# Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression

Frederic Bigey, Karine Tuery, Daisy Bougard, Jean-Marc Nicaud, Guy Moulin

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Frederic Bigey, Karine Tuery, Daisy Bougard, Jean-Marc Nicaud, Guy Moulin. Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression. *Yeast*, 2003, 20 (3), pp.233-248. 10.1002/yea.958. *hal-02165415*

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# Identification of a triacylglycerol lipase gene family in *Candida deformans*: molecular cloning and functional expression

Frédéric Bigey<fnref n="1" />*, Karine Tuery<fnref n="1" />, Daisy Bougard<fnref n="1" />#, Jean-Marc Nicaud<fnref n="2" /> and Guy Moulin<fnref n="1" />

*Correspondence to:
Frédéric Bigey, UFR de
Microbiologie Industrielle et
Génétique des Microorganismes,
INRA-ENSAM, 2 Place Viala,
F-34060 Montpellier Cedex
1, France.

E-mail:
Frédéric.Bigey@ensam.inra.fr

\# Present address: Laboratoire de Biotechnologie de l'Environnement, INRA, Avenue des Etangs, F-11100 Narbonne, France.

## Abstract

The yeast *Candida deformans* CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, *Saccharomyces cerevisiae* was transformed with *C. deformans* genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (*CdLIP1*, *CdLIP2* and *CdLIP3*) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the *CdLIP1* gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species *Yarrowia lipolytica*. Significant homologies were also found with several yeast and fungal lipases. As *C. deformans* CBS 2071 was previously considered to be synonymous with *Y. lipolytica*, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that *C. deformans* is a separate species. The nucleotide sequences of the *CdLIP1*, *CdLIP2* and *CdLIP3* genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively. Copyright © 2003 John Wiley & Sons, Ltd.

**Keywords:** triacylglycerol lipase; gene family; MALDI-TOF mass spectrometry; 26S rDNA gene; *Candida deformans*; *Saccharomyces cerevisiae*; *Yarrowia lipolytica*

Received: 13 March 2002

Accepted: 2 November 2002

## Introduction

Lipases or triacylglycerol lipases (E.C. 3.1.1.3) are enzymes capable of catalysing the hydrolysis of the ester bond between fatty acids and glycerol. They are, however, generally capable of hydrolysing esters of fatty acids and many alcohols other than glycerol. Triacylglycerols (triglycerides), having very low solubility in water, are their preferential substrates. Under natural conditions, they catalyse the hydrolysis of ester bonds at

the interface between an insoluble substrate phase and the aqueous phase in which the enzyme is dissolved. Therefore, they differ from esterases, which hydrolyse only soluble esters of fatty acids with short carbon chains. Under certain experimental conditions, some lipases can also catalyse ester synthesis via the esterification reaction (reversal of the hydrolysis reaction) or the transesterification reaction (transferring the acyl moiety of an ester to a suitable nucleophile other than water via alcoholysis or ester exchange). The amount of

<footnote n="1">¹ UFR de Microbiologie Industrielle et Génétique des Microorganismes, INRA-ENSAM, 2 Place Viala, F-34060 Montpellier Cedex 1, France</footnote>

<footnote n="2">² Laboratoire de Microbiologie et Génétique Moléculaire, INRA-CNRS, Centre de Grignon, F-78850 Thiverval-Grignon, France</footnote>
water in the reaction mixture is a critical param-
eter that determines the direction of the lipase-
catalysed reaction. In nearly anhydrous organic sol-
vents, esterification and transesterification reactions
are generally favoured while, in the presence of
water, the reaction equilibrium is shifted towards
hydrolysis and ester synthesis is limited.

Lipases are widespread in the animal, plant
and microbial kingdoms. The first studies focused
mainly on animal and plant lipases because of
their physiological importance. Recently, lipases of
microbial origin have attracted much interest due
to their diverse properties, availability and rela-
tively easy methods of preparation. Several review
articles presenting comprehensive overviews of
the biochemical properties and biotechnological
applications of these enzymes have been pub-
lished (Jaeger et al., 1994; Jaeger and Reetz, 1998;
Pandey et al., 1998; Saxena et al., 1999). Impor-
tant uses in biotechnology include their significant
role in the medical and therapeutic fields (supple-
ments in patients with pancreatic insufficiencies,
plasmatic triglyceride analysis, stereospecific syn-
thesis of pharmacologically active products), chem-
istry of fats and oils (fat and oil hydrolysis, syn-
thesis of esters with surfactant properties), cleaning
(washing powders) and food technology (modifi-
cation of flavour, improvements in conservation,
acceleration of fermentation, formulation of oils).

Our laboratory previously reported that the yeast *C. deformans* CBS 2071 produces an extracellular 1,3-regiospecific lipase which was used for palm oil modification (Muderhwa *et al.*, 1985). Subsequently, our laboratory demonstrated that this enzyme was very effective in catalysing methyl ester synthesis from triacylglycerols or free fatty acids in a high water activity medium (Boutur *et al.*, 1994). In fact, it was determined that the enzyme catalysed ester production in aqueous media, not by alcoholysis but by the esterification of free fatty acids (Boutur, 1995; Boutur *et al.*, 1995).

In this study, in an attempt to clone the
extracellular lipase gene from *C. deformans*,
we used a complementation strategy employing
several strains of *S. cerevisiae* transformed with
*C. deformans* genomic libraries. By this approach,
we have cloned and sequenced three *C. deformans*
genes that imparted a positive response to
*S. cerevisiae* cells.

**Materials and methods**

Strains and culture conditions

Strains used in this study are described in Table 1.
The media and techniques used to grow the strains
were as follows. *C. deformans* CBS 2071 (CBS
stands for Centraalbureau voor Schimmelcultures,
Utrecht, The Netherlands) was cultivated aero-
bically at 28°C in Erlenmeyer flasks filled to
one-tenth of their capacity. For lipase produc-
tion, the strain was grown in synthetic G medium
(Galzy, 1964) buffered to pH 6.5 with 100 mM
NaH2PO4/Na2HPO4, and supplemented with 5 g/l
rapeseed oil as the carbon source and inductor. Rich
medium YEG, composed of yeast extract (5 g/l,
Difco Becton Dickinson France SA, Le Pont de
Claix, France) and glucose (10 g/l), was used for
*C. deformans* chromosomal DNA extraction. Rich
YPD medium, yeast extract (10 g/l), Bacto pep-
tone (20 g/l, Difco), glucose (20 g/l), was used
for *S. cerevisiae* cultivation at 30°C. *S. cerevisiae*
transformants were selected on YNB medium con-
taining yeast nitrogen base without amino acids
(6.7 g/l, Difco) and glucose (20 g/l). Adenine, his-
tidine, tryptophan (20 mg/l) and leucine (30 mg/l)
were added when required. For solid media, 20 g/l
agar was added.

YNB-rhodamine B plates, used for lipase
detection in *S. cerevisiae* transformants, were
derived using the plate assay described by
Kouker and Jaeger (1987). The medium contained
glucose (5 g/l), rapeseed oil (5 g/l), agar (20 g/l)
and was buffered to pH 6.5 with 100 mM
NaH2PO4/Na2HPO4. The medium was autoclaved
and cooled down to 60 °C. Then, 10 ml rhodamine
B solution (1 mg/ml) and 100 ml yeast nitrogen
base without amino acids (67 g/l, Difco) were
added per litre. The medium was emulsified
by mixing with Ultra-Turrax disperser (Janke &
Kunkel KG, Staufen, Germany) and poured into
Petri dishes. After incubation, strains producing
extracellular lipase show, upon UV illumination,
an orange fluorescent halo around the colony.

For lipase detection in *Y. lipolytica*, YNBH and YNBT plates were composed as follow: 50 mM KH2PO4/Na2HPO4 (pH 6.8), yeast nitrogen base without amino acids (6.7 g/l, Difco), agar (20 g/l), olive oil (YNBH, 10 g/l), tributyrine (YNBT, 10 g/l). Rhodamine B (10 mg/l) was added to YNBH for detection of free fatty acids. The media were emulsified as above.
<table><caption>**Table 1.** Strains and plasmids used in this study</caption><thead><tr><th>Strains or plasmids</th><th>Description or relevant feature(s)</th><th>Reference or source</th></tr></thead><tbody><tr><td>Strains</td><td></td><td></td></tr><tr><td>C. deformans</td><td>(Zach) Langeron and Guerra; lipase-producing</td><td>CBS 2071</td></tr><tr><td>S. cerevisiae</td><td></td><td></td></tr><tr><td>W303-1a</td><td>MATα ura3-1 trp1-1 ade2-1 leu2-3,112 his3-11,15 can1-100</td><td></td></tr><tr><td>OL1</td><td>MATα ura3-251,373 leu2-3,112 his3-15</td><td></td></tr><tr><td>LL-20</td><td>MATα leu2-3,112 his3-11,15 can1</td><td></td></tr><tr><td>Y. lipolytica</td><td></td><td></td></tr><tr><td>JMY277</td><td>MATa ura3-302 leu2-270 xpr2-322 lip2::LEU2</td><td>Pignede et al. (2000a)</td></tr><tr><td>JMY277(62)</td><td>JMY277 transformed to Ura<sup>+</sup> by integration of JMP62</td><td>This work</td></tr><tr><td>JMY277(62CdLIP1)</td><td>JMY277 transformed to Ura<sup>+</sup> by integration of JMP62CdLIP1</td><td>This work</td></tr><tr><td>JMY277(62CdLIP2)</td><td>JMY277 transformed to Ura<sup>+</sup> by integration of JMP62CdLIP2</td><td>This work</td></tr><tr><td>JMY277(62CdLIP3)</td><td>JMY277 transformed to Ura<sup>+</sup> by integration of JMP62CdLIP3</td><td>This work</td></tr><tr><td>Plasmids</td><td></td><td></td></tr><tr><td>YEp352</td><td>Ap<sup>r</sup> E. coli-S. cerevisiae shuttle vector, 2 µm, URA3</td><td>Hill et al. (1986)</td></tr><tr><td>pRS425</td><td>Ap<sup>r</sup> E. coli-S. cerevisiae shuttle vector, 2 µm, LEU2</td><td>Christianson et al. (1992)</td></tr><tr><td>YEpCw</td><td>YEp352 derivative, with 4.6 kb fragment from C. deformans containing CdLIP1</td><td>This work</td></tr><tr><td>YEpACo</td><td>YEp352 derivative, with 5.2 kb fragment from C. deformans containing CdLIP2</td><td>This work</td></tr><tr><td>pRSAL</td><td>pRS425 derivative, with 5.6 kb fragment from C. deformans containing CdLIP3</td><td>This work</td></tr><tr><td>JMP62</td><td>Ap<sup>r</sup> E. coli, Y. lipolytica integrative vector, LIP2 promoter, URA3</td><td>Nicaud et al. (2002)</td></tr><tr><td>JMP62CdLIP1</td><td>JMP62 derivative, CdLIP1</td><td>This work</td></tr><tr><td>JMP62CdLIP2</td><td>JMP62 derivative, CdLIP2</td><td>This work</td></tr><tr><td>JMP62CdLIP3</td><td>JMP62 derivative, CdLIP3</td><td>This work</td></tr></tbody></table>

*Escherichia coli* XL1 Blue MRF' (Stratagene) used for plasmid preparation was grown in Luria-Bertani (LB) medium with ampicillin (100 mg/l) or kanamycin (40 mg/l).

Microbial growth was monitored by measuring culture absorbance at 600 nm.

## Genomic DNA isolation

Cells from 2 ml *C. deformans* culture (ca 1.5 × 10⁹ cells) were harvested by centrifugation and washed in 2 ml SDE buffer (0.9 M sorbitol, 0.1 M EDTA, 50 mM dithiothreitol, pH 7.5). Cells were resuspended in 0.5 ml SDE buffer containing 200 units of lyticase (Sigma) and incubated for 2 h at 37 °C. After centrifugation, the cell pellet was dispersed in 0.5 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM EDTA, 0.5 mg/ml proteinase K). Cell lysis was induced by adding 50 µl 10% (wt/vol) SDS. After overnight incubation at 55 °C,

proteins were extracted successively with one volume phenol (Tris-HCl saturated phenol, pH 8.0), one volume phenol:chloroform:isoamyl alcohol (25:24:1, vol:vol:vol) and one volume chloroform:isoamyl alcohol (24:1, vol:vol). DNA was precipitated with two volumes of ethanol and then dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

## Genomic library construction

Genomic DNA from *C. deformans* was partially digested with *Sau3A*, and fragments were separated by agarose gel electrophoresis. The resulting DNA fragments of 5–10 kb were recovered from agarose using the Geneclean kit (Bio 101 Inc., Vista, CA, USA), ligated into *Bam*HI-digested and dephosphorylated YEp352 and pRS425 plasmids, then introduced into *E. coli* XL1 Blue
MRF'. About 60 000 and 30 000 *E. coli* transformants were obtained for the YEp352 and the pRS425 libraries, respectively. For each library, analysis of 20 randomly selected transformants indicated that 85% of the plasmid contained inserts (mean size 5.7 kb). Plasmids from the pooled *E. coli* transformants were isolated and used to transform S. cerevisiae strains OL1, W303-1a (YEp352 library) and LL-20 (pRS425 library) to yield 18 000, 23 000 and 6200 transformants, respectively. After incubation for 4 days at 30 °C, S. cerevisiae transformants were recovered in saline solution (NaCl 9 g/l). The cells suspensions were diluted and spread on YNB-rhodamine plates and incubated at 30 °C in the dark.

## Lipase activity assay and enzyme preparation

The production of extracellular and cell-bound lipases during the growth of *C. deformans* was analysed as follows: cells from 2 ml culture medium were harvested by centrifugation; the culture supernatant was conserved for extracellular lipase assay. The cell pellet was washed three times with 0.4 ml sodium phosphate buffer (20 mM NaH₂PO₄/Na₂HPO₄, pH 7.0); the washing supernatants were pooled. Lipase activity in both the culture supernatant and cell washes was determined. Lipase activity was determined by measuring the initial rate of triolein hydrolysis at 40 °C, pH 7.0, as described by Boutur *et al.* (1995).

Lipase preparation was performed as follows: cells from 0.75 l *C. deformans* culture grown in G medium were harvested at the early stationary phase by centrifugation. The cell pellet was extracted three times with 75 ml 20 mM sodium phosphate buffer (pH 7.0). Two volumes of cold acetone were added slowly to the supernatant. The mixture was kept at 4 °C for 30 min, and then centrifuged at 8000 × g for 15 min. The protein pellet was suspended in 40 ml 50 mM sodium phosphate (pH 7.0) and concentrated down to 0.7 ml on a regenerated cellulose ultrafiltration membrane (Millipore, YM10 membrane, cut-off 10 000 Da).

## Protein analysis

When required, proteins were deglycosylated using endoglycosidase H (endo H, New England Biolabs Inc., Beverly, MA, USA) in accordance with

the manufacturer's instructions. Protein concentrations were determined using the Pierce BCA Protein Assay (Pierce, PerBio France, Bezons, France), with bovine serum albumin as the standard. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of Laemmli (1970). Low-range protein markers (Bio-Rad SA, Ivry sur Seine, France) and proteins were visualized by staining with Coomassie brilliant blue R-250.

For MALDI-TOF mass spectrometry analysis, proteins were separated by SDS-PAGE. The proteolytic protocol used for the trypsin digestion was adapted from Jensen *et al.* (1999). The 38 kDa band was excised following SDS-PAGE, washed, dehydrated in pure acetonitrile and dried under vacuum. After reduction of the disulphide bridges with dithiothreitol, the cysteines were alkylated with iodoacetamide. The proteins were digested overnight with 0.125 µg sequencing grade trypsin (Promega) at 37 °C. One volume of the medium, containing the peptides that diffused freely out of the gel slice, was mixed with an equal volume of saturated solution of α-cyano 4-hydroxycinnamic acid. A deposit of the mixture was made on a clean MALDI target and air-dried. Mass spectrometry analysis was performed on a MALDI-TOF BiFlex III spectrometer (Bruker, Breme, Germany). The masses were measured with an accuracy of 0.1 g/mol for a peptide of 1000 g/mol. Peptides are ionized [M + H]⁺ so the measured mass is increased by 1.0078 g/mol.

## Lipase expression in *Y. lipolytica*

The expression vectors JMP62CdLIP1, JMP62-CdLIP2 and JMP62CdLIP3 (Table 1) were constructed as follows: the coding regions of *CdLIP1*, *CdLIP2* and *CdLIP3* were inserted into the expression vector JMP62 under the *POX2* promoter (Nicaud *et al.*, 2002). JMP62CdLIP1 was obtained by inserting together a 200 bp *Hind*III-*Kpn*I fragment and a 1200 bp *Kpn*I-*Sph*I (*Sph*I was blunt-ended with T4 DNA polymerase) fragment from plasmid YEpCw (Figure 1A) into the *Hind*III and *Eco*RI (blunt-ended) sites of JMP62. JMP62CdLIP2 was obtained by inserting a 1500 bp *Bam*HI-*Eco*RI PCR fragment into the corresponding sites of JMP62. The *Bam*HI and *Eco*RI sites were created at the ATG initiation codon and in the 3'-untranslated region of *CdLIP2* by
<figure><img src="image_2.png" /><figcaption>**Figure 1.** (A) Restriction map of the 4.6 kb fragment from plasmid *YEPCW*. The segment for which the 2981 nt sequence is reported is represented at the top. The ORF identified in the sequence is represented by a black arrow, and restriction sites used in the construction of expression vectors are in bold. (B) Nucleotide sequence of the *CdLIP1* gene and its derived amino acid sequence. Nucleotides and amino acids are numbered from the translation start codon. In the 5'-untranslated region, a potential TATA-box is in bold and underlined. The putative polyadenylation site at the 3'-untranslated region is in bold and underlined. The black triangle indicates a predicted signal peptidase cleavage site inside the three X-Ala or X-Pro dipeptides, and the open triangle a KEX2-like endopeptidase cleavage site after the Lys-Arg sequence. Potential N-glycosylation sites are shown in bold as well as the conserved GHSLGGA sequence containing the catalytic serine residue. Peptides identified by the MALDI-TOF mass spectrometry analysis of proteolytic digests of the secreted lipase produced by *C. deformans* are boxed</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<table><thead><tr><th>Position</th><th>Strand</th><th>Base</th><th>Change</th><th>Effect</th></tr></thead><tbody><tr><td>ggtgtgctacaagtacttgtacaaattgaggtagtagacacgatttcatactaaatccgtccagagtctggactcgcagtctaaacggttattggacagggaaacaagcaacaccggaag</td><td>gtaaa</td><td>-961</td><td></td><td></td></tr><tr><td>gatttgtgggcccgcgtggcattggagattgggtgacgctgtttcacccctgctggaaagagatctagtttacttcggtgtgggaaagacagttagtagagtagagtgtccac</td><td>gaa</td><td>-841</td><td></td><td></td></tr><tr><td>gaaagaatcacgtgaatgggtgtggtggtacgacttgtcgactgggtggaaagataattggagacggttggtgatgtctgaagaccacttccatctgccaagaagtgtatcat</td><td>gttcgaggccttggcagatggaatagatagtcgtattaaggaggtggtcagttgaagaaaaatcgacggatgcaaacacccccgacgctgttttagcgggtctatacggtgctata</td><td>-601</td><td></td><td></td></tr><tr><td>caaggtgtactattgtatcacccgtggtgtgcctggatgtaatgataactgtgattagctaatttagtggcactgtgatgattagtagaaacacccaataatcacgtggaaaaaaaa</td><td>agaggatccttgtattaagtcagttgaccacggagaatccgatggtgaagtccccgaggcagattaggggtgtttgggtgagtttttgcatatattgacggaaactagaccactgcgtcaga</td><td>-361</td><td></td><td></td></tr><tr><td>ttctccaaacacaccgttctacgaacagtgtctctccaagaatataaccaaaaaggtgatacaggggcccgtgctagacatttcgcatccgaaaccgtctcacactatttttagccgggtatgg</td><td>tagtgctggttgtgctggtcatgacaataacactgggtcccctctttggcaagtgtggtattat</td><td>-121</td><td></td><td></td></tr><tr><td>ATG GTC AAC TTT GGA GCT CGG ATC AAG GAC TTC TTT TCG GTG CTG CTC TTT GGA GCA GCT TCT ACC TCC TCC TCC TCT AAG ACA GCT CTC</td><td>M V N F G A R I K D F F S V L L F G A A S T S S S</td><td>90</td><td>S</td><td>K T A L</td></tr><tr><td>GTC TCT CAG GGC TTC TAT GAC GCA GCT CTG GAC TTT TCG CAC CTG TCC AAC GTG GCC TAC TGC GTT CAT GCT CCA ATT ACT CCG TTG AAA</td><td>V S Q G F Y D A A L D F S H L S N V A Y C V H A P I T P L K</td><td>180</td><td></td><td></td></tr><tr><td>GAC GAC TTT TCC TGT GGC CAG TCG TGT GTG CAT TTT CCC GAT ATG GAG CTG GTT ACC ACG TTT GGA GGT GAT CAG TTC TCG ACG TCC ATT</td><td>D D F S C G Q S C V H F P D M E L V T T F G G D Q F S T S I</td><td>270</td><td></td><td></td></tr><tr><td>ACC GGC TAC CTG GCT CTG GAT CAC GTG AAG GAA GAA AAG TAC GTG GTT TTC CGC GGC ACG TTT TCC ATT CCC GAC GCT ATC ACG GAT ATT</td><td>T G Y L A L D H V K K E K Y V V F R G T F S I P D A I T D I</td><td>360</td><td></td><td></td></tr><tr><td>CGG TTC CAG CAG TCG TCC TTT CTC GTC AAC GTG CCT GCT CTC AAC ACG TTT GCA CCC AAC GAC CCT TCT GGC GAG GCC CAG ATC GAC TGC</td><td>R F Q Q S S F L V N V P A L N T F A P N D P S G E A Q I D C</td><td>450</td><td></td><td></td></tr><tr><td>AAG GAG TGC AAA ATC CAC GAC GGC TTT TCG CAC GCC TTC ACC GAG ACC CTC CAT AAC ATT GGA CCC GTG CTC CAG CAG CAT CTC GAT TCA</td><td>K E C K I H D G F S R A F T E T L H N I G P V L Q Q H L D S</td><td>540</td><td></td><td></td></tr><tr><td>TAT CCG GAG TAC CAG CTC TAT GTC ACA GGA CAC TCT CTG GGA GCT GCC ATG GCT CTG TTG GCA GGC ACA TCC ATC AAG CTA CAG GGC TAC</td><td>Y P E Y Q L Y V T G H S L G A A M A L L A G T S I K L Q G Y</td><td>630</td><td></td><td></td></tr><tr><td>GAT CCC ATT GTG ATC AAC TAC GGA CAG CCC CGG GTC GGA AAC AGG GCG TTT GCA GAC TAC ATT TCG ACC CTA TGG TTC GGT AAC GGA GAC</td><td>D P I V I N Y G Q P R V G N R A F A D Y I S T L W F G N G D</td><td>720</td><td></td><td></td></tr><tr><td>GGG CTG GAA ATC AAC CGA CAA CGA CGA ATG TAT CGA ATG ACC CAT TGG AAT GAT GTC TTT GTG GGT CTG CCC AAT TGG GAC GGG TAT ACT</td><td>G L E I N R Q R R M Y R M T H W N D V F V G L P N W D G Y T</td><td>810</td><td></td><td></td></tr><tr><td>CAT TCC AGT GGA GAA GTG TAC ATT AAG GGC AAG TGG GTT AAT CCA GCT CTG AGA GAT GTC TTT TCC TGT GCT GGA GGA GAG AAC CCG GAA</td><td>H S S G E V Y I K G K W V N P A L R D V F S C A G G E N P E</td><td>900</td><td></td><td></td></tr><tr><td>TGT TAC CGG TCG ACT TTC AAT TTG TTG GCC CAG ATC AAT TTG CTG CAG AAC CAT TTG TGC TAC ATT GAT TAC ATT GGA TTC TGT GCT CTC</td><td>C Y R S T F N L L A Q I N L L Q N H L C Y I D Y I G F C A L</td><td>990</td><td></td><td></td></tr><tr><td>AAT GTG GGA AGG AGA GAG GTC AAC GAA CTA CAG ACG GAT CTG CCT AGT TAT ACC GGT CCG TAT CGG TAC GGA AAT AAG ACG GAG GAG GAT</td><td>N V G R R E V N E L Q T D L P S Y T G P Y R Y G N K T E E D</td><td>1080</td><td></td><td></td></tr><tr><td>TTT GTC AGG GAG GGG TTG GAG TTG GCG CAG TGA gtgcgaaatacgaactgcgagatgataccactgcagcaagatactgacctctaaccctcttttagactgatctc</td><td>F V R E G L E L A Q *</td><td>1188</td><td></td><td></td></tr></tbody></table>

aacaacccttccaattaatacactaactgatagtcctgtgagagtattgtaattgtgtagtagccgtgtatttttccagcagcccatcaaaaaatgattattgagatagacaccaatccaga
acttgtcatcaaccgtagttttgttgacggacagtggtgggctgaaaaagaaaaagcaaaaaagaaacatatataaattagtctgtggacggagtggttttgactctatcatgtctctcca
agctggtgatcatggtggttgagctattgttaaatggtgtgtgatacttattgaggttagctcggtattgtattaacgcggtaatcctatgattaagtaattcacacgatcagtgat
tcctaccaactactgcaacaccttcgatcgccaaaaccatttcatgtgtgcagtctatgtgcctccttaatcatcaattttttaaatgcagaacgtagtagcatttttgggcccgccttttc
caggcatctgtactgagccagatcttgatggatgctcctgatttgtagccattctatcattcaagtgtccgctacaggttggacaaggactgactggtacttgtagcttgaggtcagttc
ctcgcgtgtaaaaaaaagccaagaagaaaaatacaagtagaaatacgtcttttgtgctgtctttgacctattagaaatcgtagcctagaggtgtcaaaagga
agcttagagacagctc

**Figure 2.** (A) Restriction map of the 5.2 kb fragment from plasmid *YEpACO*. The segment for which the 2889 nt sequence is reported is represented at the top. The ORF identified in the sequence is represented by a black arrow. (B) Nucleotide sequence of the *CdLIP2* gene and its derived amino acid sequence. The features are presented according to Figure 1

PCR amplification using *YEpACO* as the template. JMP62CdLIP3 was constructed by inserting a 1500 bp *Nco*I (blunt-ended)–*Eco*RI fragment from pRSAL (Figure 3A) into the *Bam*HI (blunt-ended) and *Eco*RI sites of JMP62.

The expression cassettes were introduced into the *Y. lipolytica* strain JMY277 by transformation using the lithium acetate method, as described previously (Le Dall *et al.*, 1994). The vectors were digested by *Not*I to liberate the expression cassette
A

<figure><img src="image_4.png" /></figure>

B

<table><caption>**Figure 3.** (A) Restriction map of the 5.6 kb fragment from plasmid pRSAL. The segment for which the 2845 nt sequence is reported is represented at the top. The ORF identified in the sequence is represented by a black arrow, and restriction sites used in the construction of expression vectors are in bold. (B) Nucleotide sequence of the CdLIP3 gene and its derived amino acid sequence. The features are presented according to Figure 1</caption>
  <tbody>
    <tr>
      <td>caacccctcaacaacagtcatatatccaattatgCGttctgcacatggacctagtGCGtgacgttgagatGCGttct</td>
      <td>-841</td>
    </tr>
    <tr>
      <td>tccGCGaggattctCGgtgaaactCGgtCACCGtttGttGtatGCTgagacaagagtCGtGtttGtCTgGCGgatattatGGGGttGCGtgatatGCGCtattGtGtCtaccatttca</td>
      <td>-721</td>
    </tr>
    <tr>
      <td>tttCatttcatttcagCCgttcaaccaacaacagCCgtCCagCCgtCCattCaccatttCtCCaccactagtCtataCTaccGTctCCCtcaacGatCCCGttaatGCAactCCacca</td>
      <td>-601</td>
    </tr>
    <tr>
      <td>ccGCTGGtCGGaaaaaaacaacaGcaaaactCtCagaacGGatGCTCACCTGcaagTACagCTacagCTacagCTacagacacatGCAagaGCTtCACGtGCTggaatGCTGGCGgat</td>
      <td>-481</td>
    </tr>
    <tr>
      <td>caCTCACgtCTttGttGGGGtCGgtatGacaataatGataCTCGgAACGtgatCTttACatGGCTgacCTTTGaacACACTACCCatGACTagCTCCACagCTagCTCCACCGtCTa</td>
      <td>-361</td>
    </tr>
    <tr>
      <td>agACaggGACCACCGttAGCCGACGGCatttCCCGactGCAacGCGGgtCTtatCCGCTaatGACgtttagatCCGcagGAacccACcagCTCttGtCAGGttCGtgatGta</td>
      <td>-241</td>
    </tr>
    <tr>
      <td>ttGGCagagatGTGGCGGGgatCGataacaggagacaagagCagCCaggACgtGaaACGCTTTAACCGACGGtCacaAGGTGGTactGCACagatGATTGGGGataaGAGCGGT</td>
      <td>-121</td>
    </tr>
    <tr>
      <td>gtCGCtCTCATaAGCTGtCTACagCCTtACagCTtCTCagCCTtCCTACtCTCagCCCTCattACTCTCagCCCTCACtCTACGCTatGCAacagCCTtACTGCC</td>
      <td>-1</td>
    </tr>
    <tr>
      <td>ATG GTA ACC CTC TCT GCT CGA CTC AAA GAC TTC TTT TCG GTG CTT CTG CTC GGA GCA GCC CAG ATC ACC CCG TCT ACC CAA ACA GCA GGC</td>
      <td>90</td>
    </tr>
    <tr>
      <td>M V T L S A R L K D F F S V L L L G A A Q I T P S T Q T A G</td>
      <td>30</td>
    </tr>
    <tr>
      <td>GTG TCC CAG GGC TTC TAT GAC TTT GCG CGC GAC TTT GCC CAT CTT TCC AAC ATT GCT TAC TGT GTG GAC GCC CCC ATC ACT CCG CTG AAT</td>
      <td>180</td>
    </tr>
    <tr>
      <td>V S Q G F Y D F A R D F A H L S N I A Y C V D A P I T P L N</td>
      <td>60</td>
    </tr>
    <tr>
      <td>CCA GAC TTC ACC TGC GGA AAC TCG TGC AAG TAC TTT CCG GAC ATT GAG CTG GTC AAG ACG TTT GGA GGT GAC TTT TTT GAA ACG TCC ATC</td>
      <td>270</td>
    </tr>
    <tr>
      <td>P D F T C G N S C K Y F P D I E L V K T F G G D F F E T S I</td>
      <td>90</td>
    </tr>
    <tr>
      <td>ACG GGC TAC CTG GCC GTG GAC CAT GTC AAG AAG GAA AAG TAC GTC GTC TTC CGA GGC ACC TTT TCT CTG GGA GAC GCC ATC ACG GAC TTT</td>
      <td>360</td>
    </tr>
    <tr>
      <td>T G Y L A V D H V K K E K Y V V F R G T F S L G D A I T D F</td>
      <td>120</td>
    </tr>
    <tr>
      <td>CAG TTC CAG CAG TCT CCC TTT CTA GTT GAT GTT CCT GCC CTC AAC ACC TTT TCT GCA AAC GAT ACT TCT GCC GAG GCC CAG ATC CAA TGT</td>
      <td>450</td>
    </tr>
    <tr>
      <td>Q F Q Q S P F L V D V P A L N T F S A N D T S A E A Q I Q C</td>
      <td>150</td>
    </tr>
    <tr>
      <td>GAG GGC TGC AAG ATC CAC GAC GGC TTC TCC AAG GCC TTC ACC GAG ACC TGG GGC AAC ATT GGC GAG GAT CTC AAG AAA CAT CTC GAC TCC</td>
      <td>540</td>
    </tr>
    <tr>
      <td>E G C K I H D G F S K A F T E T E T W G N I G E D L K K H L D S</td>
      <td>180</td>
    </tr>
    <tr>
      <td>AAC CCG GAC TAC CAG CTC TAC GTG TCT GGA CAC TCT CTG GGA GCT GCC ATG TCT CTT TTG GGA GCC ACC TCG TTC AAG CTG AAG GGC TAC</td>
      <td>630</td>
    </tr>
    <tr>
      <td>N P D Y Q L Y V S G H S L G A A M S L L G A T S F K L K G Y</td>
      <td>210</td>
    </tr>
    <tr>
      <td>GAT CCC ATC CTG ATC AAC TAC GGC CAG CCC CGA GTT GGA AAC AAA CCC TTT TCC GAG TTC ATC AAC AAG CTG TGG TTC GGA GAC GGC AAC</td>
      <td>720</td>
    </tr>
    <tr>
      <td>D P I L I N Y G Q P R V G N K P F S E F I N K L W F G D G N</td>
      <td>240</td>
    </tr>
    <tr>
      <td>GGT CTG GAA ATC ACC CCC GAA CGA AGA CTC TAC CGA ATG ACT CAC TGG AAT GAC ATC TTT GTG GGG CTG CCC AAC TGG GAG GGA TAC ACC</td>
      <td>810</td>
    </tr>
    <tr>
      <td>G L E I T P E R R L Y R M T H W N D I F V G L P N W E G Y T</td>
      <td>270</td>
    </tr>
    <tr>
      <td>CAT TCC AAC GGC GAG GTT TAC ATC AAG AAC CGG TTC ATC AAC CCG CCA GTC AGT GAC GTC ATC TCC TGT GCC GGA GGA GAA AAC TCC CAG</td>
      <td>900</td>
    </tr>
    <tr>
      <td>H S N G E V Y I K N R F I N P P V S D V I S C A G G E N S Q</td>
      <td>300</td>
    </tr>
    <tr>
      <td>TGC TAC CGA TCC TCG TTT AAC ATC CTG TCT CAG ATC AAC CTG CTC CAG AAC CAT CTA GCG TAC ATT GAC TAC ATC GGA TAC TGT GCT CTG</td>
      <td>990</td>
    </tr>
    <tr>
      <td>C Y R S S F N I L S Q I N L L Q N H L A Y I D Y I G Y C A L</td>
      <td>330</td>
    </tr>
    <tr>
      <td>AC ATT GGA CGG CGA GAG TTG GCT GAT CAG AAA AAG TAC ACG GGT AAT TAT TAC TAT GCT CAT AGA ACC GAG GAG GAT TTC AAG AAG TTG</td>
      <td>1080</td>
    </tr>
    <tr>
      <td>N I G R R E L A D Q K K Y T G N Y Y Y A H R T E E D F K K L</td>
      <td>360</td>
    </tr>
    <tr>
      <td>GGT TTG GAG CTA GGT CCA CGA GCT GCG AAG CAG TGA gtGGataaCacGAagCCTgacaatCCAagtCTTtCACCTaaAgtCTTtCAacCAAagaattatCAagCCATC</td>
      <td>1187</td>
    </tr>
    <tr>
      <td>G L E L G P R A A K Q *</td>
      <td>371</td>
    </tr>
    <tr>
      <td>aagattGGTtCTGaccACTCTTGtCAGttACCTTTtCAacttCTaATAATTGTataACTAACCAACTCATCTCTTCattCTCACatGACTACagTCCTataACCAACAGCAatacaa</td>
      <td>1307</td>
    </tr>
    <tr>
      <td>ctCACGGtagATTAGCacaAGACTGACTtaATGGGtATATTAATCCCGGtTatCTAACACCCGaatAGTCCCTCgAGatTAAGAGTCCTACTCCACACATACTTCGATCCT</td>
      <td>1427</td>
    </tr>
    <tr>
      <td>gTAGatGACGtCCatCTaATagGCGCttATAGACTCCAGGtCTagATGGTataAAAGCAACAAAGCTatGCTtCTACatCCTgAGatCTGTGGAattCTaATGGTAGCAagaATG</td>
      <td>1547</td>
    </tr>
    <tr>
      <td>ACCGTTTatCGCCAGtaATTGCCGtGTAataAGTCTACTCCTTCAatCTatGCAACCGataACCCCGACCAGGtGGtCGAAACCTAGCACAAGACAACTCCTCAGGGACCGAGTCA</td>
      <td>1667</td>
    </tr>
    <tr>
      <td>CGTGCAaaAACAAGATCACATTCTACACTGaaATGTGGTtCTCTGATTCCAAACATACTGAAATTTCTTCCTGATTGatATGCAATAACAGGACTGCCCCAACTAAAGCT</td>
      <td>1787</td>
    </tr>
    <tr>
      <td>aAGTaaATCAAAACAATTCTCAATCCAGCAATTACAGCAGAAATAACATCTTCCCAAATTTCATTAATAAAACTGCTCACATTCACAGCAAATCACCGGTataAACTCCAGTCT</td>
      <td>1907</td>
    </tr>
    <tr>
      <td>ttGTGagACatCTGgAACA</td>
      <td>1926</td>
    </tr>
  </tbody>
</table>
prior to transformation. Lipase production was
analysed on YNBH and YNBT plates.

Other genetic methods, DNA sequencing and
sequence analysis

Plasmid isolation, DNA manipulations, and plas-
mid transformation into *E. coli* XL1 Blue MRF'
cells were performed as described by Sambrook
*et al*. (1989).

For sequence determination, the fluorescence-
based dideoxy DNA cycle sequencing method was
used. Sequencing reactions were performed by
Genome Express (Grenoble, France), using Big
Dye terminator sequencing chemistry on Applied
Biosystems automated sequencers (Applied Biosys-
tems, Courtaboeuf, France). Sequence analyses
were performed with the SeqLab interface of
the Wisconsin Package (GCG package, version
10.1, Genetics Computer Group, Madison, WI,
USA) and with the Staden package. For sim-
ilarity searches, Swiss-Prot (release 40.0) and
TrEMBL (release 18.0) databases were scanned
with the FastA program (Pearson and Lipman,
1988). Sequence homologies were obtained after
pairwise comparison with the Gap program (GCG).
Multiple alignments of protein sequences were
obtained by the program ClustalW (Higgins and
Sharp, 1988).

**Results**

Isolation of clones encoding *C. deformans*
lipid-hydrolysing enzymes

To clone the lipase encoding gene from *C. deformans*, we used a specific and sensitive rhodamine B plate assay, initially developed for bacterial lipases (Kouker and Jaeger, 1987). After incubation, by irradiating plates with UV light, lipase-producing colonies show orange fluorescent halos. Preliminary experiments showed that *S. cerevisiae* strains OL1, W303-1a and LL-20, unlike *C. deformans*, did not form a halo in this assay and therefore did not secrete detectable lipases. This suggested that the lipase plate assay could be useful in cloning the lipase gene from *C. deformans* through the ability of its DNA sequences to complement *S. cerevisiae* strains.

Two Sau3A genomic DNA libraries were con-
structed in the yeast vectors YEp352 and pRS425

(Table 1) as described in Materials and methods.
The completeness of the YEp352 library was tested
by complementation of *S. cerevisiae* *leu2* mutation.
Twelve Leu<sup>+</sup> transformants were obtained, corre-
sponding to about 60 000 Ura<sup>+</sup> *S. cerevisiae* OL1
transformants. This indicates that about 1/5000
of the transformants contained the *C. deformans*
*LEU2* gene and that this gene is expressed in
*S. cerevisiae*.

To screen for clones exhibiting lipolytic activ-
ity, approximately 6700, 4100 and 2900 Ura⁺
*S. cerevisiae* transformants were spread on YNB-
rhodamine B plates for strains OL1, W303-1a and
LL-20, respectively. Four lipase-positive colonies
were isolated from strain W303-1a, and one each
from strains OL1 and LL-20. Plasmid DNA from
the positive clones were recovered and transformed
into *E. coli*. Retransformation of W303-1a and LL-
20 was performed to verify that the lipolytic pheno-
type was due to the plasmids. Transformants were
tested on YNB-rhodamine plates and compared
with transformants containing the parental plas-
mids (YEp352 and pRS425) as negative controls.
In contrast to the cells containing the parental plas-
mids, all the transformants tested showed lipoly-
tic activity.

The plasmid from the lipolytic OL1 transformant was named YEpACO and it contained a 5.2 kb genomic insert. The plasmids from the lipolytic W303-1a transformants shared common fragments (data not shown). One of these plasmids, named YEpCw, which contained a 4.6 kb genomic insert, was selected for further analysis. The plasmid from the LL-20 transformant was named pRSA, and it contained a 5.6 kb genomic insert. The genomic fragments from these plasmids were first single-strand sequenced by primer walking to detect open reading frames (ORFs), and sequenced on the second strand in regions which contained an ORF coding for proteins with high sequence similarities to yeast and fungal lipases (see below). Restriction maps, nucleotide sequences and amino acid translations are presented in Figures 1–3. The lipase coding genes from plasmids YEpCw, YEpACO and pRSA, were named CdLIP1, CdLIP2 and CdLIP3, respectively.

Sequence analysis of the three lipase encoding
genes

From plasmid YEpC<sub>w</sub>, we determined the
sequence on the two strands of a 2981 bp
fragment (Figure 1A). It contained *CdLIP1*, which is 1005 bp long. Analysis of the 5'-untranslated regions revealed a potential TATA-box, identical to the most optimal yeast consensus TATAAA (Chen and Struhl, 1988), at position -56 bp relative to the ATG codon (Figure 1B). The 3'-untranslated region of *CdLIP1* contained the sequence AATAAA (50 bp downstream of the stop codon) usually found upstream to the polyadenylated 3' terminus in most eukaryotic mRNAs. The resulting 334 amino acid (aa) sequence shared 91.6% identity with YILIP2, the extracellular lipase produced by *Y. lipolytica* (Pignede et al., 2000a). The two proteins shared similar primary structure organization (Pignede et al., 2000a), as shown in Figures 1B and 4A, composed of a prepro region with a 15 aa peptide, followed by three X-Ala or X-Pro dipeptides (four for YILIP2), substrates of a diamino peptidase (Matoba and Ogrydziak, 1989); a short 12 aa pro region ending at a Lys-Arg (KR) site, the substrate of the KEX2-like endopeptidase encoded by the *XPR6* gene in *Y. lipolytica* (Enderlin and Ogrydziak, 1994); and finally the 301 aa mature protein coding for a 33.4 kDa protein (Figure 1B). A potential cleavage site of a signal peptide is most likely to occur between aa positions 17 and 18, according to SignalP prediction (Nielsen et al., 1997), after the first X-Ala dipeptide, to give a 17 aa signal sequence. The mature CdLIP1 presents two potential N-glycosylation sites at amino acids 146 and 167 (Figure 1B). The predicted pI of CpLIP1 is 5.6.

From plasmid YEpACO, we determined the sequence on the two strands of a 2889 bp fragment (Figure 2A). It contained *CdLIP2*, which is 1113 bp long. Analysis of the 5'-untranslated region revealed a TATAATA sequence at position -56 bp relative to the ATG codon that could work as an effective TATA-box in yeast (Figure 2B). The 3'-untranslated region of *CdLIP2* contains the sequence TAG...TATGT...TTT (108, 121 and 146 bp downstream of the stop codon), corresponding to the tripartite element that is found close to the transcription termination and polyadenylation sites in many yeast genes (Zaret and Sherman, 1982). The resulting 370 aa sequence contains several processing motifs (Figure 2B). A potential signal sequence cleavage site was detected between positions 25 and 26, according to SignalP, resulting in a 345 aa (38.6 kDa) mature protein with an

estimated pI of 5.2. One potential N-glycosylation site has been identified at aa position 355.

From pRSA<sub>L</sub>, we determined the sequence on the two strands of a 2845 bp fragment (Figure 3A). *CdLIP3* is 1116 bp long and encodes a 371 aa sequence. In the 5'-untranslated region of *CdLIP3*, a potential TATA-box (sequence TATAAG) at position -135 bp relative to the ATG codon is present (Figure 3B). Several processing motifs were detected in the deduced aa sequence (Figure 3B). No clear signal peptide cleavage site could be predicted according to SignalP. Using SignalP-HMM (Nielsen and Krogh, 1998), a potential 28 aa signal sequence was detected, giving a 343 aa (38.5 kDa) mature protein with an estimated pI of 5.5. One potential N-glycosylation site has been identified at aa position 140.

## The three genes constitute a lipase family in *C. deformans*

The three *C. deformans* genes encode lipases with similarities to each other and with the same overall structure, suggesting that these three lipase genes are members of a gene family (Figure 4A). In particular, a high degree of conservation was found in the middle of the lipase sequences, where a conserved Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala motif matches the consensus Gly-X-Ser-X-Gly, in which the serine of the active site is enclosed in most lipolytic enzymes. The sequences also contain eight conserved cysteine residues, which may form disulphide bridges contributing to a similar three-dimensional structure.

Searching through databases for proteins similar to *C. deformans* lipases revealed, in addition to YILIP2, several yeast and fungal lipases (Figure 4B). The highest homologies were also found with other lipases (YILIP4, YILIP5, YILIP7 and YILIP8) produced by *Y. lipolytica* (J.-M. Nicaud, unpublished data). The highest amino acid sequence identities were 91.6% (between CdLIP1 and YILIP2), 86.9% (between CdLIP2 and YILIP7) and 89.2% (between CdLIP3 and YILIP8). When the *C. deformans* lipase sequences were compared with the remaining yeast and fungal lipase sequences from databases (Figure 4B), similarities in conserved regions were found, even if overall similarities were minor. The greatest similarities were found with the lipases from *Candida ernobii* (Accession No. Q9HDQ8) and
A

CdLIP2 ----------MVNF GARIKDFF----------S----------VLLFGAASTS----------SSSKTALVSQ----------GFYDA----------38
CdLIP3 ----------MVTLSARLKDFF----------S----------VLLLGAAQIT----------PSTQTAGVSQ----------GFYDF----------38
YLLIP2 ----------MKLSTLILTACATLAALP----------SP----------ITPSAAVLQKRVYTSTETSHIDQ----------ESYNF----------50
CdLIP1 ----------MKLSTLLTACATLVAALP----------SP----------VTPSEAALQKRVYTSTETSHINQ----------DSYNF----------50
RmLIP MVLKQRANYLGFLIVFTAFLV----------EAVPIKRQS----------NSTVDS----------LPPLIPSRTSAPSSSPSTTDPBAP----------AMSRN----------GPLPS----------DVETK----------76
RdLIP MVSFISIQGVSLCLLVSSMLGSSAVPVSGKGSSTNANTAVSASDAALPPLIISRCCAPSNKGSKSDLQABPYNMQKNTEWYESHGGNLTISKGKRDDNLV----------100
T1LIP ---------------MRSSL--------------VLFFVSAWTA----------LASPIRREVSQ----------DLFNQ----------31

D1 D2 D2
CdLIP2 --------------------------ALDFSHLNSVAYCVHAPITPLKDFSCG-QSCV--HFDMELVTTFGGDQFSTSITGYLAL 96
CdLIP3 --------------------------ARDAFHLNSIAYCVDAPITPLNPDFTC-NSCK--YFPDIELVKTFGGDFFETSITGYLAV 96
YLLIP2 --------------------------FEKYARLANIGYCV-GPGTKIFKPFNCG-LQCA--HPFNVELIIEFHDPRLIFDVSGYLAV 107
CDLIP1 --------------------------FEKYARLANIGYCV-GPGTKIFKPFNCG-LQCA--HPFNVELIQEFHDPRLIFDVSGYLAV 107
RmLIP YGMALNATS--YPDSVQAMS--IDGG-IRAATSQEINELTYTTSANSYCR--TVIPGAT-WDC--IHCD--ATEDLKIIKTWST--LIYDTNAMVAR 162
RdLIP GGMTLDLPSDAPPISLSSTNSASDGGKVVAACTTAIQEFTKYGIAAGTAYCCR--SVVPGNK-WDC--VQCQK-WVPDGIITTFTS--LLSDTGNYVLV 192
T1LIP --------------------------FNLFAQYSAAAYGCKNNNDAPAGTNITCTGNACPEVEKADATFLYSFEDS-GVGDVTGFLAL 91

D3 D3
D1 D2
CdLIP2 DVHKKEKYVVRGTFSIPDAITDIRFQOSSFLVNVPALNTFAPNDPSGEAQIDCKECKIHDGFSRAFTETLTHNIGFVLQQHLDSTEYPEQLYVTGHSLGAA----------196
CdLIP3 DVHKKEKYVVRGTFSLGDAITDFQOQSPFLVDVPALNTFSANDTSAEQAIQCEGKIHDGFSKAFTETWGNIGEDLKKHLDSNPDYQLYVSGHSLGAA----------196
YLLIP2 DHASKQIYLVIRGTHSLEDVITDIRIMQAP-LTNF---DLAAN-ISSTA--TCDDCLVHNGFIQSYNNTYNQIGPKLDSVIEQYPDYQIAVTGHSLGGA----------199
CdLIP1 DHTSKQIYLVIRGTHSLEDVITDIRILQAP-LTNF---DFAAN-ISSTA--TCDDCLVHKGFIESYNNTFNQIGPKLDSVIAEHPDYEIAVTGHSLGGA----------199
RmLIP GDSEKTTYLVIRGSSIRNWIADLTFVPVS----------YPPVS----------GTVKHGFLDSYGEVQNELVATVLDQFKQYPSYKVAVTGHSLGGA----------242
RdLIP SDKQKTTYLVFRGTNSFRSAITDIVFNFSD----------YPKV----------GAKVHAGFLSSYEQVNVNDYFPVVQEQLTAHPTYKVIVTGHSLGGA----------272
T1LIP DNTNKLIVLSFRGSRSIENWIGNLNFDLKE----------INDI----------CSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGA----------172

*
CdLIP2 MALLAGTSI--KLQGYDP--IVINYGQPRVGRNAPADYISTLWFG--NGDGLEINRQRRMYRMTHWNDFVGLPNWD-GYTHSSGEVYIKGKWVPNALR----------288
CdLIP3 MSLLGATSF--KLKGYDP--ILINYGQPRVGNKPSEFINKLWPG--DNGGLEITPERRLYRMTHWNDFVGLPNWE-GYTHSNGEVYIKNRFINPPVS----------288
YLLIP2 AALLFGINL--KVNGHDP--LVVTLQOPIVGNAGFANWVDXLFPQENPDVSKVSKDKRKYRITHRGDIVPQVPFWD-GYQHCSEGFIDWPLIHPPLS----------293
CdLIP1 AALLFGINL--KVNGHDP--LVVTLQOPIVGNAGFANWVDRLPGEENPDVSKVTKDKRKYRITHRGDIVPQIPFWD-GYQHCSEGFIDWPLIHPPLS----------293
RmLIP TALLCALDLYQREBGLSSNLFLYTQGQPRVGDPAPANYVSTGTP----------YRTVNERDIVPHLPPAAFGFLHAGEEYWITDSP--E----------324
RdLIP QALLAGMDLYQREPRLSKNLSIFTVGGPRVGNPNTFAAYVESTGTP----------FQRTVHKRDIVPHVPPSQFGLPHPGVESWIKSGT----------S----------353
T1LIP LATVAGADL-RGNGYDI--DVFSYGAPRVGNRAFAPFLETLVQTGG----------TLYRITHNTDIVPRLLPPREFGYSHSSPEYWIKSGTLVP--V----------252

D4 D4
D1
CdLIP2 DVFSAG-GENPECYRSTFN-LLAINLQNHLCYIDY-IGFCALNVRREVNELQTDLPSYTPGYRYGNKTEDFVREGLELAQ----------
CdLIP3 DVISCAG-GENSCQCYRSSFN-ILSQINLQNHLAYIDY-IGYCALNIGRREL---ADQKYYKTYNGGNYAHRTEEDFKKLLGLELGPRAAQ
YLLIP2 NVVMCGQ-OSNKQC--SAGNTLLQOQNVIGNHLQYFVT-BGVCGI---------------------------------------------
CdLIP1 NVVTCEG-QRNKQC--SAGNTLLQOQNVIGNHLQYFVT-BGVCGI---------------------------------------------
RmLIP TVQVCTSDLETSDC--SNSI--VPFTSVL-DHLSYFGINTGLCT---------------------------------------------
RdLIP NVQICTSEIETKDC--SNSI--VPFTSIL-DHLSYFDINEGSCL---------------------------------------------
T1LIP TRNDIVK-IEGIDA--TGGN-NQPNIPDIPAHLWYFGL-IGTCL---------------------------------------------

B

<figure><img src="image_5.png" /><figcaption>**Figure 4.** (A) Multiple alignment obtained by the ClustalW program of the lipases from *C. deformans* and *Y. lipolytica* with three lipases from the fungal lipase family: *Rhizomuccor miehei* (RmLIP lipase, Accession No. P19515), *Rhizopus delemar* (RdLIP lipase, P21811) and *Thermomyces lanuginosus* (TILIP lipase, O59952). The three amino acids of the catalytic triad are marked by asterisks, and the eight cysteines that may form four disulphide bridges are labelled according to sequence conservation with the fungal lipase family (D1, D2, D3, D4). The conserved GHSLGGA motif containing the Ser residue of the active site is boxed. (B) Dendrogram (guide tree) obtained by the ClustalW program (gap opening, 10; gap extension, 0.1; scoring matrix, identity) showing the homologies of the lipases from *C. deformans* with those found in protein databases. Accession Nos are in brackets except for the putative lipases from *Y. lipolytica* (J.-M. Nicaud, unpublished data)</figcaption></figure>
S. cerevisiae (Accession No. P47145). According to the multiple alignment on Figure 4A, the lipases from C. deformans and Y. lipolytica belong to the family of fungal lipases (from Rhizopus delenmar, Rhizomuccor miehei and Thermomyces lanuginosus). The aspartic and histidine residues that belong to the catalytic triad of lipases, together with the serine in the active site, were found at perfectly conserved positions.

## Evaluation of the relatedness of C. deformans and Y. lipolytica

Amino acid comparison of CdLIP1 and YILIP2 (see above) suggested that CdLIP1 would be the homologue of YILIP2 in C. deformans. Nucleotide sequence comparison between the coding regions

of these two genes revealed 88.4% sequence identity, while sequence homology is 60% and 67.4% in the 5'- and 3'-untranslated regions respectively. In order to evaluate the relatedness of C. deformans to Y. lipolytica, we determined the nucleotide sequence of the variable region (D1/D2) at the 5'-end of the large-subunit ribosomal DNA (LSU rDNA) gene for C. deformans. After pairwise comparison of the DNA sequence obtained from C. deformans to that of Y. lipolytica (Accession No. U40080), 14 nucleotide differences were found, indicating a high degree of genetic divergence between the two species (Figure 5). Searching through databases for more homologous nucleotide sequences revealed the Y. lipolytica sequence only.

<table><caption>**Figure 5.** Nucleotide divergence between C. deformans and Y. lipolytica (Genbank Accession No. U40080) in the variable region at the 5'-end of the large-subunit (26S) ribosomal DNA gene</caption><tr><td>Y.1</td><td>1</td><td>AAACCAACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAAAAGCTCA</td><td>50</td></tr><tr><td>C.d</td><td>1</td><td>aaaccaacagggattgcctcagtaacggcgagtgaagcggcaaaagctca</td><td>50</td></tr><tr><td>Y.1</td><td>51</td><td>AATTTGAAACCCTCGGGATTGTAATTTGAAGATTTGGCATTGGAGAAAGC</td><td>100</td></tr><tr><td>C.d</td><td>51</td><td>aatttgaaaccctcgggattgtaatttgaagatttggcattggagaaagc</td><td>100</td></tr><tr><td>Y.1</td><td>101</td><td>TAACCCAAGTTGCTTGGAATAGTACGTCATAGAGGGTGACAACCCCGTCT</td><td>150</td></tr><tr><td>C.d</td><td>101</td><td>taacccaagttacttgaatagtacgtcatagaggtgacaaccccgct</td><td>150</td></tr><tr><td>Y.1</td><td>151</td><td>GGCTAACCGTTCTCCATGTATTGCCTTATCAAAGAGTCGAGTTGTTTGGG</td><td>200</td></tr><tr><td>C.d</td><td>151</td><td>ggctaaccgttctccatgtattgccttatcaaagagtcagttgtttggg</td><td>200</td></tr><tr><td>Y.1</td><td>201</td><td>AATGCAGCTCAAAGTGGGTGGTAAACTCCATCTAAAGCTAAATACTGGTG</td><td>250</td></tr><tr><td>C.d</td><td>201</td><td>aatgcagctcaaagtgggtggtaaactccatctaaagctaaatactggtg</td><td>250</td></tr><tr><td>Y.1</td><td>251</td><td>AGAGACCGATAGCGAACAGTACTGTGAAGGAAAGGTGAAAAGAACTTTG</td><td>300</td></tr><tr><td>C.d</td><td>251</td><td>agagaccgatagcgacaagtactgtgaaggaaaggtgaaaagaactttg</td><td>300</td></tr><tr><td>Y.1</td><td>301</td><td>AAAAGAGAGTGAAATAGTATGTGAAATTGTTGATAGGGAAGGAAATGAGT</td><td>350</td></tr><tr><td>C.d</td><td>301</td><td>aaaagagagtgaaatagtatgtgaaattggttatagggaaaggaaatgagt</td><td>350</td></tr><tr><td>Y.1</td><td>351</td><td>GGAGAGTGGCCGAGGTTTCAGCCGCCCCTCGTGGGCGGTGTACTGCCGAC</td><td>400</td></tr><tr><td>C.d</td><td>351</td><td>ggagagtggtttaggtttcagccttctttcgaggg-ggtgcactaccaac</td><td>399</td></tr><tr><td>Y.1</td><td>401</td><td>GCCGAGTCATCGATAGCGAGACGAGGGTTACAAATGGGAGCGCCTTCGGG</td><td>450</td></tr><tr><td>C.d</td><td>400</td><td>gccgagtcattgatagcgagac</td><td>421</td></tr></table>
Analysis of the secreted lipase of *C. deformans*

Cell growth and triolein hydrolysis activity
were monitored during growth on G medium
(Figure 6A). Lipase production was maximal when
cell growth reached the stationary state and then
decreased rapidly. Lipase activity released by
washing cells with buffer represented 75% of total
culture activity, suggesting that a large proportion
of the lipase was bound to the cell wall, in
contrast to the lipase from *Y. lipolytica*, which was
secreted into the extracellular medium (Pignede
et al., 2000a).

When the cell-bound lipase was concentrated and
analysed by SDS-PAGE, a single protein band of
apparent molecular mass of 38.0 kDa was detected
(Figure 6B, lane a). Deglycosylation with endo-
glycosidase H under denaturing conditions resulted
in a 4.2 kDa decrease in the apparent molecu-
lar mass, suggesting that the protein was glyco-
sylated (Figure 6B, lane b). The apparent molec-
ular mass of 33.8 kDa is close to the predicted
33.4 kDa calculated for the mature polypeptide of
CdLIP1 (301 aa). The prepared lipase was sub-
jected to mass spectrometric peptide mapping (data

not shown). This map was compared with the the-
oretical map that one should obtain after trypsin
digestion of the three lipases, CdLIP1, CdLIP2 and
CdLIP3. Seven peptides in the spectrum (Table 2)
had a mass that was expected for peptides obtained
from CdLIP1, resulting in a sequence coverage of
42.2% (127 aa for a total size of 301 aa for the
mature CdLIP1 lipase) and suggesting that the main
extracellular lipase produced by *C. deformans* is
encoded by *CdLIP1*.

Expression of *C. deformans* lipase genes in
*Y. lipolytica*

To investigate whether *CdLIP2* and *CdLIP3* genes encoded for functional extracellular lipases, we used a previously described *Y. lipolytica* expression system (Pignede *et al.*, 2000a; Nicaud *et al.*, 2002). This system consists of an integrative vector (JMP62) and a host strain (*Y. lipolytica* JMY277) in which the extracellular lipase gene *YILIP2* has been disrupted. The vector contains a *URA3* marker for selection in *Y. lipolytica* and the *POX2* promoter for driving gene expression upon induction by triglycerides.

<figure><img src="image_6.png" /><figcaption>**Figure 6.** (A) Growth and lipase production of *C. deformans* on G medium were monitored over time. Production of lipase was assayed on the culture supernatant and washing supernatant of the cells. (B) SDS–PAGE analysis of a concentrated lipase preparation. Untreated proteins (lane a) and proteins deglycosylated with Endo H (lane b) were separated by SDS–PAGE (12% wt/vol acrylamide) and stained with Coomassie brilliant blue R-250. The positions of Endo H and the untreated (Lip) and deglycosylated (Lip-D) lipases are indicated on the left. Lane M contains molecular weight markers</figcaption></figure>
<table><caption>**Table 2.** CdLIPI peptides identified by MALDI-TOF mass spectrometric peptide mapping</caption><thead><tr><th rowspan="2">Amino acid position</th><th rowspan="2">Peptide sequence</th><th colspan="2">Monoisotopic mass [M + H]<sup>+</sup></th></tr><tr><th>Calculated</th><th>Observed</th></tr></thead><tbody><tr><td>113–119</td><td>QIYLVIR</td><td>904.6</td><td>904.6</td></tr><tr><td>237–248</td><td>LFFGEENPDVSK</td><td>1381.7</td><td>1381.8</td></tr><tr><td>120–132</td><td>GTHSLEDVITDIR</td><td>1455.7</td><td>1455.9</td></tr><tr><td>97–112</td><td>LVFDVSGYLAVDHTSK</td><td>1750.9</td><td>1751.0</td></tr><tr><td>34–53</td><td>VYTSTETSHINQDSYNFFEK</td><td>2410.1</td><td>2410.3</td></tr><tr><td>210–236</td><td>VNGHDP LVVTLGQPIVGNA GFANWVDR</td><td>2845.5</td><td>2845.8</td></tr><tr><td>177–209</td><td>LDSVIAEHPDYEIAVTGHSLGGAAALLFGINLK</td><td>3391.8</td><td>3392.3</td></tr></tbody></table>

<figure><img src="image_7.png" /><figcaption>**Figure 7.** Lipase production by Ura⁺ transformants. Lipase detection was performed by spotting 5 µl of each culture (ca. 10⁵ cells) onto YNBH and YNBT plates. The plates were incubated for 2 days at 28°C. On the YNBH plate, lipase production is revealed by irradiating the plate with UV light, with colonies showing orange fluorescence, and by the appearance of a clearing zone on the YNBT plate</figcaption></figure>

The production of extracellular lipase by Ura⁺ transformants was tested on YNBT and YNBH plates (Figure 7). In YNBT plates, tributyrine was chosen as a substrate because it yields water-soluble products upon hydrolysis; thus, emulsions clear relatively quickly in the presence of lipase. Tributyrine, however, is not a lipase-specific substrate, and additional evidence that lipolytic enzymes were produced was required. YNBH

plates, containing triglycerides from olive oil that meet the strict definition of a lipase substrate, were used to distinguish between lipases and esterases. After 2 days of incubation, lipase production was detected on YNBH plates and was particularly evident on YNBT plates for strains expressing *CdLIP1* and *CdLIP3*. Although lipase expression was recorded on the triglyceride containing medium YNBH, tributyrine hydrolysis was not observed for the *CdLIP2*-containing strain growing on YNBT plates. The control strain, JMY277, transformed with the vector JMP62 alone, did not show any fluorescence on YNBH plates, whereas a very small clearing zone was observed on YNBT plates, indicating the absence of extracellular activity in this strain.

We further studied the production of extracellular lipase by the recombinant strains growing in liquid medium (data not shown). The supernatant of the culture medium exhibited lipase activities of 15 U/ml, 0.5 U/ml and 2 U/ml for the strains expressing *CdLIP1*, *CdLIP2* and *CdLIP3*, respectively. This demonstrates that *CdLIP2* and *CdLIP3*, along with *CdLIP1*, were expressed as functional lipases.

## Discussion

The production of extracellular lipase activity by *C. deformans* has previously been reported (Muderhwa *et al.*, 1985). We have demonstrated that this lipase could be useful for methyl ester production (Boutur *et al.*, 1994). However, no study describing the encoding genes has been published to date. To produce large amounts of this enzyme, we used the genetic engineering approach rather than the selection of hyper-producer mutants.
At the very beginning of the work, the cloning of two genes encoding lipases (*YILIP1* and *YILIP3*) was reported for the related species *Y. lipolytica* (Choupina *et al.*, 1999). These lipases are similar to lipases from *Candida rugosa* and *Geotrichum candidum* and belong to the carboxyesterase family. They do not show clear signal peptides and therefore may be intracellular or membrane-bound. Since no information was available concerning an extracellular lipase, we decided to use a complementation strategy employing several strains of *S. cerevisiae*. This strategy has previously been reported for the cloning of a lipase gene from *C. albicans* by screening *S. cerevisiae* transformants on egg-yolk medium (Fu *et al.*, 1997), a medium predominantly used to screen for the production of extracellular phospholipase.

Two *C. deformans* genomic libraries were constructed corresponding to the equivalent of 20 genomes (ca 440 × 10⁶ bp). These libraries were used to transform three different non-lipase-producing *S. cerevisiae* strains. Six clones that developed an orange fluorescent halo (upon UV illumination) on a medium containing rhodamine B and emulsified rapeseed oil were isolated among the 14 000 Ura⁺ transformants tested. Restriction mapping and sequencing of the genomic inserts revealed three different lipase-encoding genes, which were named *CdLIP1*, *CdLIP2* and *CdLIP3*. This indicates that about 1/2300 of the transformants contained *LIP* genes, suggesting that all *C. deformans* lipase encoding genes that are accessible by the method have been isolated. However, we could not exclude the possibility that genes may be present but not expressed in *S. cerevisiae*, or that the protein was not secreted or detected during the screening. Nevertheless, these results indicate that this screening, which was predominantly used for the detection of bacterial lipase activity (Kouker and Jaeger, 1987), can also be used to detect lipase production in yeast.

The discovery of a lipase gene family in *C. deformans* is not surprising, since it had already been reported for other yeasts; e.g. *C. rugosa* and *C. albicans* are known to contain five and ten distinct lipases genes, respectively (Lotti *et al.*, 1993; Fu *et al.*, 1997; Hube *et al.*, 2000). The three cloned lipase genes encode for proteins that are related to the filamentous fungi lipase family. These lipases share in common the Gly-His-Ser-Leu-

Gly-(Gly/Ala)-Ala motif that encloses the serine of the active site. They present a putative signal sequence indicating that they correspond to secreted lipases. This is confirmed by their secretion when expressed in *Y. lipolytica*. The highest homology (91.6%) is observed between CdLIP1 and the extracellular lipase YILIP2 from *Y. lipolytica* (Pignede *et al.*, 2000a). Furthermore, alignment of the two protein sequences revealed the same organization of their primary structures, indicating that CdLIP1 is synthesized as a pre-pro protein with a 33 aa prepro region ending at a Lys-Arg (KR) site, similarly to YILIP2 (Pignede *et al.*, 2000a). MALDI-TOF mass spectrometry analysis after trypsin digestion of the *C. deformans* extracellular lipase demonstrated that *CdLIP1* encodes the extracellular lipase. However, contrary to YILIP2, which is secreted into the culture supernatant, CdLIP1 was mainly bound to the cell wall but could easily be released by washing the cells with phosphate buffer. Currently, despite the high homology between the two proteins, we have no explanation for the protein being predominately localized in the cell wall in *C. deformans*, whereas it is predominately in the culture supernatant in *Y. lipolytica*. The mature protein (301 aa) has a calculated 33.4 kDa molecular mass. This estimation is consistent with the 33.8 kDa obtained from the SDS-PAGE analysis of the deglycosylated lipase. Deglycosylation results in a 4.2 kDa decrease in the apparent molecular mass, suggesting that at least one and possibly two of the two possible N-glycosylation sites are glycosylated.

In *The Yeasts: a Taxonomic Study*, 4th edn (Kurtzman and Fell, 1998), *C. deformans* (Zach) Langeron and Guerra CBS 2071 is considered to be a synonym of *Candida (Yarrowia) lipolytica*. However, a close inspection of the nucleotide sequences in the coding regions of *CdLIP1* and *YILIP2* reveals only 88.4% sequence identity, while sequence homology falls to 60% and 67.4% in the 5'- and 3'-untranslated regions respectively. The differences found could have occurred either by natural genetic divergence between the two strains or because another extracellular lipase encoding gene is present in *C. deformans* and remains to be discovered. The latter hypothesis is unlikely, since Southern blot hybridization using *YILIP2* as the probe failed to reveal another lipase-encoding gene with higher sequence identity (data not shown). The former hypothesis is
strengthened by the finding of 14 nucleotide differences in the LSU rDNA region D1/D2 between *C. deformans* and *Y. lipolytica* (Figure 5). Kurtzman and Robnett (1997) have observed that members of a same species show no more than two nucleotide substitutions. These sequences have sufficiently diverged to allow the reliable separation of the two yeast species.

Maximum lipase production in *C. deformans* reaches 1 unit/ml, with 80% being cell-bound. When *CdLIP1* was expressed in *Y. lipolytica*, we were able to obtain 15 U/ml in the supernatant. This corresponds to a 15-fold increase in lipase production when integrated in a single copy. Further improvement could be expected if multicopy integrants are isolated using the amplification method developed by Pignede *et al.* (2000b). We could therefore compare the enzymatic properties of CdLIP1 expressed in *Y. lipolytica* with the enzyme produced by *C. deformans* for ester production.

In conclusion, we have successfully isolated three genes from *C. deformans* encoding proteins belonging to the lipase family. The genes were functionally expressed in *Y. lipolytica* and lipase activities encoded by the genes could be detected in the extracellular medium. Recombinant production of the newly described lipases are in progress, with the aim of studying their enzymatic properties.

## Acknowledgements

We thank Dr Jean-Louis Roustan for lipase production and preparation, Dr Nicolas Sommerer (INRA, laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Montpellier) for performing the MALDI-TOF mass spectrometric peptide map of the lipase, and Catherine Chabalier for technical assistance. This work was supported in part by the Institut National de la Recherche Agronomique and by the Centre National de la Recherche Scientifique.

## References

Boutur O. 1995. Essais de production d'émulsifiants par bioconversion. Thèse de doctorat d'université (PhD). ENSA. M: Montpellier.

Boutur O, Dubreucq E, Galzy P. 1994. Methyl esters production in aqueous medium by an enzymatic extract from *Candida deformans* (Zach) Langeron and Guerra. *Biotechnol Lett* **16**: 1179-1182.

Boutur O, Dubreucq E, Galzy P. 1995. Factors influencing ester synthesis catalysed in aqueous media by the lipase from *Candida deformans* (Zach) Langeron and Guerra. *J Biotechnol* **42**: 23-33.

Chen W, Struhl K. 1988. Saturation mutagenesis of a yeast *HIS* 'TATA element': genetic evidence for a specific TATA binding protein. *Proc Natl Acad Sci USA* **85**: 2691.

Choupina A, Gonzalez FJ, Morin M, et al. 1999. The lipase system of *Yarrowia lipolytica*. *Curr Genet* **35**: 297.

Christianson TW, Sikorski RS, Dante M, Shern JH, Hieter P. 1992. Multifunctional yeast high-copy-number shuttle vectors. *Gene* **110**: 119-122.

Enderlin CS, Ogrydziak DM. 1994. Cloning, nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast *Yarrowia lipolytica*. *Yeast* **10**: 67-79.

Fu Y, Ibrahim AS, Fonzi W, et al. 1997. Cloning and characterization of a gene (*LIP1*) which encodes a lipase from the pathogenic yeast *Candida albicans*. *Microbiology* **143**: 331-340.

Galzy P. 1964. Etude génétique et physiologique du métabolisme de l'acide lactique chez *Saccharomyces cerevisiae*. *Ann Technol Agric* **13**: 109-259.

Higgins D, Sharp P. 1988. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. *Gene* **73**: 237-244.

Hill JE, Myers AM, Koerner TJ, Tzagoloff A. 1986. Yeast/*E. coli* shuttle vectors with multiple unique restriction sites. *Yeast* **2**: 163-167.

Hube B, Stehr F, Bossenz M, et al. 2000. Secreted lipases of *Candida albicans*: cloning, characterisation and expression analysis of a new gene family with at least ten members. *Arch Microbiol* **174**: 362-374.

Jaeger K-E, Ransac S, Dijkstra BW, et al. 1994. Bacterial lipases. *FEMS Microbiol Rev* **15**: 29-63.

Jaeger K-E, Reetz MT. 1998. Microbial lipases form versatile tools for biotechnology. *Trends Biotechnol* **16**: 396-403.

Jensen ON, Wilm M, Shevchenko A, Mann M. 1999. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. In *Proteome Analysis Protocols*, Link AJ (ed.). Humana: Totowa, NJ; 513-530.

Kouker G, Jaeger K-E. 1987. Specific and sensitive plate assay for bacterial lipases. *Appl Environ Microbiol* **59**: 211-213.

Kurtzman CP, Fell JW. 1998. *The Yeasts: a Taxonomic Study*, 4th edn. Elsevier Science: Amsterdam.

Kurtzman CP, Robnett CJ. 1997. Identification of clinically important ascomycetous yeast based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. *J Clin Microbiol* **35**: 1216-1223.

Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. *Nature* **227**: 680-685.

Le Dall MT, Nicaud JM, Gaillardin C. 1994. Multiple-copy integration in the yeast *Yarrowia lipolytica*. *Curr Genet* **26**: 38-44.

Lotti M, Grandori R, Fusetti F, et al. 1993. Cloning and analysis of *Candida cylindracea* lipase sequences. *Gene* **124**: 45-55.

Matoba S, Ogrydziak DM. 1989. A novel location for dipeptidyl aminopeptidase processing sites in the alkaline extracellular protease of *Yarrowia lipolytica*. *J Biol Chem* **264**: 6037-6043.

Muderhwa JM, Ratomahenina R, Pina M, Graille J, Galzy P. 1985. Purification and properties of the lipase from *Candida deformans* (Zach) Langeron and Guerra. *J Am Oil Chem Soc* **62**: 1031-1036.
Nicaud J-M, Madzak C, van den Broek P, et al. 2002. Protein expression and secretion in the yeast *Yarrowia lipolytica*. *FEMS Yeast Res* 2: 371-379.

Nielsen H, Engelbrecht J, Brunak S, von Heijne G. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. *Protein Eng.* **10**: 1-6.

Nielsen H, Krogh A. 1998. Prediction of signal peptides and signal anchors by hidden Markov model. Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), Menlo Park, CA. pp. 122-130.

Pandey A, Benjamin S, Soocol CR, et al. 1998. The realm of microbial lipases in biotechnology. *Biotechnol Appl Biochem* **29**: 119-131.

Pearson W, Lipman D. 1988. Improved tools for biological sequence comparison. *Proc Natl Acad Sci USA* **85**: 2444-2448.

Pignede G, Wang H, Fudalej F, et al. 2000a. Characterization of an extracellular lipase encoded by *LIP2* in *Yarrowia lipolytica*. *J Bacteriol* **182**: 2802-2810.

Pignede G, Wang HJ, Fudalej F, et al. 2000b. Autocloning and amplification of *LIP2* in *Yarrowia lipolytica*. *Appl Environ Microbiol* **66**: 3283-3289.

Sambrook J, Fritsch EF, Maniatis T. 1989. *Molecular Cloning: a Laboratory Manual*, 2nd edn. Cold Spring Harbor Laboratory Press: New York.

Saxena RK, Ghosh PK, Gupta R, et al. 1999. Microbial lipases: potential biocatalysts for the future industry. *Curr Sci* **77**: 101-115.

Zaret KS, Sherman F. 1982. DNA sequence required for efficient transcription termination in yeast. *Cell* **28**: 563-573.