Extracellular secretion of free fatty acids by disruption of a fatty acyl-CoA synthetase gene in Saccharomyces cerevisiae

JOURNALOF BIOSCIENCEAND BIOENGINEERWG

Vol. 95, No. 5, 435440. 2003

Extracellular Secretion of Free Fatty Acids by Disruption of a Fatty Acyl-CoA Synthetase Gene in Saccharomyces cerevisiae YASUNARI MICHINAKA,’ TOSHITSUGU SHIMAUCHI,2 TSUNEHIRO AKI,‘* TOSHIAKI NAKAJIMA,2 SEIJI KAWAMOTO,’ SEIKO SHIGETA,’ OSAMU SUZUKI,@ AND KAZUHISA ONO’ Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima Universify, l-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan’ and Idemitsu Technofine Co., l-6-1 Yokoami, Sumida-ku, Tokyo 108-0014, Japan’ Received 25 October 2002iAccepted 27 December 2002

To elucidate the molecular mechanism governing fatty acid transport across the cell membrane, we first isolated a Saccharomyces cerevisiae mutant, B-l, that exhibits a reduced acyl-CoA oxidase activity and an increase in free fatty acid accumulation. Following mutagenesis of B-l, a mutant, YTSSl, which secretes free fatty acids, was isolated. The concentration of free fatty acids in the YTSSl culture medium was about 17 times higher than that in B-l. The mutation that causes the fatty acid secretion phenotype occurred at a single allele, and this phenotype was suppressed by the introduction of a single copy of FAAl, a gene for acyl-CoA synthetase, to the mutant. Although the mutation expressing this phenotype was not within FAA1 in YTS51, the disruption of FAA1 in the wild-type strain resulted in fatty acid secretion even though the level of fatty acid secretion was less than that in YTS51. We consider that YTS51 is a suitable model to elucidate the molecular basis of the fatty acid transport process. [Key words: acyl-CoA oxidase, acyl-CoA synthetase,

free fatty acids, Saccharomyces cerevisiae, secretion]

Long-chain fatty acids are major components of membrane phospholipids and substances that store and produce energy necessary to perform various cellular events. Unsaturated fatty acids in particular are responsible for the regulation of the fluidity, permeability, and stability of biological membranes (1). Certain types of polyunsaturated fatty acids also serve as precursors of eicosanoids and have positive effects on diseases such as arteriosclerosis, depression, and some cancers (2). Reflecting the accumulated evidence that shows the notable bioactivity of unsaturated fatty acids, they have attracted commercial attention for use as food materials and supplements. In recent years, industrial systems for microbial production have been developed to produce polyunsaturated fatty acids, recovery of which from higher plants and animals is otherwise unprofitable. Examples include y-linolenic acid production by the genera Mortierella and Mucor (3), arachidonic acid and eicosapentaenoic acid by the genus Martierella (4,5), and docosahexaenoic acid by thraustochytrids (6, 7). In general, economically efficient fermentation re-

quires the isolation of an excellent producer, the use of cheap raw materials, and the optimization of culture conditions. These requirements have been met in the above examples. However, because the lipids of interest are accumulated into cells or mycelia in each case, a process to extract the lipids is indispensable for their purification. The use of microorganisms that secrete lipids into the culture medium would therefore be one of the most effective ways to simplify the downstream processing while reducing the production cost. A few reports on the secretion of lipids in microbial production have been described: Miyakawa et al. (8) isolated a mutant strain of Candida lipolytica which excretes longchain fatty acids in non-esterified (free) form. The production of triacylglycerols in the culture medium was achieved by mutants of the yeasts Trichosporon sp. (9, 10) and Saccharomyces cerevisiae (11). Only in the latter report was the molecular mechanism underlying the lipid secretion investigated via the isolation of wild-type genomic DNA fragments that complemented the mutation. However, no gene(s) the mutation of which induces lipid secretion has yet been identified because the mutation is thought to lie separately on various chromosomal loci (11). In yeast, the catabolism of fatty acids through P-oxidation occurs exclusively in peroxisomes and fatty acyl-CoA oxidase is the rate-limiting enzyme in this system (12). Thus, dysfunction of this enzyme leading to the inactivation of the P-oxidation process will result in the accumulation of cellu-

* Corresponding author. e-mail: [email protected] nhone: +81-(0)824-24-7755 fax: +81-(0X324-24-7754 BPresent ad&&s: Biological Resources ‘Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki 305-8566, Japan. Abbreviations: ATCC, American Type Culture Collection; bp, basepairs; EMS, ethylmethane sulfonate. The first two authors equally contributed to this work. 435

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MICHINAKA ETAL

lar fatty acids. therefore they Based on this mutant strain characterized mechanism of

An excess of fatty acids is toxic to cells, and will be evacuated by certain mechanisms. concept, we isolated a fatty acid-secreting of S. cerevisiae in this study and genetically it to provide information on the molecular the lipid secretion process.

MATERIALS

AND METHODS

Strains, media, and growth conditions S. cerevisiae strain ura3-52 can1 ga12) was obtained as a Cl-6b (MATcl prbl-Al.6R segregant from a cross of CG378 (MATa ura3-52 leu2-3, 112 trpl289 ade5 can 1 gaZ2; American Type Culture Collection [ATCC] no. 204664) and BJ3501 (MTclpep4::HIS3prbl-Al.6R his38200 ura3-52 can1 ga12; no. 208280). Strain KDl15 (MTcr olel; [ 131) was obtained from the ATCC (no. 204991). These strains were cultivated in a rich medium consisting of 2% Polypepton, 1% Bacto yeast extract, and 2% glucose (YPD) or in a YNB minimal medium (0.7% yeast nitrogen base) supplemented with a specified carbon source and auxotrophic requirements. For fatty acid secretion experiments, a YPD medium supplemented with 1% Brij-58 and 5% glycerol (YPGD) was used. To prepare a seed culture of KDl15, oleic acid (0.2%) and Brij-58 (1%) were added to the YPD medium. These cultivations were undertaken at 28°C in a rotary shaker at 170 rpm in 200-ml Erlenmeyer flasks containing 50 ml of medium. Strain C l -6b Isolation of a j&oxidation-deficient mutant grown in YPGD medium for 12 h was harvested by centrifugation and washed in 0.2 M phosphate buffer, pH 8.0. The cells were resuspended in 0.2 M phosphate buffer, pH 8.0, containing 3% ethylmethane sulfonate (EMS) and 2% glucose, and then incubated at 30°C for 30 min. A half the volume of 6% sodium thiosulfate solution was added to the suspension and incubated for 10 min to neutralize it. To fix the mutations, the cells were cultured overnight in YNB medium supplemented with 0.5% glucose and 20 &ml uracil. After washing the cells in sterile distilled water, they were cultivated overnight in YNB medium without any additives. Oleic acid and Brij-58 were subsequently added to the culture to a final concentration of 0.2% and l%, respectively. After a 6-h incubation at 28”C, the cells were treated with nystatin (50 pg/ml) for 1 h to enrich for mutants. The cells were plated on YPD agar and replicated onto YPD plates containing either 0.2% oleic acid or 2% glucose. An isolate that grew in the presence of glucose but not of oleic acid was selected and designated as B-l. The acyl-CoA oxidase activity of B-l was determined by measuring the palmitoylCoA-dependent reduction of oxygen as previously described (14). Briefly, cells were grown in a medium containing 2% Polypepton, 1% Bacto yeast extract, 0.5% oleic acid, and 1% Brij-58 for 1 d, harvested, and homogenized with glass beads. The reaction mixture consisted of 50 mM potassium phosphate, pH 7.4, 0.85 mM 4_aminoantipyrine, 22 mM phenol, 4 units of horseradish peroxidase, the cell-free extract, and 0.1 mM palmitoyl-CoA that was added finally to start the reaction. The absorbance at 5 10 nm was measured to determine the formation of H,Oz. Mutagenesis of Isolation of fatty-acid secreting mutant strain B-l was performed by treatment with EMS as described above. The mutagenized cells were suspended in 2% glucose at a concentration of 2 x 1ORcells/ml. A 0.2-ml aliquot was placed on top of a discontinuous Percoll density gradient consisting of 1OO%, 90%, 80%, and 70% (v/v) Percoll (Amersham Bioscience, Uppsala, Sweden) diluted with 0.25 M sucrose. The cells were separated by centrifugation at 5OOOxg for 5 h. Cells located in the boundary between 90% and 100% Percoll were collected, as their density may have increased due to secretion of intracellular lipids, and cultivated on YPD plates for 3 d. The plates were exposed to

UV light ( 15 W) for 15 min, covered by 10 ml of melted Y PD agar mixed with 0.5% Brij-58 and 1O”cells of KDI 15 (olel), and cultivated for 2 d. An isolate that gave a ‘halo’ of KDl15 colonies was selected as a fatty acid secreting mutant, and designated YTS5 I_ Tetrad analysis was performed as described previously (I 5). Screening of genes that complement the halo-forming mutation Genomic DNA purified with zymolyase from S. cerevisiae Cl-6b was partially digested with Sau3A1, then fractionated by centrifugation in a sucrose density gradient as previously described (15). The fractions containing -10 kbp fragments were pooled, and the fragments were purified and inserted into the BarnHI site of plasmid YEp24 (ATCC no. 3705 1). The resulting genomic library was introduced into YTSSI by the lithium acetate method ( 15). The transformants were tested for fatty acid secreting ability by the olel overlay assay as described above. The colonies that did not give a halo were isolated, and their plasmids were recovered. Partial fragments generated by digestion of the isolated gene with restriction enzymes and/or ExolII were recloned into plasmid YEp352 (ATCC; [ 161) to identify the minimum region essential to the complementation by the olef overlay assay. Integration of lJRA3 at FAA1 chromosomal locus To obtain a fragment containing the FAA1 gene, the following two primers located in the 600-bp flanking region of the FAA1 gene; 5’CCGCTAGCAAACAGACCTAGCTAGCGGAC-3’ and 5’-CCG CTAGCTGGAGAAGACAGGCACGCAGA-3’, generating Nhel sites (underlined), were synthesized. The PCR fragment amplified with the above primers was digested with Nhel and then ligated into the same site of the yeast integration vector Ylp5 containing the URA3 gene (ATCC no. 37061; [ 171). The resulting plasmid was linearized by digestion with Xhol, located within the IX4/ gene, and was used to transform strain B-l a (n/lATa; genetic background is identical to that of B-l, but its mating-type was switched by transient expression of the HO gene [IS]). A transformant designated B-l aEAA1 was used for tetrad analysis. Disruption of FAA1 To obtain a fragment of the li‘l.4/ (YOR317w) gene, the genomic DNA was PCR-amplified using oligonucleotide primers FAA 1F, 5’-CCCAGATCTTTTGTGGGC AATACCGACCGT-3’ (forward), and FAAlR, S’-CCCAGATCT CCTTGGTCTTTACCTGTCTTC-3’ (reverse), introducing Bglll sites (underlined). The PCR product was cloned on a pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced using a DYEnamic ET terminator sequencing kit (Amersham Bioscience) on a genetic analyzer (AB13 10; Applied Biosystems, Foster City, CA, USA). The fragment was isolated by digestion of the plasmid with Bglll and inserted into the BamHI site of Ylp5. The resulting plasmid was linearized by digestion with BgflI, then transformed into either the Cl -6b or B-l cells. Ura+ isolates were tested for correct integration of URA3 by Southern blot analysis using a fluorescein-labeled probe that was generated via PCR with the FAA/ primers and the Gene Images labeling module (Amersham Bioscience). The probe was hybridized and detected on a blot of Draldigested genomic DNA that had been separated by agarose gel electrophoresis, with a Gene Images CDP-Star detection module (Amersham Bioscience). Lipid analysis The culture broth was separated into supernatant and cells by centrifugation. Lipids in the supernatant were extracted twice with 30 ml of chloroform and concentrated, and then the intracellular lipids were extracted as previously described (19). The lipids were resolved on a thin-layer silica gel plate (Kieselgel 60; Merck, Darmstadt, Germany) with hexane/diethyl ether/acetic acid (80: 30: 1, v/v) as the developing solvent. Lipid fractions were visualized by copper sulfate and their concentration were determined with a densitometer (5). For fatty acid analysis, the lipid fractions were eluted with chloroform and methylesterified by the addition of an equal volume of 10% methanolic hydrochloride (Tokyo Kasei, Tokyo) then heating at 60°C‘ for 3 h. Fatt!

VorJ. 95,2003

FATTY ACID SECRETION

acid methyl esters were analyzed on a gas-liquid chromatograph as previously described (5).

RESULTS Isolation of a mutant secreting free fatty acids It has been considered that the repression of fatty acid catabolism might accelerate the accumulation of free fatty acids in cells. Thus, we first aimed to isolate an S. cerevisiae mutant that had reduced P-oxidation activity. After mutagenization of the Cl-6b cells with EMS, a mutant, B-l, that could assimilate glucose but not oleic acid, was selected with growth characteristics similar to those of the parent cells. Figure 1A shows that the acyl-CoA oxidase activity in B-l was significantly (~25 times) lower than that in Cl-6b. Al-

0.10

0.05 0 CT 8 0

437

though a notable amount of lipid secretion was not observed, the intracellular lipids from B-l contained 2.6 times more free fatty acids than those from C 1-6b (Fig. 18). Next, we mutagenized B-l with EMS, and a free fatty acid-secreting mutant was selected from high-density cells by the olel overlay assay (Fig. 2A). Since lipase hydrolyzing acylglycerides was not supplied for the assay, we expected that the secreted lipids would contain free fatty acids (oleic and/or palmitoleic acids), which olel cells can incorporate to support growth. The lipid analysis on the isolate, YTS5 1, indicates that a major component of the extracellular fraction was free fatty acids (-95% of total lipids in culture supernatant), which constituted about 18% of the intracellular fraction (Fig. 2B and Table 1). We could not observe any significant differences in the intra- and extracellular fatty acid compositions in the corresponding lipid classes from YTS5 1. Moreover, deformed cells and cell debris were not found in the culture medium by microscopic observation. Diploid cells generated by mating YTSSI and B-l were deficient in the halo formation, indicating that the mutation causing the free fatty acid secretion is recessive (data not shown). Moreover, sporulation followed by tetrad dissection of the diploid, revealed that this phenotype segregates into 2 : 2 for each of 20 asci tested. This result suggests that the mutation of interest occurs in a single chromosomal allele, which could be complemented by the wild-type gene. Screening and identification of genes that complement Through two the mutation for free fatty acid secretion

A

-0.05 4

IN YEAST

6

8

Reaction time (min)

SE

.Front

B-l

MS51

FAME

B

TG

-Front

TG FFA

MG

FFA .Origin

FIG. 1. Characterization of a @oxidation-deficient mutant B-l. (A) Acyl-CoA oxidase activities in S. cerevisiae strains Cl-6b (wild-type; circles) and B-l (triangles). Palmitoyl-CoA was added to the reaction after pre-incubation for 3 min. (B) TLC analysis of intracellular lipids. Lipid fractions identified in the marker (TLC mix 34; Larodan Fine Chemicals, Malmii, Sweden) are specified on the left. An arrow indicates a band of free fatty acids from B- 1. SE, Sterol ester (identified by R,value); TG triacylglycerol; DG diacylglycerol; MG, monoacylglycerol; FAME, fatty acid methyl ester; FFA, free fatty acids.

-Origin

B-l

YTS51

FIG. 2. Characterization of a free fatty acid-secreting mutant YTS5 1. (A) Formation of a halo by fatty acid auxotrophic mutants (olel) around a colony of YTS5 1 on an agar plate. (B) TLC analysis of extracellular lipids. Bands indicated by an asterisk are derived from Brij-58. TG, Triacylglycerol; FFA, free fatty acids.

43X

1. Blosc 1. Blol-ruc,

MICHINAKA ET AL. TABLE I. Lipid and fatty acid composition ofS cerevisiue mutant strain Y IS5 1 Major intracellular lipids-

Lipid (%) Fatty acid (%) Cl4:O C16:O C16: I C18:O C18: 1

Extracellular lipids

Triacylglycerol

Free fatty acids

20.3

18.2

18.6

1.4 20.4 31.1 8.7 38.4

2.1 25.2 27.9 10.0 34.9

6.9 25.3 22.8 15.4 29.7

Free fatty acids

Triacylglycerol

Sterol ester

3.8

wi.7

4.3 30.4 36.6 7.x 20.8

7.9 25.1 33.6 x.0 29.7

C 14 : 0, Myristic acid; C I6 : 0, palmitic acid; C 16 : I, palmitoleic acid; C 18 : 0, stearic acid; C I 8 : I, oleic acid.

rounds of screening of -20,000 clones from the genomic DNA library, we isolated two plasmids, SHC39 and SHC 1, that could suppress the halo formation by YTSS 1 completely and incompletely, respectively. However, since a multicopy plasmid (YEp24) was employed as a library vector, the isolated genes might be able to suppress the fatty acid secretion phenotype only when their copy numbers are high. To test this possibility, another set of complementation tests was performed using the isolated genes re-cloned in a plasmid with copy numbers of l-2 (pRS3 16). Under this condition, the halo formation by YTS51 was suppressed by the inserted DNA fragment from SHC39, but not by that from SHC 1. This observation suggests that SHC39 carries a gene that complements or suppresses the mutation in low copy number, while the gene in SHC 1 is a multi-copy suppressor. To identify the target genes, each overlapping partial DNA fragment from the plasmids SHC39 and SHCl (Fig. 3) was subcloned into plasmid YEp352 and examined for its ability to suppress the halo formation by introduction into YTSS 1. Consequently, a minimum essential region in SHC39 of -2.4 kb was identified (Fig. 3A), whereas that in SHCl was -2.1 kb (Fig. 3B). Sequencing analyses for these regions revealed the existence of open reading frames of 2 139 and 2121 bp, respectively. A database search indicated that these genes are FAA1 (YOR317w) and FAA3 (YZL009W), both of which encode a long-chain fatty acyl-CoA synthetase. In S. Role of FAAI for fatty acid secretion in YTS51 cerevisiae, four separate genes encoding acyl-CoA synthetase have been identified: FAA I, FAAZ, FAA3 and FAA4 (20). The acyl-CoA synthetases encoded by FAA1 and FAA4 are involved in the incorporation and activation of exogenous long-chain fatty acids, whereas those encoded by FAA2 and FAA3 appear to act on endogenous ones. Disruption of FAA/ gives rise to a greater reduction in fatty acid import than disruption of FAA4 (2 1). Thus, in YTSS 1, the mutation that induces the secretion of free fatty acids could occur in FAAI. To examine this possibility, FAAI and URA3 genes were introduced to the vicinity of the FAA1 locus in the chromosome of B-la, and segregation patterns of the fatty acid secretion phenotype were determined using tetrads from the cross of YTSS 1 and B- 1aEAA1. In 9 asci from the cross, however, the segregation ratios of halo-forming segregants to non-halo forming ones were 2 for 2 : 2, 6 for 1: 3, and 1 for 0:4. This suggests that the FAA1 gene product is a suppressor of the fatty acid secretion phenotype and that mutations in FAAI, even if they occur, are not the primary cause of that phenotype in YTSS 1.

Disruption of FAAZ causes free fatty acid secretion The fact that the expression of acyl-CoA synthetase in YTSS 1 suppressed the fatty acid secretion indicated that the free fatty acid catabolic system would functionally link to the fatty acid secretion process in YTSS 1. Thus, we chose FAA1 to investigate its participation in free fatty acid secretion, and lipid analysis was conducted for B-l carrying a disrupted FAA1 (B-lhFAA1). The results shown in Table 2 indicate that the extracellular fraction from B-1 AEAAI (0.72 mg/lO” cells) contained a higher level of free fatty acids than that from B-l (0.24 mg/lO” cells). The increase in the free fatty acid content due to FAA1 disruption was also observed in the intracellular fraction, and accompanied the dccrease in the content of esterified lipids such as acylglycerols and sterol esters. Moreover, the secretion of free fatty acids was observed even in an FAAl-disrupted strain with

Complementabon of halo formation P

S

1 kb H

B

S

Sm

5

B

X

L-.---Y *

P

I

/

6 S

1kb k--i

Sm

H

PS

X

H

B

E H

,

Complementatlon of halo formation

S I

e I

+

FIG 3. Complementation of fatty acid secretion phenotype (halo formation) of YTS51 by partial DNA fragments from plasmids SHC39 (A) and SHCI (B). Genomic DNA fragments inserted into the plasmids are shown by thick bars. Restriction enzymes used to generate the partial fragments are as follows: B, BumHI; E, EcoRI; H, liindI11; Ps, Pstl; P, P&I; S, &cl; Sm, &al; Xb, X&l; X, Xhol. Asterisks indicate an ExoIlI-generated end of the fragment.

VOL.95,2003

FATTY ACID SECRETION IN YEAST

TABLE 2. Composition of intra- and extracellular lipids in wild-type and free fatty acid-secreting mutant strains Strain Cell growth Intracellular lipids Sterol ester Triacylglycerol Diacylglycerol Monoacylglycerol Free fatty acids Sterol Others Extracellular lipids Triacylglycerol Free fatty acids

Cl-6b

B-l

YTS51

B-IAFAAI

8.28

(cellsx 1OVml) 8.40 5.88

9.48

12.32 3.33 5.10 0.53 0.24 0.56 0.92 1.64 0.34 0.10 0.24

(mg/109 cells) 14.31 13.57 3.33 2.52 3.76 2.76 0.88 0.85 0.40 0.27 I .45 2.48 1.64 2.11 2.85 2.58 0.35 4.16 0.11 0.16 0.24 4.00

13.06 2.91 2.63 0.68 0.36 2.11 1.35 3.02 0.74 0.02 0.72

Cells were grown in 50 ml of YPGD medium for 3 d. Harvested cells were counted and analyzed for their intra- and extracellular lipids.

intact acyl-CoA oxidase activity, although the secretion level decreased to about 40% of that of B- lAE4A I (data not shown). From these results, we conclude that the null disruption of FAA1 induces the accumulation and secretion of free fatty acids, the concentration of which further increases by attenuation of the acyl-CoA oxidase activity. DISCUSSION In this study, we first isolated a mutant strain of S. cerevisiae which had a reduced acyl-CoA oxidase activity. Since this enzyme catalyzes a reaction of the first, rate-limiting step in the pathway of peroxisomal P-oxidation (22), we expected to find de novo synthesized and incorporated fatty acids accumulating in the mutant cells. The mutant strain B-l exhibited an increased free fatty acid content in the cells as expected (Fig. 1). We thought that this characteristic would be advantageous for the screening of a lipid-secreting mutant due to the increased lipid levels. Using B-l as a parent strain, a fatty acid-secreting mutant, YTS5 1, was isolated through the selection of high-density cells followed by an overlay assay using an unsaturated fatty acid auxotrophic (olel) mutant. The concentration of free fatty acids in the culture medium of YTSS 1 was 17 times higher than that in the culture medium of B-l (Table 2). The lipid profile of the extracellular fraction was clearly different from that of the intracellular profile (Table 1). Moreover, we did not observe deformed cells or cell debris in the culture medium. Therefore, the lipids observed in the culture medium were not considered to have been released from the cells by autolysis and physical disruption. The following genetic analyses indicated that the mutation that expresses the fatty acid secretion phenotype occurs at a single chromosomal allele, and that this phenotype is suppressed by the introduction of a single copy of FAA1 to the mutant. Although the mutation inducing fatty acid secretion in YTSSl was not located in FAAI, the disruption of FAA1 in the wild-type strains result in the secretion of fatty acids. Fatty acyl-CoA synthetase is ubiquitous in organisms and is involved in the utilization of fatty acids that have been synthesized de novo by fatty acid synthase, released from

439

membrane phospholipids, and incorporated from outside the cells, by coupling with the CoA molecule. In yeast, four fatty acyl-CoA synthetases encoded by unlinked FAA genes differ in their sub-cellular locations and their specificities for fatty acids of different chain lengths (20, 23). FAAlp and FAA4p, associated with the cytoplasm, act preferentially on the long-chain fatty acids. The reduction in the quantities of esterified lipids, including acylglycerols and sterol esters, in YTS51 compared to its parent strain, could be due to the disruption of FAA gene(s) (Table 2). Interestingly, it was recently reported that both FAAlp and FAA4p may form a complex with FAT 1p, a homologue of the mammalian fatty acid transport protein, which is involved in the import in addition to the activation of fatty acids (21). The fatty acid transport system is thus functionally linked to the intracellular utilization of fatty acids. Therefore, the inactivation of acyl-CoA synthetase would result in dysfunction of both fatty acid import and activation. It is considered that the import and export of fatty acids across the membrane is achieved in two ways, including simple diffusion and the process mediated by membranebound proteins. These mechanisms may not be mutually exclusive: when secretion of fatty acids occurs by simple diffusion, the import system may be activated to maintain the normal level of intracellular fatty acids. Impairment of the transport system would cause the cell to lose the ability to regulate its fatty acid level. In addition, the inactivity of acyl-CoA synthetase results in the accumulation of free fatty acids, making it difficult for the transport system to diminish the efflux of excess fatty acids. Accordingly, it is reasonable that, in YTS5 1, the mutation in FAA1 triggers the secretion of free fatty acids. On the other hand, although FAA3p does not contribute significantly to the fatty acid transport system (21), the repression of fatty acid secretion by expression of multi-copy FAA3 could be due to the promotion of the activation and metabolism of the excess fatty acids. The hypothesis, however, does not exclude the possibility that attenuation of acyl-CoA synthetase activity elicits an active export of fatty acids for cellular homeostasis. Previously, a fatty acid-secreting mutant of C. lipolytica was isolated by mutagenization in an acyl-CoA synthetase I-defective background (8). This work was done based on the concept that the mutation in the gene for acyl-CoA synthetase does not exclusively cause fatty acid secretion, which seems to contradict our conclusion. However, at least two types (I and II) of acyl-CoA synthetase exist in C. lipolytica (24), and their involvement and functional differentiation in fatty acid transport have not yet been determined. Therefore, the deactivated enzyme in the isolated mutant might not be the one that functions for fatty acid transport. Moreover, the detection limit for secreted lipids in the olel overlay assay performed to isolate the mutant in the study of Miyakawa et al. (8) might have been higher than ours. Indeed, we observed a low level of fatty acid secretion in the FAA]-disrupted mutant by TLC analysis (B-IAEAAI; Table 2), but this mutant did not produce a visible halo in the olel overlay assay. It is very likely that C. lipolytica carries another unidentified mutation causing an increase in the amount of extracellular fatty acids, which makes the mutants positive in the overlay assay. Mutation of acyl-CoA

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MICHINAKA ET AL.

oxidase may be one of the causes of the elevated amounts of fatty acids to be excreted (B-l ; Table 2). When the level of fatty acid secretion is reasonably high, it is not easy to distinguish any quantitative differences by an overlay assay. This may be the reason why the gene(s) affecting the secretion levels could not be isolated in the screening of the genomic DNA library. It would be interesting to identify such a gene(s) and determine whether it functionally relates to FAA genes. The yeast S. cerevisiae produces a relatively low level of lipids; yields of total lipids and secreted fatty acids in YTSS 1 were only 0.10 mg/ml and 0.024 mg/ml, respectively (Table 2). Although the culture conditions were not optimized in our case, productivity was far below those reported in other lipid-secreting mutants of yeasts such as C. lipolytica (8) and Tricosporon spp. (9, 10). However, using S. cerevisiae as a model organism on which genetic analyses can be readily performed, this study revealed at least a part of the molecular mechanism governing lipid transport. Research on the mechanism of lipid secretion is important not only for biotechnological use, but also for understanding cellular events mediated by the transport of lipid molecules. Although cells such as adipocytes, for example, export storage lipids according to physiological requirements, little is known about the mechanisms governing lipid transport across cell membranes. Through further research, the findings would be applicable to the effective production of valuable lipids by oleaginous microorganisms, and would help us to better understand the process of lipid transport. ACKNOWLEDGMENTS We are grateful to Prof. E. Tsuchiya for technical helpful discussion.

8.

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