis XDASH II genomic library using DNA fragments derived from the previously cloned genes CYP52A1 and CYP52A2, five additional members of the CYP52 gene family could be isolated and character-

776

SEGHEZZI ET AL.

420

nm

AA

=

0.02

~I 400

nanometers

(nm)

~i-1-1-

400

nanometers

(nm)

500

~~i 500

400

nanometers

(nm)

500

r~ 400

nanometers

(nm)

500

CO-difference spectra of microsomal fractions of S. cerevisiae transformed with the P450 expression plasmids (A, alkl; B, alk2; C, alk3; D, alk4; E, alk5; F, alk7; H, S. cerevisiae transformed with parent vector YEp51; G, C. tropicalis grown on tetradecane). The spectra were recorded in 0.1 M phosphate buffer pH 7.5 according to Omura and Sato (1962). Protein concentrations (mg of protein/ml of buffer) in each cuvette were as follows: alkl, 1.7; alk2, 3.9; alk3, 1.0; alk4, 2.4; alk5, 1.3; alk7, 0.9; YEp51, 1.4; C. tropicalis, 2.2. AA indicates the scale of absorption difference and was 0.02 for spectra A-G, and 0.01 for spectrum H. Expression plasmids and specific P450 contents are listed in Table 2. FIG. 6.

ized. The complete pattern of cross-hybridizing bands obtained with Eco RI-restricted C. tropicalis DNA and a CYP52A1 probe under low-stringency conditions (Sanglard and Fiechter, 1989) can now be assigned to the isolated additional CYP52 genes. This leads us to conclude that, based on this low-stringency detection method, we have isolated almost all CYP52 genes of this yeast. Presently the possibility cannot be excluded that additional

CYP52-related genes are present in the yeast genome. Specific disruption of the genes described in this study by genetic methods and the resulting growth phenotype on aliphatic substrates of such strains could help to address this

problem.

The CYP52 gene family of C. tropicalis is thus extended members, with two pairs being tandemly arranged on the genome. There are different elements that tend to to seven

IDENTIFICATION OF P450 GENES

777

-z.

~

M

5

-S

-S

U

t.

[^

(fl

tÔ

"1

I

53.5 kDa

57.9 kDa

alk

Coomassie staining

antibody

B

alk7

A. Coomassie-stained NaDodS04-polyacrylamide gel of microsomes from yeast transformants producing alkl tropicalis grown on alkane. B. Immunoblot analysis. The molecular sizes of the stained bands were calculated by their migration relative to protein size standards and are shown by arrows (53.5 kDa, C. tropicalis main reactive band; 57.9 kDa, alkl from S. cerevisiae) or mentioned in the text. C. Enlarged reproduction (1.8-fold) of the NaDodS04-polyacrylamide gel showing the S. cerevisiae control and the microsomal fraction of yeast producing alk7. The arrow indicates the band representing the alk7 protein.

FIG. 7.

to alk7 and from C.

Table 3. In Vitro Assays

of

Type of microsomal

fraction isolated from strain

[plasmid)'a

YS18[pYEp51] GRF18[pDS509-6(alkl)] GRF18[pWSl(alk2)] YS18[pRRl(alk3)] YS18[pWS4(alk4)] YS18[pWS5(alk5)] C. tropicalis on tetradecane et

Lauric Acid

and

Hexadecane Hydroxylation Acttvittes

of

Microsomal Fractions

Lauric acid

Hexadecane

P450

CPR

hydroxylation (pmoles/min/ nmoles P450)b

hydroxylation (pmoles/min/ nmoles P450)b

specific content fpmoles P450/ mg protein)^

specific activity

22 126 23 163

2,503 2,712 8,619

12 308 335

1,224 133 201

1,490

201 111 570 102 240 171

aThe plasmids pDS509-6 and pWSl, and the S. cerevisiae strain GRF18 were described previously (Sanglard and Loper,

at, 1991).

(mU/mg protein) & 98 110 115 108 112 107 478

1989; Seghezzi

bLauric acid and hexadecane hydroxylation activities were determined as described (Seghezzi et al, 1991). cThe P450 concentration was determined as described by Omura and Sato (1962). Protein concentrations were measured using the BioRad protein assay. dThe specific activity determination of NADPH-cytochrome P450 reducíase, CPR (Sutter et al., 1990) was described by Sanglard et al.

(1984).

indicate that these genes are nonallelic variants. First, restriction mapping of the C. tropicalis CYP52 genes revealed completely different patterns for all restriction sites tested, and not only minor variations as would be expected for allelic forms. In fact, this C. tropicalis strain has a particular high degree of homozygosity, since no restriction site polymorphism has been detected in other unrelated genes (see below). Second, nucleotide sequence analysis showed that the highest homologies between two CYP52

genes was 73.1%, a value far below, for example, the 98.1% reported by Ohkuma et al (1991b) for two allelic P450 genes isolated from a diploid strain of C. maltosa. Third, using gene-specific probes the seven genes could be located on four different chromosomes of the organism, and not on individual homologs as it would have been expected for allelic variants. The specific gene probes for CYP52A7, CYP52A8, and CYP52B1 generated two signals of equal intensity corre-

778

SEGHEZZI ET AL.

sponding to chromosomes VI and VIII. Kamiryo (1991) reported a similar phenomenon for the

et

al

genes and POX8B. This pattern could be ex-

POX6A, POX8A, plained by different hypotheses. First, the electrophoresis

procedure (CHEF) could lead to artifactual chromosomal separations. This explanation appears however very unlikely, because in our case the observed pattern was reproducible. Furthermore, because Candida are diploid or aneuploid microorganisms, either one chromosomal homolog could have lost or gained a DNA segment resulting in a different migration in gel electrophoresis, or a single allelic sequence could have been translocated heterochromosomally, for example by mitotic recombination through repeated sequences. A deletion in one chromosomal homolog of chromosome VIII resulting in its comigration with chromosomal band VI could also explain the observed discrepancy between the apparent band intensities of chromosomes VI and VIII in the stained agarose gel (Fig. 3, left) and the corresponding hybridization signal intensities (Fig. 3, right, lanes 3 and 4). Heterogenous homologs could be maintained in the asporogenic C. tropicalis, which has no haploid generation and thus does not depend on precise pairing of homologs or the integrity of each set

of chromosomes. While the existence of P450 multigene families in higher eukaryotes is often observed (Nebert and Gonzalez, 1987; Nebert et al, 1991), it constitutes an exception for lower eukaryotes such as yeast. To date the CYP52 multigene family of C. tropicalis and the one recently reported in C. maltosa (Ohkuma et al, 1991b; Schunck et al, 1989; Tagaki et al, 1989) are the only known examples. The second type of P450 enzyme known in yeast, namely 14DM, is encoded by only one gene in S. cerevisiae (Kalb et al., 1987) and in C. tropicalis (Chen et al, 1987). Other known genes of C. tropicalis, such as ADE2 (Sanglard and Fiechter, 1992) or the gene ACP encoding the extracellular protease (Togni et al, 1991; Sanglard et al, 1992) and URA3 (unpublished data), are present in two identical allelic forms because this yeast strain is diploid. The POX gene family coding for acyl-coenzyme A oxidase isoforms contains only three members, POX2, POX4, and POX5 (Okazaki et al, 1986, 1987). In addition, the known gene families of S. cerevisiae are also smaller with, for example, only four structural genes encoding the acid phosphatases (Rogers et al, 1982; Bajwa et al, 1984). The size of the CYP52 gene family therefore represents an interesting starting point for evolutionary studies. Additionally it should be mentioned that the tandem arrangement, including the intergenic distance of the two genes CYP52A2 and CYP52A1, is also found for their most closely related counterparts in C. maltosa and could thus mean that the duplication of an ancestral gene had occurred even before the two species diverged, as speculated by Ohkuma et al

(1991b).

Six of the seven CYP52 genes were shown to be inducible by different aliphatic carbon sources and repressed by glucose. The degree of induction not only varies substantially from gene to gene but also from substrate to substrate. In addition it was shown that apart from the carbon source the culture parameters also influence the transcrip-

tional control and/or the stability of the transcripts. One such parameter is the oxygen partial pressure that has previously been shown to be involved in the transcriptional regulation of S. cerevisiae genes such as ERG11 encoding the cytochrome P450 lanosterol 14a-demethylase (Turi and Loper, 1992), CYC7 (Zitomer et al, 1987), COX5 (Hodge et al, 1989), the gene encoding a catalase (Hörtner et al, 1982) or an antioxidant protein (Kim et al, 1989). CYP52A 7 could be successfully expressed in S. cerevisiae and thus could not be considered as a pseudogene, as originally concluded from the induction experiments (see Results). It is possible that this gene is induced in C. tropicalis under conditions different from those chosen in our experiments. The deduced amino acid sequences of all CYP52 genes were found to contain the characteristic heme binding region of P450s, called HR2 or proximal heme binding site, with the well-conserved cysteine residue as the fifth ligand to the prosthetic group, as well as the distal heme binding site. Differences were observed in the central and aminoterminal portion of the proteins, the latter containing a putative transmembrane domain (Fig. 2, underlined). This amino-terminal hydrophobic domain seems to be absent in the case of alk6 and reduced in the case of alk7. This reduced domain seems to be still sufficient for the targeting of alk7 to the endoplasmic reticulum membrane, because alk7 could be detected in the endoplasmic reticulum membrane fraction (see Results). However, no typical P450 spectrum was recorded in this fraction, thus suggesting that alk7 is present in a denatured form. Whether or not the weak amino-terminal hydrophobic domain of alk7 contributed to this phenomenon is still unclear. In an attempt to characterize the products encoded by the five additional CYP52 genes presented in this work, alk3, alk4, and alk5 were successfully expressed heterologously in S. cerevisiae and the presence of P450 proteins determined by spectral and immunological analysis. With alk3 it could be shown that, when using the inducible expression system (GALIO promoter), an almost four-fold increase in product yield was achieved as compared to the constitutive expression system (ADH1 promoter) applied earlier for the production of alkl and alk2 (data not shown). Using isolated microsomes, alk3 was shown to exert a strong hydroxylation activity toward hexadecane with the corresponding gene CYP52A6 being most strongly induced by alkanes. Therefore a correlation between induction and in vitro function seems to exist as found earlier for alkl and alk2. However, the product of CYP52A8, which is only weakly induced by substituted aliphatic substrates, displays a clear substrate preference for lauric acid. A different approach to study the functions of the diverse gene products would be to sequentially disrupt each of the genes, as it has been done with the POX genes of C. tropicalis (Picataggio et al, 1991) by making use of a recently developed transformation system. This would allow to establish a simple screening system for the disrupted mutants when grown on different aliphatic substrates. A similar approach was chosen to provide evidence for the CYP52A multigene family of C. maltosa (Ohkuma et al,

IDENTIFICATION OF P450 GENES

1991a), where the two-step disruption of the first P450 gene led to a mutant which still assimilated n-alkane and

779

Saccharomyces cerevisiae by glucose, oxygen and heme. Eur. J. Biochem. 128, 179-184. HUG, H., BLANCK, H.W., and FIECHTER, A. (1974). The still contained «-alkane-inducible P450. functional role of lipids in hydrocarbon assimilation. Biotechnol. Bioeng. 16, 965-985. KALB, V.F., WOODS, C.W., TURI, T.G., DEY, C.R., SUTACKNOWLEDGMENT TER, T.R., and LOPER, J.C. (1987). Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cereThis work was supported by a Swiss Research National visiae. DNA 6, 529-537. Foundation grant 20-28794.00 to D.S. KAMIRYO, T., MITO, N., NIKI, T., and SUZUKI, T. (1991). Assignment of most genes encoding major peroxisomal polypeptides to chromosomal band V of the asporogenic yeast CanREFERENCES dida tropicalis. Yeast 7, 503-511. KIM, I.H., KIM, K., and REE, S.G. (1989). Induction of an BAJWA, W., MEYHACK, B., RUDOLPH, H., SCHWEINantioxidant protein of Saccharomyces cerevisiae by 02, Fe3* or GRUBER, A.-M., and HINNEN, A. (1984). Structural analy2-mercaptoethanol. Proc. Nati. Acad. Sei. USA 86, 6018-6022. sis of the two tendemly repeated acid phosphatase genes in KUROIWA, T., SAKAGUCHI, M., KATSUYOSHI, M., and yeast. Nucleic Acids Res. 12, 7721-7739. OMURA, T. (1991). Systematic analysis of stop-transfer seBORCK, K., BEGGS, J.D., BRAMMAR, W.J., HOPKINS, quence for microsomal membrane. J. Biol. Chem. 266, 9251A.S., and MURRAY, N.E. (1976). The construction in vitro of 9255. transducing derivatives of phage lambda. Mol. Gen. Genet. KYTE, J., and DOOLITTLE, R.F. (1982). A simple method for 146, 199-206. displaying the hydropathic character of a protein. J. Mol. Biol. BROACH, J.R., LI, Y.Y., WU, L.-C.C, and JAYARAM, M. 157, 105-132. (1983). Vectors for high-level, inducible expression of cloned LOPER, J.C, CHEN, C, and DEY, C.R. (1985). Gene engigenes in yeast. In Experimental Manipulation of Gene Expresneering of yeast for biodégradation: Immunological cross-reacsion. M. Inouye, ed. (Academic Press, New York) pp. 83-117. tivity amon cytochrome P450 system proteins of SaccharomyBÜHLER, M., and SCHINDLER, J. (1984). Aliphatic hydrocarces cerevisiae and Candida tropicalis. Hazardous Waste bons. In Biotechnology, vol. 6a. K. Riesling, ed. (Verlag Hazardous Mat. 2, 131-141. Chemie, Basel) pp. 329-385. MONOD, M., PORCHET, S., ROSSELET-BAUDRAZ, F., and CHEN, C, TURI, T.G., SANGLARD, D., and LOPER, J.C. FRENK, E. (1990). The identification of pathogenic yeast strains by electrophoretic analysis of their chromosomes. J. (1987). Isolation of the Candida tropicalis gene for P450 lanosterol demethylase and its expression in Saccharomyces Med. Microbiol. 32, 123-129. cerevisiae. Biochem. Biophys. Res. Commun. 146, 1311-1317. MURRAY, N.E., BRAMMAR, W.J., and MURRAY, K. (1977). Lambdoid phages that simplify the recovery of in vitro recomCHIRGWIN, J.M., PRZYBYLA, A.E., MacDONALD, R.J., and RUTTER, W.J. (1979). Isolation of biologically active binants. Mol. Gen. Genet. 150, 53-59. ribonucleic acid from sources enriched in ribonuclease. Bio- NEBERT, D.W., and GONZALEZ, F.J. (1987). P450 genes: chemistry 18, 5294-5299. Structure, evolution and regulation. Annu. Rev. Biochem. 56, DAVIS, L.G., DIBNER, M.D., and BATTEY, J.F. (1986). Prep945-993. aration and analysis of RNA from eukaryotic cells. In Basic NEBERT, D.W., NELSON, D.R., ADESNIK, M., COON, Methods in Molecular Biology. (Elsevier, Amsterdam) pp. 129M.J., ESTABROOK, R.W., GONZALEZ, J.J., GUEN152. GERICH, F.P., GUNSALUS, I.C., JOHNSON, E.F., KEMFEINBERG, A.P., and VOGELSTEIN, B. (1983). A technique PER, B., LEVIN, W., PHILLIPS, I.R., SATO, R., and for radiolabeling DNA restriction endonuclease fragments to WATERMAN, M.R. (1989). The P450 gene superfamily. Uphigh specific activity. Anal. Biochem. 132, 6-13. dated listing of all genes and recommended nomenclature for FUKUDA, Y., ATOMI, H., KURIHARA, T., HIKIDA, M., the chromosomal loci. DNA 8, 1-13. UEDA, M., and TANAKA, A. (1991). Genes encoding peroxi- NEBERT, D.W., NELSON, D.R., COON, M.J., ESTABROOK, somal enzymes are not necessarily assigned on the same chroR.W., FEYEREISEN, R., FUJII-KURIYAMA, Y.A., GONmosome of an alkane-utilizable yeast Candida tropicalis. FEBS ZALEZ, F.J., GUENGERICH, F.P., GUNSALUS, I.C., Lett. 286, 61-63. JOHNSON, E.F., LOPER, J.C, SATO, R., WATERMAN, GMÜNDER, F.K., KÄPPELI, O., and FIECHTER, A. (1981). M.R., and WAXMAN, D.J. (1991). The P450 superfamily: Chemostat studies on the hexadecane assimilation by the yeast Update on new sequences, gene mapping and recommended Candida tropicalis, II. Regulation of cytochromes and ennomenclature. DNA Cell Biol. 10, 1-14. zymes. Eur. J. Appl. Microbiol. Biotechnol. 12, 135-142. NEEDLEMAN, S.B., and WUNSCH, CD. (1970). A general HAMILTON, R., WANATABE, C.K., and DE BOER, H.A. method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 48, 443-453. (1987). Compilation and comparison of the sequence context around the AUG start codons in Saccharomyces cerevisiae OHKUMA, M., HIKIJI, T., TANIMOTO, T., SCHUNCK, mRNAs. Nucleic Acids Res. 15, 3581-3593. W.-H., MUELLER, H.-G., YANO, K., and TAKAGI, M. HANAHAN, D. (1985). Techniques for transformation of E. (1991a). Evidence that more than one gene encodes /i-alkane-inducible cytochrome P-450s in Candida maltosa, found by twocoli. In DNA Cloning, vol. I. D. Glover, ed. (IRL Press, Oxford) pp. 109-135. step gene disruption. Agrie. Biol. Chem. 55, 1757-1764. HODGE, M.R., KIM, G., SINGH, K., and CUMSKY, M.G. OHKUMA, M., TANIMOTO, T., YANO, K., and TAKAGI, M. (1989). Inverse regulation of the yeast COX5 genes by oxygen (1991b). CYP52 (Cytochrome P450alk) multigene family in and heme. Mol. Cell. Biol. 9, 1958-1964. Candida maltosa: Molecular cloning and nucleotide sequence HÖRTNER, H., AMMERER, G„ GARTTER, E., HAMILof the two tandemly arranged genes. DNA Cell Biol. 10, 271TON, B., RYTKA, J., BILINSKI, T., and RUIS, H. (1982). 282. Regulation of synthesis of catalase and iso-1-cytochrome c in OKAZAKI, K., TAKECHI, T., KAMBARA, N., FUKUI, S.,

SEGHEZZI ET AL.

780

KUBOTA, I., and KAMIRYO, T. (1986). Two acyl-coenzyme A oxidases in peroxisomes of the yeast Candida tropicalis: Primary structures deduced from genomic DNA sequence. Proc. Nati. Acad. Sei. USA 83, 1232-1236. OKAZAKI, K., TAN, H., FUKUI, S., KUBOTA, I., and KAMIRYO, T. (1987). Peroxisomal Acyl-coenzyme A oxidase multigene family of the yeast Candida tropicalis: Nucleotide sequences of a third gene and its protein product. Gene 58, 3744. OMURA, T., and SATO, R. (1962). The carbon monoxide-binding pigment of liver microsomes. J. Biol. Chem. 237, 1376. PICATAGGIO, S., DEANDA, K., and MIELENZ, J. (1991). Determination of Candida tropicalis acyl coenzyme A oxidase isoenzyme function by sequential gene disruption. Mol. Cell. Biol. 11, 4333-4339. ROGERS, D.T., LEMIRE, J.W., and BOSTIAN, D.A. (1982). Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family. Proc. Nati. Acad. Sei. USA 79, 2157-2161. ROTHSTEIN, R. (1985). Cloning in yeast. In DNA Cloning. A Practical Approach, vol. II. D. Glover, ed. (IRL Press, Oxford) pp. 45-66. SAIKI, R., GELFAND, D., STOFLET, S., SCHAFT, S., HIGUCHI, R., HORN, G., MULLÍS, K., and ERLICH, H. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. SAMBROOK, J., FRITSCH, E.F., and MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). SANGLARD, D., and FIECHTER, A. (1989). Heterogeneity within the alkane-inducible cytochrome P450 gene family of the yeast Candida tropicalis. FEBS Lett. 256, 128-134. SANGLARD, D., and LOPER, J.C. (1989). Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: Identification of a new P450 gene family. Gene 76, 121-136. SANGLARD, D., and FIECHTER, A. (1992). DNA transformation of Candida tropicalis with replicative and integrative vectors. Yeast (in press). SANGLARD, D., KÄPPELI, O., and FIECHTER, A. (1984). Metabolic conditions determining the composition and catalytic activity of cytochrome P450 monoxygenases in Candida tropicalis. J. Bacteriol. 157, 297-302. SANGLARD, D., CHEN, C, and LOPER, J.C. (1987). Isolation of the alkane inducible P450 (P450alk) gene from the yeast Candida tropicalis. Biochem. Biophys. Res. Commun. 144, 251-257. SANGLARD, D., TOGNI, G., DE VIRAGH, P.A., and MONOD, M. (1992). Disruption of the gene encoding the secreted acid protease (ACP) in the yeast Candida tropicalis. FEMS Microbiol. Lett. 95, 149-156. SANTOS, M., COLTHURST, D.R., WILLS, N., McLAUGH-

LIN, C.S., and TUITE, M.F. (1990). Efficient translation of the UAG termination codon in Candida species. Curr. Genet. 17, 487-491. SCHUNCK, W.-H., KAERGEL, E., GROSS, B., WIEDMANN, B., MAUERSBERGER, S., KOPKE, K., KIEBLING, U., STRAUSS, M., GAESTEL, M., and MUELLER, H.-G. (1989). Molecular cloning and characterization of the primary structure of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa. Biochem. Biophys. Res. Commun. 161, 843-850. SEGHEZZI, W., SANGLARD, D., and FIECHTER, A. (1991). Characterization of a second alkane-inducible cytochrome P450-encoding gene, CYP52A2, from Candida tropicalis. Gene 106, 51-60. SENGSTAG, C, and HINNEN, A. (1987). The sequence of the Saccharomyces cerevisiae gene PH02 codes for a regulatory protein with unusual amino acid composition. Nucleic Acids Res. 15, 233-246. SUTTER, T.R., SANGLARD, D., and LOPER, J.C. (1990). Isolation and characterization of the alkane-inducible NADPHcytochrome P-450 oxidoreductase gene from Candida tropicalis. J. Biol. Chem. 265, 16428-16436.

TAKAGI, M., OHKUMA, M., KOBAYASHI, N., WATANABE, M., and YANO, K. (1989). Purification of cytochrome P450alk from n-alkane-grown cells of Candida maltosa, and

cloning and nucleotide sequencing of the coding gene. Agrie. Biol. Chem. 53, 335-342. TOGNI, G., SANGLARD, D., FALCHETTO, R., and MONOD, M. (1991). Isolation and nucleotide sequence of the extracellular acid protease gene (ACP) from the yeast Candida tropicalis. FEBS Lett. 286, 181-185. TURI, G.T., and LOPER, J.C. (1992). Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14a-demethylase (ERG11). J. Biol. Chem. 267, 2046-2056. ZITOMER, R.S., SELLERS, J.W., McCARTER, D.W., HASTINGS, G.A., WICK, P., and LOWRY, C.V. (1987). Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene. Mol. Cell. Biol. 7, 2212-2220. Address reprint requests to: Dr. D. Sanglard Institute of Biotechnology Swiss Federal Institute of Technology ETH

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publication July 14, 1992, and in revised form September 10, 1992. Received for