TY - JOUR
AU - Nicaud, Jean-Marc
AB - Abstract The oleaginous yeast Yarrowia lipolytica efficiently metabolizes hydrophobic substrates such as alkanes, fatty acids or triacylglycerol. This yeast has been identified in oil-polluted water and in lipid-rich food. The enzymes involved in lipid breakdown, for use as a carbon source, are known, but the molecular mechanisms controlling the expression of the genes encoding these enzymes are still poorly understood. The study of mRNAs obtained from cells grown on oleic acid identified a new group of genes called SOA genes (specific for oleic acid). SOA1 and SOA2 are two small genes coding for proteins with no known homologs. Single- and double-disrupted strains were constructed. Wild-type and mutant strains were grown on dextrose, oleic acid and triacylglycerols. The double mutant presents a clear phenotype consisting of a growth defect on tributyrin and triolein, but not on dextrose or oleic acid media. Lipase activity was 50-fold lower in this mutant than in the wild-type strain. The impact of SOA deletion on the expression of the main extracellular lipase gene (LIP2) was monitored using a LIP2-β-galactosidase promoter fusion protein. These data suggest that Soa proteins are components of a molecular mechanism controlling lipase gene expression in response to extracellular triacylglycerol. hydrophobic substrates, lipids, oleaginous yeast, transcriptional regulation Introduction The oleaginous yeast Yarrowia lipolytica is able to use hydrophobic substrates as a carbon source. The first reports on this ability were published in 1948 (Peters & Nelson, 1948). Strains have been isolated from soil, sewage or petroleum-polluted water, in which paraffin is a carbon source. This organism is also present on cheeses, such as Camembert, in which fatty acids are the most abundant carbon source (Corsetti, 2001; Bonaiti, 2006; Mounier, 2009). The ability of Y. lipolytica to grow on alkanes, to hydrolyze extracellular triacylglycerols and to utilize free fatty acids (FFA) as carbon sources make this oleaginous yeast a good model for studying fatty acid metabolism. Lipases from this yeast have biotechnological applications, such as waste treatment and food industry (for review see Pandey, 1999). They are also useful in some therapeutic approaches, notably as substitutes for pancreatic lipases (Zentler-Munro, 1992). The position of Y. lipolytica as the eukaryotic model for hydrophobic substrate utilization is reinforced by the availability of its whole-genome sequence (Dujon, 2004), and therefore the identification of the genes involved in the fatty acid metabolism. Although fatty acids have a simple structure, they can be subdivided into a large number of families depending on the specificities of their hydrophobic chain. One of the criteria for this classification is the chain length, which determines both their physico-chemical and biological properties. Fatty acids are used as an energy source through the β-oxidation process, catalyzed in four steps by peroxisomal enzymes. In Y. lipolytica, the first step is catalyzed by acyl CoA oxidase (Aox) encoded by the POX1–POX6 genes (Wang, 1999; Fickers, 2005a). The second and third reactions are catalyzed by a multifunctional enzyme (MFE) encoded by the MFE gene (Hiltunen, 1992; Qin, 1999). The last step is catalyzed by the thiolase (THIO gene) (Pagot, 1998). Adaptation to the use of fatty acids with various chain lengths is reflected in Y. lipolytica by the expansion of the acyl CoA oxidase protein family. Two Aox proteins were shown to present specificity towards fatty acids of particular chain lengths: Aox2p encoded by POX2 showed specificity toward long-chain fatty acids (Luo, 2002) and Aox3p (POX3 gene) toward short-chain fatty acids (Wang, 1999; Luo, 2000). Oils mostly contain triacylglycerols, i.e. fatty acids esterified on glycerol. However, they also contain a few FFA. Triglycerides first need to be hydrolyzed into FFA and glycerol by lipolytic enzymes, such as lipases. Owing to its lipophilic lifestyle, Y. lipolytica seems to be well adapted for the efficient use of triglycerides, as suggested by the presence of 16 lipase-encoding genes. This expansion in the protein family appears to be species specific, as the hemiascomycetous yeasts sequenced so far generally only have one or two lipases. The lipase family GL3R0084 defined by the Génolevures consortium (formerly family GLS.94) contains 16 Y. lipolytica members encoded by the genes LIP2 (YALI0A20350g), LIP4, LIP5 and LIP7–LIP19. The esterase family GL3C3695 (formerly GLS.95) presents four Y. lipolytica members: LIP1 (YALI0E10659g), LIP3 (YALI0B08030g), LIP6 (YALI0C00231g) and LIP20 (YALI0E05995g) (Thevenieau, 2009). Some of the Y. lipolytica lipases have been characterized, including the extracellular lipases lip2p, lip7p and lip8p. These three lipases have different substrate specificities, ranging from medium- to long-chain fatty acids (Fickers, 2005b). The LIP2 gene codes for a pre-pro-protein containing a Lys–Arg (KR) cleavage site (Pignede, 2000). The mature protein represents the lipase that is secreted the most; it preferentially hydrolyzes long-chain fatty acid esters (C18). The LIP7 and LIP8 genes encode lipases that are partially secreted. Lip7p has substrate specificity toward C6 esters and Lip8p toward C10 esters (Fickers, 2005b). Some lipases present different expression patterns; e.g. LIP2 was shown to be induced by oleic acid, whereas LIP11 was the only lipase expressed on dextrose (J.-M. Nicaud et al., unpublished data). In addition, specific media or growth conditions eliciting strong induction of the lipase and Aox genes have been identified (Wang, 1999). The availability and the type of carbon and nitrogen sources play an important role in lipase expression (Fickers, 2004). However, the mechanisms controlling the production of lipases and acyl CoA oxidase are still poorly understood, and the molecular link between growth conditions and gene induction is still unknown in yeasts. Data obtained from multicellular organisms demonstrated the impact of the endocrinal context on lipase gene induction (for a review, see Jaworski, 2007) and identified the sterol response element binding protein (SREBP) as the main regulator of lipogenesis in mammalian cells. Mammalian SREBP is an endocellular lipid sensor protein detecting modification in membrane fluidity at the endoplasmic reticulum level (for a review, see Thewke, 2000). The molecular mechanism of mammalian SREBP activation is reminiscent of that of the yeast proteins Spt23p and Mga2p. However, Spt23p and Mga2p do not appear to be involved in the carbon source perception (Hoppe, 2000). To the best of our knowledge, there are no reports relating to molecular processes that control lipase and Aox gene expression in response to extracellular triacylglycerols. To understand the control of hydrophobic substrate utilization in Y. lipolytica, for use as a carbon source, three cDNA libraries were constructed: one from exponentially growing cells on oleic acid and two from exponential or stationary-phase cells growing on dextrose (M. Mekouar et al., unpublished data). From this analysis, new genes exclusively expressed on oleic acid medium (specific oleic acid genes) were identified and were named SOA for genes specific of oleic acid. These genes escaped previous annotation due to their short length and the absence of similarity to other previously identified genes. The aim of this study was to characterize the function of two of these new genes: SOA1 and SOA2. Material and methods Strains and culture conditions The yeast and bacterial strains used in this study are reported in Table 1. Bacterial strains, Mach1 T1® (Invitrogen, Cergy Pontoise, France) and DH5α (Gibco BRL, Rockville, MD) used for the amplification of recombinant plasmids were grown at 37 °C in Luria–Bertani medium supplemented with 100 μg L−1 ampicillin or 40 μg L−1 kanamycin if required. 1 Strains and plasmids used in this study Strain  Genotype/plasmid  Reference/source  E. coli  Mach1T1  ΔrecA1398 endA1 tonAΦ80ΔlacM15ΔlacX74 hsdR(rK- mK+)  Invitrogen  DH5α  F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupGΦ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(rK−mK+),λ−  Promega  JME459  Mach1T1/pBluescript II KS+ (ColE1 ori, LacZ, bla)  Stratagene  JME507  DH5α/JMP113 (1.2-kb ylURA3 fragment in pBluescript II KS+)  Fickers (2003a)  JME508  DH5α/JMP114 (1.8-kb ylLEU2 fragment in pBluescript II KS+)  Fickers (2003a)  JME951  Mach1T1/JMP951 (pBluescript II KS+–P-URA3-T SOA1)  This work  JME952  Mach1T1/JMP952 (pBluescript II KS+–P-URA3-T SOA2)  This work  JME979  Mach1T1/JMP979 (pBluescript II KS+–P-LEU2-T SOA1)  This work  JME455  DH5α/JMP104 (proLIP2-LacZ/LEU2,URA3)  Fickers (2003b)  JME454  DH5α/JMP103 (proPOX2-LacZ/LEU2,URA3)  Fickers (2003b)  JME461  DH5α/pRRQ2 (Cre ARS68 LEU2 in pBluescript II KS+)  Richard (2001)  Y. lipolytica  W29  MATA, wild type  Barth & Gaillardin (1996)  PO1d  MATA, ura3-302, leu2-270, xpr2-322  Barth & Gaillardin (1996)  JMY1510  PO1d, soa1∷URA3  This work  JMY1512  PO1d, soa2∷URA3  This work  JMY1525  JMY1510, LEU2  This work  JMY1527  JMY1512, LEU2  This work  JMY1531  PO1d, soa1∷URA3, soa2∷LEU2  This work  JMY1539  PO1d, soa1Δ, soa2∷LEU2  This work  JMY1760  PO1d, JMP104  Fickers (2003b)  JMY1761  PO1d, JMP103  Fickers (2003b)  JMY1762  PO1d, soa1∷URA3, JMP104,  This work  JMY1763  PO1d, soa1∷URA3, JMP103  This work  JMY1764  PO1d, soa2∷URA3, JMP104  This work  JMY1765  PO1d, soa2∷URA3, JMP103  This work  JMY1766  PO1d, soa1Δ, soa2∷LEU2, JMP104  This work  JMY1767  PO1d, soa1Δ, soa2∷LEU2, JMP103  This work  Strain  Genotype/plasmid  Reference/source  E. coli  Mach1T1  ΔrecA1398 endA1 tonAΦ80ΔlacM15ΔlacX74 hsdR(rK- mK+)  Invitrogen  DH5α  F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupGΦ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(rK−mK+),λ−  Promega  JME459  Mach1T1/pBluescript II KS+ (ColE1 ori, LacZ, bla)  Stratagene  JME507  DH5α/JMP113 (1.2-kb ylURA3 fragment in pBluescript II KS+)  Fickers (2003a)  JME508  DH5α/JMP114 (1.8-kb ylLEU2 fragment in pBluescript II KS+)  Fickers (2003a)  JME951  Mach1T1/JMP951 (pBluescript II KS+–P-URA3-T SOA1)  This work  JME952  Mach1T1/JMP952 (pBluescript II KS+–P-URA3-T SOA2)  This work  JME979  Mach1T1/JMP979 (pBluescript II KS+–P-LEU2-T SOA1)  This work  JME455  DH5α/JMP104 (proLIP2-LacZ/LEU2,URA3)  Fickers (2003b)  JME454  DH5α/JMP103 (proPOX2-LacZ/LEU2,URA3)  Fickers (2003b)  JME461  DH5α/pRRQ2 (Cre ARS68 LEU2 in pBluescript II KS+)  Richard (2001)  Y. lipolytica  W29  MATA, wild type  Barth & Gaillardin (1996)  PO1d  MATA, ura3-302, leu2-270, xpr2-322  Barth & Gaillardin (1996)  JMY1510  PO1d, soa1∷URA3  This work  JMY1512  PO1d, soa2∷URA3  This work  JMY1525  JMY1510, LEU2  This work  JMY1527  JMY1512, LEU2  This work  JMY1531  PO1d, soa1∷URA3, soa2∷LEU2  This work  JMY1539  PO1d, soa1Δ, soa2∷LEU2  This work  JMY1760  PO1d, JMP104  Fickers (2003b)  JMY1761  PO1d, JMP103  Fickers (2003b)  JMY1762  PO1d, soa1∷URA3, JMP104,  This work  JMY1763  PO1d, soa1∷URA3, JMP103  This work  JMY1764  PO1d, soa2∷URA3, JMP104  This work  JMY1765  PO1d, soa2∷URA3, JMP103  This work  JMY1766  PO1d, soa1Δ, soa2∷LEU2, JMP104  This work  JMY1767  PO1d, soa1Δ, soa2∷LEU2, JMP103  This work  View Large 1 Strains and plasmids used in this study Strain  Genotype/plasmid  Reference/source  E. coli  Mach1T1  ΔrecA1398 endA1 tonAΦ80ΔlacM15ΔlacX74 hsdR(rK- mK+)  Invitrogen  DH5α  F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupGΦ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(rK−mK+),λ−  Promega  JME459  Mach1T1/pBluescript II KS+ (ColE1 ori, LacZ, bla)  Stratagene  JME507  DH5α/JMP113 (1.2-kb ylURA3 fragment in pBluescript II KS+)  Fickers (2003a)  JME508  DH5α/JMP114 (1.8-kb ylLEU2 fragment in pBluescript II KS+)  Fickers (2003a)  JME951  Mach1T1/JMP951 (pBluescript II KS+–P-URA3-T SOA1)  This work  JME952  Mach1T1/JMP952 (pBluescript II KS+–P-URA3-T SOA2)  This work  JME979  Mach1T1/JMP979 (pBluescript II KS+–P-LEU2-T SOA1)  This work  JME455  DH5α/JMP104 (proLIP2-LacZ/LEU2,URA3)  Fickers (2003b)  JME454  DH5α/JMP103 (proPOX2-LacZ/LEU2,URA3)  Fickers (2003b)  JME461  DH5α/pRRQ2 (Cre ARS68 LEU2 in pBluescript II KS+)  Richard (2001)  Y. lipolytica  W29  MATA, wild type  Barth & Gaillardin (1996)  PO1d  MATA, ura3-302, leu2-270, xpr2-322  Barth & Gaillardin (1996)  JMY1510  PO1d, soa1∷URA3  This work  JMY1512  PO1d, soa2∷URA3  This work  JMY1525  JMY1510, LEU2  This work  JMY1527  JMY1512, LEU2  This work  JMY1531  PO1d, soa1∷URA3, soa2∷LEU2  This work  JMY1539  PO1d, soa1Δ, soa2∷LEU2  This work  JMY1760  PO1d, JMP104  Fickers (2003b)  JMY1761  PO1d, JMP103  Fickers (2003b)  JMY1762  PO1d, soa1∷URA3, JMP104,  This work  JMY1763  PO1d, soa1∷URA3, JMP103  This work  JMY1764  PO1d, soa2∷URA3, JMP104  This work  JMY1765  PO1d, soa2∷URA3, JMP103  This work  JMY1766  PO1d, soa1Δ, soa2∷LEU2, JMP104  This work  JMY1767  PO1d, soa1Δ, soa2∷LEU2, JMP103  This work  Strain  Genotype/plasmid  Reference/source  E. coli  Mach1T1  ΔrecA1398 endA1 tonAΦ80ΔlacM15ΔlacX74 hsdR(rK- mK+)  Invitrogen  DH5α  F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupGΦ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(rK−mK+),λ−  Promega  JME459  Mach1T1/pBluescript II KS+ (ColE1 ori, LacZ, bla)  Stratagene  JME507  DH5α/JMP113 (1.2-kb ylURA3 fragment in pBluescript II KS+)  Fickers (2003a)  JME508  DH5α/JMP114 (1.8-kb ylLEU2 fragment in pBluescript II KS+)  Fickers (2003a)  JME951  Mach1T1/JMP951 (pBluescript II KS+–P-URA3-T SOA1)  This work  JME952  Mach1T1/JMP952 (pBluescript II KS+–P-URA3-T SOA2)  This work  JME979  Mach1T1/JMP979 (pBluescript II KS+–P-LEU2-T SOA1)  This work  JME455  DH5α/JMP104 (proLIP2-LacZ/LEU2,URA3)  Fickers (2003b)  JME454  DH5α/JMP103 (proPOX2-LacZ/LEU2,URA3)  Fickers (2003b)  JME461  DH5α/pRRQ2 (Cre ARS68 LEU2 in pBluescript II KS+)  Richard (2001)  Y. lipolytica  W29  MATA, wild type  Barth & Gaillardin (1996)  PO1d  MATA, ura3-302, leu2-270, xpr2-322  Barth & Gaillardin (1996)  JMY1510  PO1d, soa1∷URA3  This work  JMY1512  PO1d, soa2∷URA3  This work  JMY1525  JMY1510, LEU2  This work  JMY1527  JMY1512, LEU2  This work  JMY1531  PO1d, soa1∷URA3, soa2∷LEU2  This work  JMY1539  PO1d, soa1Δ, soa2∷LEU2  This work  JMY1760  PO1d, JMP104  Fickers (2003b)  JMY1761  PO1d, JMP103  Fickers (2003b)  JMY1762  PO1d, soa1∷URA3, JMP104,  This work  JMY1763  PO1d, soa1∷URA3, JMP103  This work  JMY1764  PO1d, soa2∷URA3, JMP104  This work  JMY1765  PO1d, soa2∷URA3, JMP103  This work  JMY1766  PO1d, soa1Δ, soa2∷LEU2, JMP104  This work  JMY1767  PO1d, soa1Δ, soa2∷LEU2, JMP103  This work  View Large The growth media and conditions used for Y. lipolytica have been described previously by Barth & Gaillardin (1996). The yeasts were grown on YPD and YNB complemented with 0.2% (w/v) casa-amino acids or 0.1% (w/v) uracil, if required. Carbon sources were dextrose (D), oleic acid (OA), tributyrin (TB) or triolein (TO) at 5% (w/v). The hydrophobic substrates were emulsified by sonication in a mixture containing 20% (w/v) of OA, TB or TO and 0.625% (v/v) of Tween 40. Standard molecular biology techniques Standard molecular biology techniques were used throughout this study (Sambrook, 1989). Restriction enzymes were obtained from Ozyme (Saint-Quentin-en-Yvelines, France). Genomic DNA from yeast was prepared according to a previous study (Querol, 1992). PCR amplifications were performed on an Eppendorf 2720 thermal cycler with Pyrobest DNA polymerase (Lonza, Levallois-Perret, France). PCR fragments were purified using the QIAquick PCR Purification Kit (Qiagen, Courtaboeuf, France), and DNA fragments were recovered from agarose gels using the QIAquick Gel Extraction Kit (Qiagen). Genetic modifications SOA1 and SOA2 genes were deleted by replacing the coding region by a cassette containing URA3 or LEU2 as the selectable marker, as described previously (Fickers, 2003a). The promoter (P) and the terminator (T) of SOA1 and SOA2 were obtained by PCR amplification using Y. lipolytica W29 genomic DNA as the template and the primers listed in Table 2. The resulting amplicons were ligated, and inserted into pBluescript II KS+ (Stratagene) previously restriction digested with EcoRV. The loxP-URA3-loxR and loxP-LEU2-loxR cassettes containing the selectable markers (Fickers, 2003a) were inserted into PT-containing plasmids. The resulting constructs, designated JMP951, JMP952 and JMP979, carried P-URA3-T from SOA1, P-URA3-T from SOA2 and P-LEU2-T from SOA2, respectively (Table 1). Then, deletion cassettes were generated by PCR. The SOA1 and SOA2 deletions were performed in strain PO1d, auxotrophic for uracil and leucine, using the lithium acetate method (Le Dall, 1994). Yeast transformants were selected on appropriate minimal media. Deletion of SOA1 and SOA2 was verified by PCR using primers Ver1 and Ver2 (Table 2). 2 Primers used in this study for PCR amplification Primer name  Sequences  P1-SOA1  5′-CCCTTGAATATGGTCCATCAGACTCTCTTCAGG-3′  P2-SOA1  5′-CGATTACCCTGTTATCCCTACCGCTTAGTGGTGGATATGTAGCAATGCGGTGGTG-3′  T1-SOA1  5′-GGTAGGGATAACAGGGTAATCGGACTAGACGCAGGACGGGTGAGGTGCCGGACGG-3′  T2-SOA1  5′-CCGATCAGAGTCGAAACTTGCCCTAGAGCTC-3′  P1-SOA2  5′-CTAAAGATGTCTCAACGTAGAGGGTGATACGG-3′  P2-SOA2  5′-CGATTACCCTGTTATCCCTACCAGTTGTAGTTGTAATGTGTGGTGTAGTGTGGGGTGAG-3′  T1-SOA2  5′-GGTAGGGATAACAGGGTAATCGGCGGTGCCTAATCTGTGATTATCCGGTCC-3′  T2-SOA2  5′-GCCGAACTAGGTCATAACCCCTGAGGTTCGGAG-3′  Ver1SOA1  5′-GAACCGTCAGGTGTGGAAGAACGACCAAGG-3′  Ver2SOA1  5′-GTCCCTAAACATGGTACACACGACGACTGCGC-3′  Ver1SOA2  5′-CAGAAGAGATATGCACACAGTCGTATTGTACTCG-3′  Ver2SOA2  5′-GGCCGTCTCTAGACAGAGTTGGCTGCTCAAGG-3′  Primer name  Sequences  P1-SOA1  5′-CCCTTGAATATGGTCCATCAGACTCTCTTCAGG-3′  P2-SOA1  5′-CGATTACCCTGTTATCCCTACCGCTTAGTGGTGGATATGTAGCAATGCGGTGGTG-3′  T1-SOA1  5′-GGTAGGGATAACAGGGTAATCGGACTAGACGCAGGACGGGTGAGGTGCCGGACGG-3′  T2-SOA1  5′-CCGATCAGAGTCGAAACTTGCCCTAGAGCTC-3′  P1-SOA2  5′-CTAAAGATGTCTCAACGTAGAGGGTGATACGG-3′  P2-SOA2  5′-CGATTACCCTGTTATCCCTACCAGTTGTAGTTGTAATGTGTGGTGTAGTGTGGGGTGAG-3′  T1-SOA2  5′-GGTAGGGATAACAGGGTAATCGGCGGTGCCTAATCTGTGATTATCCGGTCC-3′  T2-SOA2  5′-GCCGAACTAGGTCATAACCCCTGAGGTTCGGAG-3′  Ver1SOA1  5′-GAACCGTCAGGTGTGGAAGAACGACCAAGG-3′  Ver2SOA1  5′-GTCCCTAAACATGGTACACACGACGACTGCGC-3′  Ver1SOA2  5′-CAGAAGAGATATGCACACAGTCGTATTGTACTCG-3′  Ver2SOA2  5′-GGCCGTCTCTAGACAGAGTTGGCTGCTCAAGG-3′  View Large 2 Primers used in this study for PCR amplification Primer name  Sequences  P1-SOA1  5′-CCCTTGAATATGGTCCATCAGACTCTCTTCAGG-3′  P2-SOA1  5′-CGATTACCCTGTTATCCCTACCGCTTAGTGGTGGATATGTAGCAATGCGGTGGTG-3′  T1-SOA1  5′-GGTAGGGATAACAGGGTAATCGGACTAGACGCAGGACGGGTGAGGTGCCGGACGG-3′  T2-SOA1  5′-CCGATCAGAGTCGAAACTTGCCCTAGAGCTC-3′  P1-SOA2  5′-CTAAAGATGTCTCAACGTAGAGGGTGATACGG-3′  P2-SOA2  5′-CGATTACCCTGTTATCCCTACCAGTTGTAGTTGTAATGTGTGGTGTAGTGTGGGGTGAG-3′  T1-SOA2  5′-GGTAGGGATAACAGGGTAATCGGCGGTGCCTAATCTGTGATTATCCGGTCC-3′  T2-SOA2  5′-GCCGAACTAGGTCATAACCCCTGAGGTTCGGAG-3′  Ver1SOA1  5′-GAACCGTCAGGTGTGGAAGAACGACCAAGG-3′  Ver2SOA1  5′-GTCCCTAAACATGGTACACACGACGACTGCGC-3′  Ver1SOA2  5′-CAGAAGAGATATGCACACAGTCGTATTGTACTCG-3′  Ver2SOA2  5′-GGCCGTCTCTAGACAGAGTTGGCTGCTCAAGG-3′  Primer name  Sequences  P1-SOA1  5′-CCCTTGAATATGGTCCATCAGACTCTCTTCAGG-3′  P2-SOA1  5′-CGATTACCCTGTTATCCCTACCGCTTAGTGGTGGATATGTAGCAATGCGGTGGTG-3′  T1-SOA1  5′-GGTAGGGATAACAGGGTAATCGGACTAGACGCAGGACGGGTGAGGTGCCGGACGG-3′  T2-SOA1  5′-CCGATCAGAGTCGAAACTTGCCCTAGAGCTC-3′  P1-SOA2  5′-CTAAAGATGTCTCAACGTAGAGGGTGATACGG-3′  P2-SOA2  5′-CGATTACCCTGTTATCCCTACCAGTTGTAGTTGTAATGTGTGGTGTAGTGTGGGGTGAG-3′  T1-SOA2  5′-GGTAGGGATAACAGGGTAATCGGCGGTGCCTAATCTGTGATTATCCGGTCC-3′  T2-SOA2  5′-GCCGAACTAGGTCATAACCCCTGAGGTTCGGAG-3′  Ver1SOA1  5′-GAACCGTCAGGTGTGGAAGAACGACCAAGG-3′  Ver2SOA1  5′-GTCCCTAAACATGGTACACACGACGACTGCGC-3′  Ver1SOA2  5′-CAGAAGAGATATGCACACAGTCGTATTGTACTCG-3′  Ver2SOA2  5′-GGCCGTCTCTAGACAGAGTTGGCTGCTCAAGG-3′  View Large URA3 marker rescue was performed using the pRRQ2 plasmid in the single disrupted strain: JMY1510 (Richard, 2001). Briefly, this vector contains the CRE recombinase gene under the control of the hp4d promoter and the LEU2 ORF. The pRRQ2 transformants were selected on YNB containing uracil. LIP2 and POX2 expression was monitored using a fusion between their promoter and the β-galactosidase reporter gene. PO1d and the various SOA mutants were transformed with plasmids JMP103 and JMP104 constructed by Fickers (2003b). This reporter plasmid is randomly integrated into nuclear chromosomes. Enzymatic assay Extracellular lipase activity in liquid medium was determined by spectrophotometry, by monitoring p-nitro phenol formation resulting from the hydrolysis of p-nitro phenyl-butyrate (pNPB) as described previously (Fickers, 2003b). This method was optimized using 2-methyl butan-2-ol (2M2B) as a solvent for pNPB solubilization (Bordes, 2007). Lipase activity was measured in 96-well plates using 20 μL of yeast culture supernatant and 280 μL of reaction buffer (60 mM phosphate buffer pH=7.2, 100 mM NaCl and 5 μL of pNBP in 40 mM 2M2B). Activity was determined at 30 °C by monitoring the A405 nm for at least 5 min in a plate reader and agitator (Sinergy II, BioTek®). One lipase activity unit was defined as the amount of enzyme releasing 1 μmol of p-nitro phenol per min at 30 °C. β-Galactosidase assays were performed after chloroform permeabilization, as described previously (Gaillardin & Ribet, 1987). This activity was expressed in Miller units. In both enzymatic essays, cultures were inoculated at a OD600 nm of 0.25. Results SOA genes, new ORF identified in oleic acid cDNA library To monitor gene expression during the growth of Y. lipolytica on hydrophobic substrates, a cDNA library was constructed using mRNA extracted from cells grown on oleic acid media in the exponential phase. For comparison, two other cDNA libraries were constructed using mRNA extracted from cells grown on dextrose in the exponential and stationary phases. For each library, 10 000 isolates were sequenced (M. Mekouar et al., unpublished data). There is a direct correlation between the number of cDNA molecules per gene and its expression level. Values for genes of the β-oxidation, the lipase family GL3R0084 and the SOA genes are given in Table 3. As expected on oleic acid, the main genes involved in the β-oxidation pathway were highly represented. We found 21 cDNAs for POX2 (YALI0F10857g) and 27 cDNAs for POX3 (YALI0D24750g) in the oleic acid cDNA library, whereas only one and zero molecules of cDNA were found for POX2 and POX3 on dextrose in the exponential phase and 1 and 6 cDNA molecules in the stationary phase, respectively. For the gene encoding the MFE (YALI0E15378g), cDNAs were identified 33 times on oleic acid, but, on dextrose, cDNAs were identified only five times for cells in the exponential phase and 20 times for those in the stationary phase. For the thiolase-encoding gene (POT1, YALI0E18568g), cDNAs were identified 105 times on OA, zero times in exponential-phase cells grown on dextrose and 11 times in stationary-phase cells grown on dextrose. This shows that although the β-oxidation pathway is strongly expressed on oleic acid, it is also expressed in cells grown on dextrose at the stationary phase (1–6, 20 and 11 for POX2–POX3, MFE and POT1, respectively). 3 Number of clones in the cDNA libraries for genes of the β-oxidation, the lipase family GL3R0084 and the SOA genes Gene name  No. of clones in cDNA libraries  Systematic  Common  AO  GLUexp  GLUstat  β-oxidation  YALI0F10857g  POX2  21  1  1  YALI0D24750g  POX3  27  0  6  YALI0E15378g  MFE  33  5  20  YALI0E18568g  POT1  105  0  11  Lipases  YALI0A20350g  LIP2  15  0  0  YALI0E08492g  LIP4  0  0  0  YALI0E02640g  LIP5  0  0  0  YALI0D19184g  LIP7  0  0  0  YALI0B09361g  LIP8  0  0  0  YALI0E34507g  LIP9  0  0  0  YALI0F11429g  LIP10  0  0  0  YALI0D09064g  LIP11  0  0  0  YALI0D15906g  LIP12  0  0  0  YALI0E00286g  LIP13  0  0  1  YALI0B11858g  LIP14  0  0  0  YALI0E11561g  LIP15  0  0  0  YALI0D18480g  LIP16  0  0  0  YALI0F32131g  LIP17  0  0  0  YALI0B20350g  LIP18  0  0  0  YALI0A10439g  LIP19  0  0  0  SOA genes  YALI0C19096g  SOA1  13  0  0  YALI0D04884g  SOA2  20  0  0  Gene name  No. of clones in cDNA libraries  Systematic  Common  AO  GLUexp  GLUstat  β-oxidation  YALI0F10857g  POX2  21  1  1  YALI0D24750g  POX3  27  0  6  YALI0E15378g  MFE  33  5  20  YALI0E18568g  POT1  105  0  11  Lipases  YALI0A20350g  LIP2  15  0  0  YALI0E08492g  LIP4  0  0  0  YALI0E02640g  LIP5  0  0  0  YALI0D19184g  LIP7  0  0  0  YALI0B09361g  LIP8  0  0  0  YALI0E34507g  LIP9  0  0  0  YALI0F11429g  LIP10  0  0  0  YALI0D09064g  LIP11  0  0  0  YALI0D15906g  LIP12  0  0  0  YALI0E00286g  LIP13  0  0  1  YALI0B11858g  LIP14  0  0  0  YALI0E11561g  LIP15  0  0  0  YALI0D18480g  LIP16  0  0  0  YALI0F32131g  LIP17  0  0  0  YALI0B20350g  LIP18  0  0  0  YALI0A10439g  LIP19  0  0  0  SOA genes  YALI0C19096g  SOA1  13  0  0  YALI0D04884g  SOA2  20  0  0  LIP genes are numbered according to Pignede (2000); Fickers (2005b) and Thevenieau (2009). GLUexp, glucose in exponential phase; GLUstat, glucose in stationary phase. View Large 3 Number of clones in the cDNA libraries for genes of the β-oxidation, the lipase family GL3R0084 and the SOA genes Gene name  No. of clones in cDNA libraries  Systematic  Common  AO  GLUexp  GLUstat  β-oxidation  YALI0F10857g  POX2  21  1  1  YALI0D24750g  POX3  27  0  6  YALI0E15378g  MFE  33  5  20  YALI0E18568g  POT1  105  0  11  Lipases  YALI0A20350g  LIP2  15  0  0  YALI0E08492g  LIP4  0  0  0  YALI0E02640g  LIP5  0  0  0  YALI0D19184g  LIP7  0  0  0  YALI0B09361g  LIP8  0  0  0  YALI0E34507g  LIP9  0  0  0  YALI0F11429g  LIP10  0  0  0  YALI0D09064g  LIP11  0  0  0  YALI0D15906g  LIP12  0  0  0  YALI0E00286g  LIP13  0  0  1  YALI0B11858g  LIP14  0  0  0  YALI0E11561g  LIP15  0  0  0  YALI0D18480g  LIP16  0  0  0  YALI0F32131g  LIP17  0  0  0  YALI0B20350g  LIP18  0  0  0  YALI0A10439g  LIP19  0  0  0  SOA genes  YALI0C19096g  SOA1  13  0  0  YALI0D04884g  SOA2  20  0  0  Gene name  No. of clones in cDNA libraries  Systematic  Common  AO  GLUexp  GLUstat  β-oxidation  YALI0F10857g  POX2  21  1  1  YALI0D24750g  POX3  27  0  6  YALI0E15378g  MFE  33  5  20  YALI0E18568g  POT1  105  0  11  Lipases  YALI0A20350g  LIP2  15  0  0  YALI0E08492g  LIP4  0  0  0  YALI0E02640g  LIP5  0  0  0  YALI0D19184g  LIP7  0  0  0  YALI0B09361g  LIP8  0  0  0  YALI0E34507g  LIP9  0  0  0  YALI0F11429g  LIP10  0  0  0  YALI0D09064g  LIP11  0  0  0  YALI0D15906g  LIP12  0  0  0  YALI0E00286g  LIP13  0  0  1  YALI0B11858g  LIP14  0  0  0  YALI0E11561g  LIP15  0  0  0  YALI0D18480g  LIP16  0  0  0  YALI0F32131g  LIP17  0  0  0  YALI0B20350g  LIP18  0  0  0  YALI0A10439g  LIP19  0  0  0  SOA genes  YALI0C19096g  SOA1  13  0  0  YALI0D04884g  SOA2  20  0  0  LIP genes are numbered according to Pignede (2000); Fickers (2005b) and Thevenieau (2009). GLUexp, glucose in exponential phase; GLUstat, glucose in stationary phase. View Large Transcripts corresponding to new genes encoding proteins smaller than 100 amino acids were found. Three genes were expressed in the oleic acid library alone and were called SOA genes for specific oleic acid genes. These genes were not identified during Y. lipolytica genome annotation, due to their small size and the absence of known homologs (Dujon, 2004). Here, we focused on SOA1 and SOA2 genes. SOA1 (YALI0C19096g) transcripts were found 13 times and SOA2 (YALI0D04884g) transcripts were found 21 times in this library. For SOA1, nine of the 13 cDNAs were full length. Putative transcription start sites were found between 165 and 125 nt upstream from the methionine initiation codon (Supporting Information, Fig. S1). For the SOA2 gene, 18 of the 21 cDNAs were full length. The transcription start sites were located 99 and 94 nt upstream from the first ATG (Fig. S2). SOA1 (http://www.genolevures.org/elt/YALI/YALI0C19096g) codes for a 96 amino acid protein (Fig. S1) rich in charged amino acids; it contains 27 positively charged [20 lysine (K) and seven arginine (R)] and 17 negatively charged amino acids [14 glutamate (E) and three aspartate (D)]. SOA2 (http://www.genolevures.org/elt/YALI/YALI0D04884g) codes for a 72 amino acid protein (Fig. S2). Soa2p is also rich in charged amino acids, with 15 positively charged [eight lysine (K), two arginine (R) and five histidine (H)] and 14 negatively charged amino acids [11 glutamate (E) and three aspartate (D)]. To determine Soa1p and Soa2p structure, putative conserved two-dimensional (2D) structures were searched using the predict protein webserver (http://www.predictprotein.org/) (Rost, 2004). The profile network prediction (Rost & Sander, 1993) indicated that Soa1p is organized as an α-helix. The representation of Soa1p in this conformation suggested the presence of two domains (Fig. 1). Hydrophobic amino acid residues are generally predicted to lie mostly on one side of the α-helix, whereas hydrophilic amino acid residues constitute the other side. No conserved 2D structure was predicted for Soa2p. 1 View largeDownload slide Graphical representation of Soa1p as an α-helix. The red or green boxes indicate, respectively, the acidic and basic residues of amino acid at physiological pH. The blue boxes indicate a hydrophobic amino acid. The left part of the helix mostly contains hydrophobic amino acids. By contrast, the right part contains more charged amino acids. 1 View largeDownload slide Graphical representation of Soa1p as an α-helix. The red or green boxes indicate, respectively, the acidic and basic residues of amino acid at physiological pH. The blue boxes indicate a hydrophobic amino acid. The left part of the helix mostly contains hydrophobic amino acids. By contrast, the right part contains more charged amino acids. SOA gene deletion reduces growth on triacylglycerols medium To elucidate the function of Soa1p and Soa2p, single disrupted mutants were constructed in the PO1d strain: soa1∷URA3 (JMY1510) and soa2∷URA3 (JMY1512). Assuming that strains presenting an auxotrophy, especially for leucine, present growth defects on hydrophobic substrate (Mauersberger, 2001), the mono disruptants were transformed with a genomic fragment carrying the LEU2 gene. Prototroph mono disruptants were selected (JMY1525 and JMY1527, respectively) and showed no apparent growth defect on dextrose-containing medium (Figs 2a and 3a). 2 View largeDownload slide Analysis of growth and lipase production based on the carbon source of wild-type and SOA-deleted strains cultured on plates. Wild-type strain W29, the single-deleted strains JMY1525 (soa1Δ) and JMY1527 (soa2Δ), and the double-deleted strain JMY1531 (soa1Δsoa2Δ) were pregrown overnight in YPD, centrifuged, washed and resuspended at 1 OD600 nm mL−1. Cell suspension drops (10 μL) of different dilutions (nondiluted, 10−1, 10−2, 10−3 and 10−4) were deposited onto minimal medium containing various carbon sources: (a) dextrose, (b) oleic acid, (c) tributyrin and (d) triolein. The plates were incubated 48 h at 28°C. The size of the clear zone around the colonies reflects lipase production. Only dilutions 10−3 and 10−4 are shown. 2 View largeDownload slide Analysis of growth and lipase production based on the carbon source of wild-type and SOA-deleted strains cultured on plates. Wild-type strain W29, the single-deleted strains JMY1525 (soa1Δ) and JMY1527 (soa2Δ), and the double-deleted strain JMY1531 (soa1Δsoa2Δ) were pregrown overnight in YPD, centrifuged, washed and resuspended at 1 OD600 nm mL−1. Cell suspension drops (10 μL) of different dilutions (nondiluted, 10−1, 10−2, 10−3 and 10−4) were deposited onto minimal medium containing various carbon sources: (a) dextrose, (b) oleic acid, (c) tributyrin and (d) triolein. The plates were incubated 48 h at 28°C. The size of the clear zone around the colonies reflects lipase production. Only dilutions 10−3 and 10−4 are shown. 3 View largeDownload slide Time course of cell growth of Yarrowia lipolytica wild-type strain and SOA-deleted mutants based on the carbon source. Wild-type strain W29 (●), the single-deleted strains JMY1525 (soa1Δ, ▪) and JMY1527 (soa2Δ, ▲), and the double-deleted strain JMY1531 (soa1Δsoa2Δ, ×) were grown in minimal medium containing, as carbon source, dextrose (a, YNBD), oleic acid (b, YNBO), tributyrin (c, YNBTB) and triolein (d, YNBTO). Cultures in 50 mL of YNB in 250-mL baffled flasks were inoculated with washed cells from overnight preculture at an initial OD600 nm of 0.25. YNB supplemented with 3% fatty acid or oil as carbon sources. The results are mean values of three independent experiments. SD were <10% of the average value. 3 View largeDownload slide Time course of cell growth of Yarrowia lipolytica wild-type strain and SOA-deleted mutants based on the carbon source. Wild-type strain W29 (●), the single-deleted strains JMY1525 (soa1Δ, ▪) and JMY1527 (soa2Δ, ▲), and the double-deleted strain JMY1531 (soa1Δsoa2Δ, ×) were grown in minimal medium containing, as carbon source, dextrose (a, YNBD), oleic acid (b, YNBO), tributyrin (c, YNBTB) and triolein (d, YNBTO). Cultures in 50 mL of YNB in 250-mL baffled flasks were inoculated with washed cells from overnight preculture at an initial OD600 nm of 0.25. YNB supplemented with 3% fatty acid or oil as carbon sources. The results are mean values of three independent experiments. SD were <10% of the average value. Even so, SOA1 and SOA2 present no sequence homology; they present similar features, which may indicate redundant properties. Therefore, a double mutant deleted for both SOA1 and SOA2 was constructed, soa1∷URA3 soa2∷LEU2 (JMY1539). SOA genes are overexpressed if cells use hydrophobic substrates as carbon sources. To determine the existence of a phenotype associated with the deletion of the SOA genes, mono and double disruptants were drop-tested on plates containing two hydrophobic substrates: fatty acid (oleic acid) and triacylglycerols (tributyrin and thiolein). No apparent growth defect could be observed for the mono-disrupted strains on the media tested (Fig. 2). By contrast, on tributyrin medium, we observed a slight decrease in colony size of the double mutant. In addition, the hydrolysis halo around the colony of the double mutant was smaller than that around a wild-type colony (Fig. 2c). This suggested a reduction in extracellular lipase activity. A similar phenotype was previously reported for the double mutant lip2Δlip8Δ (Fickers, 2005b). Although no growth defect was associated with the LIP2 and LIP8 double deletion in cultures grown on plates, the growth rate was significantly reduced for the lip2Δlip8Δ mutant during liquid culture in triacylglycerols-containing medium (Fickers, 2005b). This led us to investigate the ability of the soa1Δsoa2Δ double mutant to grow in liquid medium containing triacylglycerols as the carbon source. Growth curves (see Fig. 3) showed no difference between the four strains on dextrose or oleic acid media. However, the double mutant exhibited a clear growth defect in media containing triacylglycerols. This is independent from the fatty acid chain length of the triacylglycerols, as the growth defect was equally marked on tributyrin (C4:0) (Fig. 3c) and triolein (C18:1) (Fig. 3d). These results confirmed that soa1Δsoa2Δ double mutants are impaired in the use of these substrates. However, the absence of growth defects on oleic acid medium suggested that strains deleted for SOA can use hydrophobic substrates if the fatty acids are nonesterified. SOA gene deletion leads to a decrease in lipase activity To determine how well the various mutants hydrolyze triacylglycerols into glycerol and FFA, cells were grown on dextrose medium to stationary phase and were shifted to minimal medium containing tributyrin and triolein; oleic acid medium was also used, as a control. After 24 or 48 h under these conditions, the supernatants were collected and the lipase activity was monitored. Extracellular lipase was similarly induced in wild type and mutants grown on oleic acid medium, with about 50 U observed at 24 h and 100 U at 48 h, similar to that observed in a previous report (Fickers, 2003b; Bordes, 2007). By contrast, clear differences were observed on tributyrin and triolein media. We observed a large decrease in lipase activity in the single mutants after 24 h (Fig. 4). Thus, lipase activity in soa1Δ and soa2Δ mutants was, respectively, 48% and 45% that of wild type. The double mutant had very low lipase activity (5% of the wild type). 4 View largeDownload slide Lipase production during growth of wild-type strain and SOA mutants. (a) Lipase production on minimal media with triolein (YNBTO). (b) Lipase production on minimal media containing tributyrin (YNBTB). Lipase activity was measured at 24 h (gray) and 48 h (black) in the culture medium. Wild-type strain (W29), the single-deleted strains soa1Δ (JMY1525) and soa2Δ (JMY1527), and the double-deleted strain soa1Δsoa2Δ (JMY1531) were pregrown overnight in YPD, centrifuged, washed and used to inoculate medium at an initial OD600 nm of 0.25. The results are mean values of three independent experiments. The error bar indicates mean SD. 4 View largeDownload slide Lipase production during growth of wild-type strain and SOA mutants. (a) Lipase production on minimal media with triolein (YNBTO). (b) Lipase production on minimal media containing tributyrin (YNBTB). Lipase activity was measured at 24 h (gray) and 48 h (black) in the culture medium. Wild-type strain (W29), the single-deleted strains soa1Δ (JMY1525) and soa2Δ (JMY1527), and the double-deleted strain soa1Δsoa2Δ (JMY1531) were pregrown overnight in YPD, centrifuged, washed and used to inoculate medium at an initial OD600 nm of 0.25. The results are mean values of three independent experiments. The error bar indicates mean SD. The difference was even greater when lipase was monitored after 48 h of growth in triacylglycerols media. A fivefold reduction in extracellular lipase activity was observed for the single mutant and a 100-fold reduction for the double mutant (Fig. 4b). Similar changes were observed in triolein and tributyrin media, suggesting similar effects under both conditions. These results suggest a defect in lipase expression or secretion. SOA1 and/or SOA2 deletion reduces expression of LIP2 but not of POX2 LIP2 promoter induction was analyzed using the β-galactosidase reporter gene in wild-type and mutant strains. In parallel, we followed the impact of SOA gene deletion on β-oxidation by monitoring the expression of the β-galactosidase gene under the POX2 promoter. We compared the expression of these two genes because both are induced on hydrophobic substrates and have specificity for long-chain fatty acids (preferentially C18). The promoter activities were determined on dextrose, oleic acid, tributyrin and triolein. In agreement with previous studies on the wild-type strain, we observed a limited activity of the LIP2 promoter in cells growing on dextrose during the exponential growth phase (24 h, Fig. 5a), and this activity was weakly induced (twofold) in stationary phase (48 h, Fig. 5b). Similar observations were made with strains expressing the reporter gene under the control of the POX2 promoter (Fig. 5c and d). 5 View largeDownload slide LIP2 or POX2 gene expression during growth of wild-type strain and SOA-deleted mutants in minimal media based on various carbon sources: dextrose, FFA and triacylglycerols media. LIP2 and POX2 promoter activity was measured by monitoring β-galactosidase activity driven by LIP2- or POX2-LacZ promoter fusions. Activities were measured at 24 h [(a) for LIP2 and (c) for POX2] and 48 h [(b) for LIP2 and (d) for POX2] after inoculation. Wild-type strain (W29, open bar), the single-deleted strains soa1Δ (JMY1525, hatched bar) and soa2Δ (JMY1527, gray bar), and the double-deleted strain soa1Δsoa2Δ (JMY1531, black bar) were pregrown overnight in YPD, centrifuged, washed and used to inoculate medium at an initial OD600 nm of 0.25. The results are means value of three independent experiments. The error bar indicates mean SD. 5 View largeDownload slide LIP2 or POX2 gene expression during growth of wild-type strain and SOA-deleted mutants in minimal media based on various carbon sources: dextrose, FFA and triacylglycerols media. LIP2 and POX2 promoter activity was measured by monitoring β-galactosidase activity driven by LIP2- or POX2-LacZ promoter fusions. Activities were measured at 24 h [(a) for LIP2 and (c) for POX2] and 48 h [(b) for LIP2 and (d) for POX2] after inoculation. Wild-type strain (W29, open bar), the single-deleted strains soa1Δ (JMY1525, hatched bar) and soa2Δ (JMY1527, gray bar), and the double-deleted strain soa1Δsoa2Δ (JMY1531, black bar) were pregrown overnight in YPD, centrifuged, washed and used to inoculate medium at an initial OD600 nm of 0.25. The results are means value of three independent experiments. The error bar indicates mean SD. LIP2 promoter induction in wild-type cells was at least threefold greater on oleic acid medium and twofold greater on tributyrin and triolein media than on dextrose (Fig. 5a). After 48 h on OA medium, the expression of the reporter gene decreased and reached the level observed with dextrose-cultivated cells, whereas the activity of the LIP2 promoter remained stable in triacylglycerols-cultivated cells (Fig. 5b). Promoter activity of POX2 and LIP2 in soa1Δ and soa2Δ strains was similar to that of the wild type on dextrose and on oleic acid. However, on both types of triacylglycerols media, induction was weaker in mutant strains (between two- and threefolds less) than in the wild-type strain at 24 and 48 h (Fig. 5a and b). LIP2 promoter activity was twofold lower in soa2Δ cells cultured on triolein for 24 h than in similarly cultured soa1Δ, suggesting that the regulation of LIP2 expression may be differently regulated by Soa1p and Soa2p. We observed no difference in POX2 expression after 24 or 48 h on triacylglycerols media (Fig. 5c and d). In the case of the double mutant (soa1Δsoa2Δ), the LIP2 promoter activity was only affected on tributyrin and triolein. After 24 h, the LIP2 promoter activity was reduced to 22% and 19% that of the wild type, respectively (Fig. 5a). This ratio continued to decrease after 48 h (11% on tributyrin and 10% on triolein), as a consequence of the sustained induction of the LIP2 promoter in the wild type (Fig. 5b). Concerning POX2 expression, no modification was observed in response to the double deletion (Fig. 5c and d). These results suggested that SOA genes were not involved in POX2 expression. Discussion SOA1 and SOA2 are two new genes experimentally detected in a cDNA library from cells grown on oleic acid as the carbon source. They were not identified during the systematic annotation of Y. lipolytica due to their short length (<300 pb) and the absence of homologs in other organisms. Considering their lipid medium specificity, we aimed to characterize their role in lipid metabolism. Soa1p and Soa2p primary structure is characterized by an alternation of hydrophilic and hydrophobic residues. The predicted 2D structure suggested that Soa1p is an α-helix. The representation of Soa1p as an α-helix exposes hydrophobic amino acid residues on one side of the helix, and hydrophilic residues on the other side. Such helical structure has been observed for the amino acid region AA199–AA223 of the 415 amino acid protein Arf-Gap1p of Saccharomyces cerevisiae. This region has a dual distribution, with one side of the helical-wheel presenting hydroxylated amino acids (serine and threonine) and the other, hydrophobic amino acids (Bigay, 2005). Bigay and colleagues suggest a possible interaction between the hydrophobic residues and the phospholipid bilayer. The lipid-packing sensor proteins of the Arf-GAP family present other structural features, such as a 120 amino acid Zn-finger domain and a specific lipid-binding domain. By contrast, the SOA1 gene product only contains an amphipathic helix structure presenting the dual distribution of hydrophobic and charged amino acids. Thus, Soa1p may be associated with the phospholipid bilayer but is most likely unable to interact with DNA, due to the absence of DNA-binding domain. To characterize the functions of Soa1p and Soa2p, we constructed single- and double-deleted strains for SOA1 and SOA2. The single-deleted strains presented no growth defect on hydrophobic substrates (oleic acid or triacylglycerols). By contrast, the double mutant (soa1Δsoa2Δ) presented a reduced ability to grow on liquid media containing triacylglycerols, but not on oleic acid. Also, the absence of a triacylglycerols hydrolysis halo on plates pointed to a defect in lipase secretion. Thus, the double mutant appeared to be less able to hydrolyze the ester bond between glycerol and fatty acids, indicating a defect in either lipase secretion or lipase expression. To confirm the extracellular lipase activity defect, we monitored lipase activity in various mutants, and compared these values with those of the wild-type strain grown on triacylglycerols media. We previously showed extracellular lipase activity in the medium of cells grown on triacylglycerols (Pignede, 2000). This activity is mainly due to the secretion of Lip2p, the major extracellular lipase produced by wild-type Y. lipolytica (Pignede, 2000). We reported lower extracellular lipase activity in both SOA single-deleted strains than in the wild type. The double mutant exhibited very low lipase activity associated with an absence of LIP2 gene induction. All these findings implied that Soa1p and Soa2p had complementary functions. However, for cells grown on triolein-containing medium, we observed a greater decrease in LIP2 promoter activity in soa2Δ strain than in the soa1Δ strain. Thus, Soa1p and Soa2p may not have completely overlapping functions (Fig. 6). Soa2p may be more specific to long-chain triacylglycerols. 6 View largeDownload slide Model for signal transduction by Soa proteins. The entry of FFA into the cytoplasm induced SOA gene expression, whereas the presence of extracellular triacylglycerols was detected by a putative sensor supposed to be located within the yeast membrane (represented by a blue polygon with a question mark). If triacylglycerols constituted the carbon source, Soa proteins associated with putative DNA-binding proteins (represented by a red box with question mark). At this stage, there are two possible explanations: either there is one particular protein for each Soa protein or there is a unique protein that interacts with both Soa proteins. The Soa protein and DNA-binding protein complex induces LIP2 expression, in turn secreting extracellular lipase. The transcription factor(s) that activate SOA expression, the sensor protein and the physical partner of Soa protein remain to be identified. 6 View largeDownload slide Model for signal transduction by Soa proteins. The entry of FFA into the cytoplasm induced SOA gene expression, whereas the presence of extracellular triacylglycerols was detected by a putative sensor supposed to be located within the yeast membrane (represented by a blue polygon with a question mark). If triacylglycerols constituted the carbon source, Soa proteins associated with putative DNA-binding proteins (represented by a red box with question mark). At this stage, there are two possible explanations: either there is one particular protein for each Soa protein or there is a unique protein that interacts with both Soa proteins. The Soa protein and DNA-binding protein complex induces LIP2 expression, in turn secreting extracellular lipase. The transcription factor(s) that activate SOA expression, the sensor protein and the physical partner of Soa protein remain to be identified. In addition, the presence of nonesterified oleic acid transiently induced LIP2 expression after 24 h of culture. The SOA gene deletion did not affect LIP2 induction on oleic acid. Thus, Soa1p and Soa2p likely regulate LIP2 gene expression exclusively in response to extracellular triacylglycerols. We observed no modification in POX2 gene expression in response to SOA gene deletion. This indicated that at least two separate systems control the triacylglycerols hydrolysis pathway and the FFA oxidation pathway. Soa1p and Soa2p represent two members of a new regulatory pathway controlling lipase gene expression. This pathway was activated in response to extracellular triacylglycerols and not oleic acid. This pathway controls at least one gene involved in triacylglycerols hydrolysis. It would be interesting to know whether other genes such as LIP7 and LIP8, which are also secreted lipases (Fickers, 2005b), are regulated via the same SOA pathway. This pathway does not regulate the expression of POX2, which is one of the genes involved in β-oxidation of FFA. Similarly, there should be further investigation into the expression of all β-oxidation genes in SOA mutants. Lipases have specific roles in triacylglycerols saponification, which represents a first step in triacylglycerols utilization by the cells. Acyl CoA oxidases, such as POX2, hydrolyze all types of FFA obtained from various hydrophobic substrates including triacylglycerols. This represents the final and common step in hydrophobic substrate utilization. Thus, it is understandable that LIP2 is regulated by a specific pathway. In conclusion, we showed that Soa proteins are involved in a new pathway controlling LIP2 transcription. A model for this pathway is represented in Fig. 6. However, the analysis of Soa protein sequence revealed no similarity with other transcription factor or DNA-binding domains. Therefore, Soa proteins may be intermediates interacting directly or indirectly with transcription factors. Further studies, such two-hybrid experiments, are obviously needed to investigate the physical interactions involving Soa proteins. Another strategy could be genetic screening to identify epistatic mutations restoring the double mutant phenotype. Future studies should also center on the identification of all genes regulated by Soa proteins by high throughput transcriptomic approaches. Acknowledgements We thank Meryem Mekouar for her help in cDNA analysis, Julie Sappa of Alex Edelman & Associates for her help in correcting the English version of the manuscript. 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TI - SOA genes encode proteins controlling lipase expression in response to triacylglycerol utilization in the yeast Yarrowia lipolytica
JO - FEMS Yeast Research
DO - 10.1111/j.1567-1364.2009.00590.x
DA - 2009-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/soa-genes-encode-proteins-controlling-lipase-expression-in-response-to-4zGX72AO0o
SP - 93
EP - 103
VL - 10
IS - 1
DP - DeepDyve
ER -