Physiological Role of Acyl Coenzyme A Synthetase Homologs in LipidMetabolism in Neurospora crassa

Christine M. Roche,a,b Harvey W. Blanch,a,b Douglas S. Clark,a,b N. Louise Glassb,c

Chemical and Biomolecular Engineering Department,a Energy Biosciences Institute,b and Plant and Microbial Biology Department,c The University of California, Berkeley,California, USA

Acyl coenzyme A (CoA) synthetase (ACS) enzymes catalyze the activation of free fatty acids (FAs) to CoA esters by a two-stepthioesterification reaction. Activated FAs participate in a variety of anabolic and catabolic lipid metabolic pathways, including denovo complex lipid biosynthesis, FA �-oxidation, and lipid membrane remodeling. Analysis of the genome sequence of the fila-mentous fungus Neurospora crassa identified seven putative fatty ACSs (ACS-1 through ACS-7). ACS-3 was found to be the ma-jor activator for exogenous FAs for anabolic lipid metabolic pathways, and consistent with this finding, ACS-3 localized to theendoplasmic reticulum, plasma membrane, and septa. Double-mutant analyses confirmed partial functional redundancy ofACS-2 and ACS-3. ACS-5 was determined to function in siderophore biosynthesis, indicating alternative functions for ACS en-zymes in addition to fatty acid metabolism. The N. crassa ACSs involved in activation of FAs for catabolism were not specificallydefined, presumably due to functional redundancy of several of ACSs for catabolism of exogenous FAs.

Lipids are essential components of all living cells. They are ma-jor components of biological membranes (e.g., phospholipids)

and serve as energy reserves (e.g., triacylglycerols) as well as sig-naling molecules (e.g., sphingolipids and lysophosphatidic acid).In fungi, fatty acids (FAs) are synthesized de novo via the FA syn-thase complex (FAS type I) present in the cytosol (1) or in themitochondria via a suite of enzymes, including an acyl carrierprotein. These mitochondrial FAS type II enzymes are not presentin a complex (2). Many fungi can also utilize exogenous FAs, bothfor incorporation into lipids and as a carbon source. In addition toincorporation into complex lipids in the endoplasmic reticulum(ER) (3, 4) and mitochondria (5), FAs can be remodeled by de-saturation and elongation (6–8) and degraded for energy produc-tion in the peroxisome (glyoxysome) (9, 10) and in the mitochon-dria (9, 11).

One of the most important catalytic activities in lipid metabo-lism is activation of FAs by acyl coenzyme A (CoA) synthetase(ACS) (EC 6.2.1.3). Activated FAs participate in a number of cel-lular metabolic pathways, including synthesis of phospholipids,triacylglycerols, and cholesterol esters, FA elongation, FA desatu-ration, and �-oxidation. ACSs catalyze the two-step ATP-depen-dent reaction of FA activation, whereby first the FA substrate isadenylated to form an acyl-AMP intermediate and subsequentlyAMP is exchanged with CoA, forming a thioester bond to yield theactivated acyl-CoA (12).

The number of carbons present in the FA substrate of ACSsranges from 2 to more than 26. The characteristic length of the FAsubstrate defines subfamilies of ACSs as short-chain (SC; 2 to 4carbons), medium-chain (MC; 4 to 12 carbons), long-chain (LC;12 to 20 carbons), very-long-chain (VLC; 18 to 26 carbons), and“bubblegum” (14 to 24 carbons) FA activators (13). All ACSs con-tain a highly conserved ATP/AMP binding motif (14), which isconserved among all adenylate-forming enzymes. A second con-served motif (fatty acyl-CoA synthetase [FACS] signature motif)has been identified in ACSs activating long-chain FAs (15). Vari-ations of the FACS motif have been described for ACSs activatingother length classes of FA: medium chain (16), very long chain(17), and bubblegum (18). Structural determination of a long-

chain ACS from the bacterium Thermus thermophilus providedinsights into four conserved regions in fatty ACSs: adenine bind-ing, linker, and gate motifs, as well as the phosphate binding sitewhich corresponds to motif I (19). The linker motif, which linksthe N- and C-terminal domains of the protein, is believed to con-trol the conformation of the substrate binding pocket and thus islikely important for substrate specificity. The linker motif, whichis part of the FACS signature motif, has various consensus se-quences for different-length class activators.

In the yeast Saccharomyces cerevisiae, six fatty ACSs have beenidentified (20). Four of these enzymes (Faa1p, Faa2p, Faa3p, andFaa4p) show activity toward medium-, long-, and very-long-chain FAs, and one (Fat1p) shows activity only toward very-long-chain FAs. An additional putative ACS (Fat2p) that is homologousto Fat1p has been shown to localize to the peroxisome; however,function and substrate specificity have not been defined. In addi-tion to very-long-chain ACS activity, Fat1p is required for uptakeof long-chain FAs. It is believed that exogenous FA utilizationoccurs via vectorial acylation, which requires the concerted activ-ities of Fat1p for transport and either Faa1p or Faa4p for activa-tion. While Faa1p has been shown to account for 90% of ACSactivity in S. cerevisiae, Faa4p provides partially redundant activ-ity. Faa2p has been shown to localize to the peroxisome and isbelieved to be involved in the �-oxidation of medium- and long-chain FAs. While the metabolic role of Faa3p has not been defined,this enzyme localizes to the cell surface and shows preference forunsaturated long-chain FAs and very-long-chain FAs.

The filamentous fungus Aspergillus nidulans is reported to con-

Received 26 March 2013 Accepted 14 July 2013

Published ahead of print 19 July 2013

Address correspondence to N. Louise Glass, Lglass@berkeley.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00079-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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tain six fatty ACSs; two (FaaA and FaaB) are homologs to the Faaproteins of S. cerevisiae, and four (FatA through FatD) are ho-mologs to Fat proteins. Three additional predicted ACSs wereidentified computationally (21). The six fatty ACSs were interro-gated for their involvement in FA activation for degradation byassessing induction of the associated genes during growth on a FAsubstrate as well as growth of individual deletion strains on FAs asa sole carbon source (21). Reiser and coworkers showed that FaaBfunctions as the major peroxisomal ACS responsible for activatinga broad range of FAs for catabolism (21), while FaaA exhibitedcytoplasmic localization. FatA, FatB, and FatD localized to theperoxisome. Strains containing a single deletion of a Fat gene, aswell as the FatA, FatB, and FatC triple deletion strain, failed toshow a difference in growth phenotype on FA substrates com-pared to the wild type (WT) (21). This result was attributed toeither functional redundancy or misidentification by in silico anal-yses.

A distantly related filamentous ascomycete fungus, Neurosporacrassa, has been used as a model system for studying the role oflipids, especially for understanding factors that control synthesisand composition of FAs and complex FAs containing lipids (7,22–25). However, little is known with regard to activation of FAsto CoA esters. Only one N. crassa ACS has been reported to date,acetyl-CoA synthetase (encoded by acu-5 [NCU06836]), whichhas been implicated in the utilization of acetate (26). We hypoth-esized that N. crassa contains a number of additional ACS en-zymes residing in various cellular compartments and exerting spe-cific functions by generating FA-CoA esters that play a role inanabolic or catabolic reactions.

The possibility of perturbing FA metabolism to induce secre-tion of free FAs for the purpose of producing biodiesel precursorsis an attractive alternative fuel strategy. In S. cerevisiae, strainscontaining a deletion of FAA1 and FAA4 exhibit a FA secretionphenotype, which was attributed to interrupted FA recycling (27,28). We hypothesized that altering the intracellular pool of acti-vated FAs by deleting fatty ACSs that function to activate FAs forspecific pathways would disrupt FA equilibrium. In this work, weinterrogated the N. crassa genome for fatty ACSs using sequencehomology to known fungal ACSs. To explore the roles of theseputative ACSs in FA activation, we characterized the respectivegene deletion strains for the ability to activate FA for anabolic andcatabolic lipid processes, as well as for variations in lipid compo-sition. We constructed double mutants to assess functional redun-dancy among the ACSs. As a final line of evidence for metabolicfunction, cellular localization was determined.

MATERIALS AND METHODSN. crassa strains and growth conditions. The N. crassa wild-type (WT)strain FGSC 2489 and the gene deletion strains available from the N. crassadeletion collection (29) were obtained from the Fungal Genetics StockCenter (FGSC; University of Missouri, Kansas City, MO) (30). A his-3::H1-dsRed strain was used for fluorescence microscopy colocalization(31). A �acs-5 homokaryotic strain was obtained through a cross of theheterokaryon available in the deletion collection (FGSC 13827) withFCSC 2489. A �acs-5 homokaryotic strain was confirmed by PCR using aflank-specific primer and a primer to the hygromycin cassette used fordeletion construction (29) (see Table S1 in the supplemental material).Double mutant strains were obtained from crosses between the singlegene deletion strains of opposite mating types; double mutant genotypeswere confirmed by PCR using a flank-specific primer and a primer to thehygromycin cassette used for deletion construction (29) (see Table S1 in

the supplemental material). Crosses were performed on Westergaard’smedium (32). Strains were precultured on Vogel’s medium (VM) agar(33) supplemented with 2% sucrose for 8 days to obtain conidia forgrowth experiments.

For time course growth experiments, 1 � 106 conidia/ml were inocu-lated into 250-ml Erlenmeyer flasks containing 100 ml of VM supple-mented with 2% (wt/vol) glucose or Tween 80 and cultured in constantlight at 25°C and 200 rpm. Refreshed spent medium was prepared byusing sterile filtered culture broth of a 2-day-old VM–2% glucose cultureof the WT strain. Whole flasks were harvested and processed as follows.Duplicate 10-ml samples were filtered, washed, and dried to constantmass on preweighed filter paper for cell concentration measurements.Glucose remaining in the culture broth supernatant was determined viahigh-performance liquid chromatography (HPLC) using a ShimadzuHPLC equipped with an RFQ fast acid column (Phenomenex Inc.) run at55°C with 0.01 N sulfuric acid pumped at 1 ml/min as the eluent. FAremaining in Tween 80 medium was determined by lipid analysis of cul-ture broth supernatant as indicated below. Fifty milliliters of culture waswashed with deionized water to remove the residual carbon source andmedium salts and centrifuged at 4,500 rpm for 10 min at 4°C. The cellpellet was frozen in liquid nitrogen and lyophilized to complete drynessfor lipid analysis (see below).

For the anabolic and catabolic pathway determination, 1 � 106

conidia/ml were inoculated into 24-well plates containing 3 ml/well ofVM supplemented with 0.5% (wt/vol) lactose and/or even-chain satu-rated FAs ranging from 4 to 24 carbons at a final concentration of 25 mMcarbon suspended in 1% Tergitol–NP-40 and cultured in constant light at25°C and 200 rpm. The FA synthase inhibitor cerulenin was added to afinal concentration of 10 �g/ml. Growth was determined by visual inspec-tion.

Lipid analysis. Total lipids were extracted and derivatized from lyoph-ilized biomass via a direct transesterification process. Briefly, 2 ml of acid-ified methanol (MeOH; 5% [vol/vol] sulfuric acid) was added to 20 to 30mg of lyophilized biomass, and the biomass was incubated at 95°C for 30min. After cooling, FA methyl esters (FAMEs) were extracted in 2 ml ofhexane via gentle agitation at 37°C for 45 min. Residual FAs in Tween 80media were extracted and derivatized from liquid culture broth by sapon-ification followed by methylation. Briefly, 1 ml of methanol saturated withKOH was added to 0.5 ml culture broth, and the mixture was incubated at100°C for 2 h. Acid-catalyzed methylation was accomplished by adding1.5 ml 1:1.2 6 N HCl-MeOH and incubating at 80°C for 6 h. FAMEs wereextracted into 2 ml hexane via gentle agitation at 37°C for 45 min.

FAME extracts were analyzed directly by gas chromatography-flameionization detection (GC-FID) using a Varian 3900 gas chromatograph.FAMEs were separated with an Omegawax 250 column (Supleco Inc.).Helium was used as the carrier gas at a flow rate of 1 ml/min. The columntemperature program was as follows: hold at 120°C for 3 min, ramp from120 to 250°C at 7°C/min, and hold at 250°C for 10 min. FAMEs wereidentified and quantified against a 37-component FAME mix externalstandard set (Supelco Inc.). Methyl tridecanote was spiked into samplesprior to transesterification, and recovery of this internal standard wasgreater than 95%.

Molecular techniques and strain construction. Standard methodsfor cloning and other molecular techniques were performed according topublished methods (34). Genomic DNA from the N. crassa WT strain wasextracted according to the method of Lee and Taylor (http://www.fgsc.net/fgn35/lee35.pdf). DNA amplification was performed using Phusionhigh-fidelity polymerase (Finnzymes) according to the manufacturer’sinstructions.

To facilitate creation of green fluorescent protein (GFP) gene fusionstrains for colocalization and complementation studies, the pCSR1 vector(35), which allows positive selection of csr-1 targeted transformants, wasmodified to contain the Myceliophthora thermophila gpdA promoter, amultiple cloning site (MCS), the synthetic GFP (sGFP) gene, and the M.

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thermophila gpdA terminator (T. Starr and N. L. Glass, personal commu-nication).

Restriction-free (RF) cloning (36) was used to insert acs into thepCSR1:gpd-gfp vector. The open reading frame of each of the ACS geneswith the exception of the stop codon was amplified from genomic DNA ofWT N. crassa (FGSC 2489) using the respective primer sets listed in TableS1 in the supplemental material. Primers were designed to contain a 24-base overlap with the vector at the desired insertion site, followed by 20 to25 bases complementary to the gene of interest. PCR products were aga-rose gel purified and extracted using a gel purification kit (Zymo Re-search). Individual PCR products were used as a “megaprimer” to amplifythe vector at the template/primer loading ratio of 40:400 nmol vector tomegaprimer. Subsequently, DpnI was added to the reaction mixture todigest the methylated parental vector, and the reaction mixture was useddirectly to transform chemically competent Escherichia coli to repair thenicks and propagate the vector. All of the resulting plasmids were se-quenced to verify correct construction.

Transformation of N. crassa by electroporation was performed as pre-viously described (35). The sGFP gene fusions were transformed into therespective gene deletion strains. Positive transformants were verified byPCR genotyping using the primer set listed in Table S1 in the supplemen-tal material.

Confocal microscopy. Mycelia for microscopy were grown on VMagar supplemented with 2% sucrose at 25°C overnight. Mycelia were ob-served using a 100�, 1.4-numerical-aperture (NA) oil immersion objec-tive on a Leica SD6000 microscope attached to a Yokogawa CSU-X1 spin-ning disc head with a 488-nm and/or 561- nm laser. Dual-color imagingwas accomplished using automated acquisition of the two wavelengths inseries with a time differential for emission filter changeover.

Bioinformatic analysis. Candidate N. crassa ACS genes and proteinswere identified using the Basic Local Alignment Search Tool (BLAST)algorithm (http://www.ncbi.nlm.nih.gov/BLAST) (37). The search strat-egy used queries of the S. cerevisiae ACS proteins (Faa1p to Faa4p, Fat1p,and Fat2p) to probe the N. crassa nonredundant protein database(BLASTp) and N. crassa nucleotide database (tBLASTn). N. crassa DNAand protein sequences were obtained from the assembled genome se-quence at the Broad Institute (http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html). The candidate ACS amino acidsequences were examined for the presence of the two highly conservedmotifs aforementioned (i.e., motif I [AMP/ATP binding] and motif II[FACS signature]) using FIMO (http://meme.sdsc.edu/meme/cgi-bin/fimo.cgi). Approximately 1,000 bp of the predicted promoter regions ofthe candidate ACS genes was analyzed for the conserved binding sequence(CCTCGG) of the FarA and FarB transcription factors, which have beenimplicated in regulation of genes involved in fatty acid degradation in A.nidulans (38).

Putative ACS orthologs of select fungal species were identified usingBLASTp against the respective protein database. Multiple protein align-ment was performed using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) (39); any sequences not including either motif 1 or 2 were dis-carded. Phylogenetic trees were generated using neighbor-joining analysisin MEGA5 (molecular evolutionary genetic analysis) program (40). Evo-lutionary distances were computed using the Poisson correction method.All positions containing gaps were eliminated from the data set. The ro-bustness of the tree topology was evaluated by bootstrap analysis using asampling size of 1,000.

RESULTSIdentification of genes encoding putative N. crassa acyl-CoAsynthetases. The amino acid sequences of six S. cerevisiae acyl-CoA synthetase proteins, Faa1p to Faa4p, Fat1p, and Fat2p, wereused in BLASTp and tBLASTn searches of the N. crassa proteinand nucleotide databases, respectively. Each identified ACS ho-molog was queried for the presence of the highly conserved AMP/ATP binding motif (14), as well as for the FACS signature motif

(41); if either was absent, the identified homolog was eliminated.Seven N. crassa homologs of the S. cerevisiae ACS proteins wereidentified. The protein encoded by NCU01654 (ACS-1) showedsignificant identity (32.2%) to S. cerevisiae Faa2p, while two otherN. crassa proteins, ACS-2 (encoded by NCU03929) and ACS-3(encoded by NCU04380), showed high identity (40.6 to 47.8%) toS. cerevisiae proteins Faa1p, Faa3p, and Faa4p. Genes encodingfour additional N. crassa proteins had high identity (26.8 to52.2%) to genes encoding the FAT proteins: NCU06032 (encod-ing ACS-6) to the well-characterized Fat1p gene and NCU00608(encoding ACS-4), NCU06063 (encoding ACS-5), andNCU08935 (encoding ACS-7) to the less-characterized Fat2pgene.

A previous phylogenetic analysis of human, mouse, zebrafish,fruit fly, nematode, and yeast ACSs revealed clades correspondingto FA substrate toward which the ACS was active (i.e., medium-,long-, or very-long-chain FAs) (42). To evaluate relationshipsamong fungal ACSs, the genome sequences and protein databasesof 11 additional fungi representing diversity across the fungalkingdom (Aspergillus nidulans, Botryotinia fuckeliana, Candida al-bicans, Cochliobolus heterostrophus, Coccidioides immitis, Crypto-coccus neoformans, Fusarium graminearum, Magnaporthe grisea,Phanerochaete chrysosporium, Puccinia graminis, and Rhizopusoryzae) were interrogated for ACSs using the same search param-eters as above. As many as 12 and as few as 3 putative ACS geneswere identified in each fungal species. Protein sequences from the94 identified fungal ACSs grouped into 4 clades (Fig. 1A; see TableS2 in the supplemental material). The clustering of the ACSs wasconsistent with the substrate specificity of characterized S. cerevi-siae ACSs and predicted FA specificity for the long-chain (LC) andvery-long-chain (VLC) clades. The FAA homologs, all predictedto have LC specificity, fell into two subclades. One subclade in-cluded N. crassa ACS-1 (Nc-ACS-1), S. cerevisiae Faa2p (Sc-Faa2p), and A. nidulans FaaB (An-FaaB), all of which localize tothe peroxisome/glyoxysome (21, 43, 44). Within the second sub-clade, S. cerevisiae has two closely related paralogs (Sc-Faa3P andSc-Faa4p), plus an additional ACS (Sc-Faa1p), while A. nidulanshas only one (An-FaaA) in this entire subclade. In contrast, N.crassa has two predicted proteins, ACS-2 (encoded byNCU03929) and ACS-3 (encoded by NCU04380).

The filamentous fungal homologs of Sc-Fat2p, for which sub-strate specificity is uncharacterized, formed two clades; we denotethese as ACS families 1 and 2 (Fig. 1A). Family 2 comprised one S.cerevisiae protein, Fat2p, one A. nidulans protein, FatD, and N.crassa ACS-7 (encoded by NCU08935). An A. fumigatus homologof one of the ACS proteins within family 1 in A. nidulans(AN0609), termed SidI, localized to the peroxisome and was re-cently characterized as a mevalonyl-CoA ligase involved in sidero-phore (triacetylfusarinine C [TAFC]) biosynthesis (45, 46). Twoadditional ACSs in family 1 (AN5272 and AN4659) were identi-fied by in silico sequence analysis and are predicted to localize tothe peroxisome (AN5272) or to mitochondria (AN4659); AN4659was predicted to have short-chain FA specificity (21). In N. crassa,two predicted proteins, ACS-4 (encoded by NCU00608) andACS-5 (encoded by NCU06063) cluster in family 1. In particular,ACS-5 is in the same clade as AN0609.

In addition to the AMP/ATP binding and the FACS signaturemotifs, four structurally significant domains have been defined forACSs: phosphate binding, linker, adenine binding, and gate (19),where the phosphate binding domain is located within the AMP/

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FIG 1 Summary of acyl-CoA synthetases in fungi. (A) Phylogenetic tree of putative ACS proteins identified in 13 fungal species (Aspergillus nidulans, Botryotiniafuckeliana, Candida albicans, Cochliobolus heterostrophus, Coccidioides immitis, Cryptococcus neoformans, Fusarium graminearum, Magnaporthe grisea, Neuros-pora crassa, Phanerochaete chrysosporium, Puccinia graminis, Rhizopus oryzae, and Saccharomyces cerevisiae). N. crassa ACSs are in boldface, and gene names areindicated with arrows. A. nidulans and S. cerevisiae ACSs are in boldface and italicized. (B) Domain organization of the N. crassa ACSs. (C) Conserved amino acidsequences of putative N. crassa ACSs. Boldface and underlined amino acids within motifs I and II show the phosphate binding and linker domains, respectively.LC, long chain; VLC, very long chain.

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ATP binding motif and the linker domain is located within theFACS signature motif. All seven of the predicted N. crassa ACSproteins contained sequences similar to those of all four domains;their alignment reveals a conserved domain structure (Fig. 1B andC). It has been proposed that the structural location of the linkermotif determines the substrate specificity for ACS enzymes (19).Three similar consensus sequences have been suggested for short-chain and medium-chain (Gly-Arg-Xaa-Asp), long-chain (Asp-Arg-Xaa-Lys), and very-long-chain (Asp-Arg-Xaa-Gly) ACSs. Asexpected, the linker motif sequences for ACS-1, ACS-2, andACS-3 match the consensus sequence for the long-chain ACS,while the linker motif sequence for ACS-6 matches the consensussequence for the very-long-chain ACS. The linker motif for theFat2p homologs (ACS-4, ACS-5, and ACS-7), however, is moresimilar to the short- and medium-chain ACS consensus motif.Our alignment showed that the S. cerevisiae and A. nidulans ACSsthat fall within the same clades as the N. crassa ACSs share therespective short-/medium-, long-, and very-long-chain linkermotifs.

In A. nidulans, two Zn2-Cys6 binuclear transcription factors,FarA and FarB, regulate genes involved in fatty acid catabolism;farA� mutants are unable to use fatty acids as a sole carbon source,while a farB� mutant is unable to utilize short-chain fatty acidsbut is unaffected in growth on longer-chain fatty acids (38). Ho-mologs of farA and farB are present in the N. crassa genome (far-1,NCU08000, and far-2, NCU03643). The binding sites for A. nidu-lans FarA and FarB have been determined by in vitro analyses andare identical for both FarA and FarB: CCGAGG/CCTCGG (38).This binding site was used to search predicted promoter regions ofacs-1 through acs-7. All but acs-4 contained one or more of theputative FAR-1/FAR-2 binding sites. Chromatin immunoprecipi-tation-DNA sequencing (ChIP-seq) analysis identified FAR-1binding within promoter regions of acs-1, acs-5, and acs-6 andFAR-2 binding sites within predicted promoters of acs-5 and acs-7(E. L. Bredeweg and M. Freitag, personal communication).

Phenotypic analysis of �acs strains grown on glucose. Themacroscopic phenotype and growth of individual strains contain-ing deletions in each of the ACS genes were evaluated on agarmedium containing glucose. The �acs-1, �acs-2, �acs-3, �acs-4,and �acs-5 deletion strains were morphologically indistinguish-able from their wild-type isogenic parental strain. However, the�acs-6 and �acs-7 strains displayed slightly longer aerial hyphaethan the wild-type strain (Fig. 2A). The maximal linear growthrates of all of the mutant strains were equivalent to that of thewild-type strain on agar with Vogel’s medium (VM)–2% glucose(data not shown). However, the ability to grow in submergedliquid VM–2% glucose was not consistent among all strains. Forexample, the �acs-5 mutant exhibited a significant growth defect(Fig. 2B) and displayed a 20-h lag in biomass accumulation duringsubmerged growth compared to the wild type. The maximalgrowth rate (0.31 g/liter/h between 36 to 60 h) and accumulatedcell mass (8 g/liter), however, were unaffected.

To further investigate the physiological role of ACS-5, we as-sessed whether the growth defect in the �acs-5 mutant was a resultof a germination defect in liquid media by evaluating the fre-quency of conidial germination in liquid VM–2% glucose for thewild type. We also included the �acs-3 and �acs-4 mutants, as theydisplayed an altered phenotype compared to the wild-type strain.The frequency of germination for the wild-type strain at 4 h post-inoculation was 79%. The �acs-4 and �acs-5 mutants germinated

with the same frequency as the wild type (78% and 76%, respec-tively), while the �acs-3 mutant exhibited slightly increased ger-mination frequency (88%) at 4 h.

To determine if an arrest in growth occurred in the �acs-5mutant after germination, the number of colonies that developedfrom individual conidia was determined and compared to that forthe wild type, as well as to those for the �acs-3 and the �acs-4mutants. Approximately 200 conidia per strain were plated ontoVogel’s medium containing sorbose and incubated at 30°C for 2days. For the wild-type strain, 100% of plated conidia formedviable, macroscopic colonies. The �acs-3, �acs-4, and �acs-5 mu-tants produced 65%, 96%, and 0.5% viable colonies, respectively.Colony formation was reassessed after 7 days, and no additionalcolonies of the �acs-3 and �acs-4 strains were observed. However,70% of �acs-5 conidia formed viable, macroscopic colonies. Thisresult suggests that the �acs-5 strain has a defect postgerminationthat resulted in an extreme lag phase. These data for the �acs-5strain are consistent with previously reported growth phenotypesfor conidia that have lost the siderophore ferricrocin (47), includ-ing an inoculum-dependent population effect (48). In A. fumiga-tus, a homolog of acs-5, termed sidI, was shown to be essential forformation of the TAFC siderophore (45), and recently it wasshown that introduction of Nc-acs-5 into an A. nidulans mutantlacking PTS1-dependent peroxisomal import (�pexE) increasedTAFC production (46).

N. crassa secretes the hydroxamate cyclic peptide siderophorecoprogen under iron-limited conditions, but also under normallaboratory culture conditions (47). To evaluate whether ACS-5 isinvolved in siderophore biosynthesis in N. crassa, wild-type and�acs-5 strains were grown in iron-replete medium (VM–2% glu-cose supplemented with1.5 mM FeSO4). Similar conditions al-lowed recovery of growth phenotypes associated with disruptionof extracellular siderophore biosynthesis in A. fumigatus (49). Thelag phase in the �acs-5 mutant was completely abolished underthese conditions (see Fig. S1 in the supplemental material). Previ-ously, it has been shown that the addition of spent medium froma wild-type culture reverted the growth phenotype of N. crassasiderophore mutants, consistent with the presence of an extracel-lular siderophore (47, 50). As predicted, the addition of spentmedium from a wild-type culture to fresh medium inoculatedwith the �acs-5 mutant also abolished the lag phase (see Fig. S1 inthe supplemental material). These data indicate that, as for SidI inA. fumigatus, ACS-5 is involved in siderophore biosynthesis in N.crassa.

Lipid phenotype of ACS mutants. Since ACSs play a pivotalrole in the majority of the lipid metabolic pathways, we deter-mined whether any of the N. crassa ACS deletion mutants hadlipid perturbations. As a proxy for lipid perturbation, we evalu-ated lipid-derived FA methyl esters (FAMEs) using gas chroma-tography-flame ionization detection (GC-FID). Lipids were ex-tracted, derivatized, and quantified from lyophilized biomass ofwild-type and �acs cultures grown under submerged conditionson VM–2% glucose over a period of 5 days (Fig. 2C). In the wild-type strain, the lipid content increased until carbon source deple-tion (36 h). Intracellular lipid content remained constant for anadditional period of 25 h, after which lipid stores began to beutilized, as indicated by the decrease in lipid content after 70 h.The lipid profiles by FAME analyses of the �acs-1, �acs-2, �acs-4,�acs-6, and �acs-7 mutants were indistinguishable from that ofthe wild-type strain. Consistent with the growth phenotype in

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liquid culture, lipid accumulation in the �acs-5 mutant showed aprolonged lag phase and reached wild-type levels only after 70 h ofgrowth. Most notably, the �acs-3 strain accumulated 75% morelipid per cell mass than the wild-type strain, with the highest lipidaccumulation at 60 h (Fig. 2C). However, no evidence of FA se-cretion was detected via FAME analysis of culture broth superna-tant for any strain.

FAME analysis was used to determine the profile of FAs inwild-type and the �acs strains at the 60-h time point, with theexception of the �acs-5 mutant, which was analyzed at the 72-htime point (Fig. 2D). Like most other fungi, N. crassa producespredominantly 16- and 18-carbon saturated and unsaturated FAs,with 18:2 accounting for 50% (wt/wt) of the total FA. A small

amount of 24-carbon FAs was detected as a part of the sterol esterclass of lipid (data not shown). Analysis of lipid distribution byFAME analysis in the �acs-3 mutant (60 h) revealed a decrease inunsaturated FAs (16:1, 18:2, and 18:3), with a corresponding in-crease in saturated 16-carbon (16:0) and saturated and monoun-saturated 18-carbon FAs (18:0 and 18:1) (Fig. 2D). This resultsuggests that ACS-3 is involved in activating FAs for desaturation.

The �acs-1, �acs-2, �acs-4, �acs-6, and �acs-7 mutantsshowed a lipid profile identical to that of the wild-type strain (Fig.2D). At the 72-h time point, the �acs-5 mutant has more 16:0 butwas otherwise more similar to the wild type for the other lipidclasses. However, at earlier time points the lipid accumulation inthe �acs-5 mutant was significantly different (Fig. 2C). We there-

WT Δacs-1 Δacs-6Δacs-5Δacs-4Δacs-3Δacs-2 Δacs-7

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FIG 2 Phenotypic analysis of N. crassa �acs strains on glucose. (A) Macroscopic morphology of the WT (FGSC 2489) and �acs strains. (B) Biomass accumu-lation in submerged liquid culture in VM–2% glucose. (C) Gas chromatography-flame ionization detection (GC-FID) quantification of fatty acid methyl esters(FAMEs) derived from total lipids during time course in VM–2% glucose. Lipid was quantified per mg of lyophilized biomass. (D) Fatty acid (FA) compositionof total cellular lipid of N. crassa �acs strains. Individual FAMEs derived from total lipids were quantified using GC-FID. Fatty acids are given as a mass fractionof total derivatized FA at the 60-h growth point for all strains, except �acs-5, which was analyzed at 72 h. Ninety-seven percent of total cellular FAs compriseC16:0, C16:1, C18:0, C18:1, C18:2, and C18:3. (E) FA profile for WT and �acs-5 strains during early time points. All values are representative of quadruplicatebiological samples. Error bars indicate standard deviations.

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fore compared FA composition in the �acs-5 mutant to that in thewild type over a time course of early growth (12 to 24 h for WT and24 to 60 h for the �acs-5 mutant) (Fig. 2E). During the lag phase ofgrowth in the wild-type strain (up to 12 h), 70% of the total FAcomprised 18:2 and 18:3 lipids in equivalent amounts. As the cul-ture aged to stationary phase, the fraction of 18:3 decreased to 5%and 18:2 increased to 60% of the total lipids. For the �acs-5 strain,18:2 constituted 50% or more of the total FAs at all time points inthe culture, although at �50 h of growth, the �acs-5 mutantshowed a rise in 18:3 FAs accompanied by a dip 18:1 FAs.

Characterization of the role of FA activation for anabolic andcatabolic pathways. The activated FAs synthesized by the ACSsare used in both anabolic pathways (i.e., complex lipid assembly,FA elongation, FA desaturation) and catabolic pathways (i.e.,�-oxidation). ACS activity is not required for activation of de novoFAs since fatty acyl-CoA esters are the end product of FA biosyn-thesis; however, ACSs will be active on endogenous FAs obtainedfrom complex lipid remodeling or exogenous FAs obtained fromthe culture medium. In this study, we probed ACS activity againstexogenous FA.

To assay for FA activation for catabolism, even-number 6- to24-carbon saturated FAs were provided as a sole carbon source tothe wild type and the �acs deletion mutants. Strains that exhibiteda growth defect under these conditions are deficient in activating aFA for catabolism. As shown in Table 1 and in Fig. S2 in thesupplemental material, no strain, including the wild type, was able

to grow on 6- to 10-carbon FAs as the sole carbon source, indicat-ing that 6- to 10-carbon FAs are not activated by any of the ACSs.Similarly, slower growth was observed for all strains on very-long-chain FAs (20:0, 22:0, and 24:0). Growth on the FAs 12:0, 14:0,16:0, 18:0 of the �acs-2, �acs-4, �acs-5, �acs-6, and �acs-7 mu-tants was identical to that of the wild type. However, the �acs-1and �acs-3 mutants did not grow on 12-carbon FAs. These resultsindicate that the medium-chain FA 12:0 is preferentially activatedfor catabolism by ACS-1 and ACS-3. The fact that no mutant wassignificantly different from the wild type in utilization of long- andvery-long-chain FAs suggests that there is functional redundancyamong the ACSs of N. crassa for activation of these FAs for catab-olism.

To assay for activation for anabolic pathways, the growth phe-notype between two feeding regimens was evaluated for eachstrain: FA and lactose-supplemented Vogel’s medium in the pres-ence or absence of cerulenin, an inhibitor of FA biosynthesis (Ta-ble 1; see Fig. S3 and S4 in the supplemental material). In theabsence of cerulenin, all strains should grow on all FAs unless theFA is inhibitory; this condition serves as the growth control. ForFA media plus lactose and cerulenin, a growth defect in the �acsstrains indicates a deficiency in activating the FA for anabolicpathways. If growth is observed under these conditions, the ACS isnot needed for anabolic pathways or the activity is compensatedfor by another ACS. As shown in Table 1 and in Fig. S3 in thesupplemental material, wild-type N. crassa is unable to grow in the

TABLE 1 Growth phenotype of N. crassa �acs mutants in the presence of fatty acids

Medium and strain

Phenotypea for:

No FA 4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0

VM–25 mM carbon (fatty acid)WT NG LG NG NG NG G G G G LG VLG VLG�acs-1 NG LG NG NG NG NG G G G LG VLG VLG�acs-2 NG LG NG NG NG G G G G LG VLG VLG�acs-3 NG LG NG NG NG NG G G G LG VLG VLG�acs-4 NG LG NG NG NG G G G G LG VLG VLG�acs-5 NG LG NG NG NG G G G G LG VLG VLG�acs-6 NG LG NG NG NG G G G G LG VLG VLG�acs-7 NG LG NG NG NG G G G G LG VLG VLG

VM–0.5% lactose, 25 mM carbon (fatty acid)WT G VLG NG NG NG VLG G G G LG LG LG�acs-1 G VLG NG NG NG NG G G G LG LG LG�acs-2 G VLG NG NG NG VLG G G G LG LG LG�acs-3 G VLG NG NG NG VLG G G G LG LG LG�acs-4 G VLG NG NG NG VLG G G G LG LG LG�acs-5 G VLG NG NG NG VLG G G G LG LG LG�acs-6 G VLG NG NG NG VLG G G G LG LG LG�acs-7 G VLG NG NG NG VLG G G G LG LG LG

VM–0.5% lactose, 25 mM carbon (fatty acid),10 �g/ml cerulenin

WT VLG VLG NG NG NG VLG G G G LG LG LG�acs-1 VLG VLG NG NG NG NG G G G LG LG LG�acs-2 VLG VLG NG NG NG NG G G G LG LG LG�acs-3 VLG NG NG NG NG NG VLG LG LG NG NG NG�acs-4 VLG VLG NG NG NG NG G G G LG LG LG�acs-5 VLG VLG NG NG NG NG G G G LG LG LG�acs-6 VLG VLG NG NG NG NG G G G LG LG LG�acs-7 VLG VLG NG NG NG NG G G G LG LG LG

a NG, no growth; VLG, very low growth; LG, low growth; G, growth. Boldface indicates differences from the respective control.

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presence of 6:0 to 10:0 FAs, even in the presence of lactose. BecauseN. crassa was also unable to activate these FAs for catabolism (Ta-ble 1; see Fig. S2 in the supplemental material), these results indi-cate that these FAs inhibit de novo biosynthesis of FAs of appro-priate chain length for membrane formation. This result isconsistent with findings where medium-length FAs inhibited FAbiosynthesis and prevented synthesis of full-length membrane lip-ids in A. nidulans and insect eggs (11, 51). In addition, the �acs-1mutant was unable to grow in the presence of 12:0 and lactose(Table 1; see Fig. S3 in the supplemental material). Among theACS mutants, only the �acs-3 mutant exhibited differentialgrowth in the presence of cerulenin, lactose, and 14:0 to 24:0 FAs(Table 1; see Fig. S4 in the supplemental material). These dataindicate that ACS-3 is the main activating ACS of exogenous FAs.Other ACSs, however, provide some functional redundancy to-ward long-chain FAs because the �acs-3 strain exhibited somegrowth on 14:0 to 18:0 FAs.

Phenotypic analysis of the �acs-3 strain grown in the pres-ence of FAs. To further probe the underlying physiological role ofACS-3, we assessed the growth and lipid phenotypes of the wildtype and the �acs-3 mutant when grown on Tween 80. This non-ionic surfactant composed of PEGylated sorbitan esterified witholeic acid (18:1) (25% by weight of Tween 80) is an ideal source forFA supplementation for filamentous fungi (24). N. crassa secreteslipases that cleave the FA from the polysorbitan backbone. Boththe FA and polysorbitan component can be used as a carbonsource by N. crassa, as is indicated by the growth yield (Fig. 3).Because Tween 80 is soluble in water and can be fully used as acarbon source by N. crassa, we can assess the influence on the lipidprofile of FA-supplemented media in the wild type versus the mu-tant.

Both the wild-type and �acs-3 strains exhibited a 40% reduc-tion in linear growth rate (0.19 g/liter/h) on VM–2% Tween 80compared to growth on glucose media (Fig. 3). The lipid contentof the wild-type versus that of the �acs-3 culture grown on VM

supplemented with Tween 80 was assessed via FAME analysis (Fig.3). For the wild-type strain, the overall lipid content at its maxi-mum was 6-fold higher than that for growth on glucose as the solecarbon source (compare Fig. 2C with 3). This result indicates that,under an excess of FA, N. crassa is either storing FA for later use orinternally compartmentalizing the FAs to mediate the toxicity ofthe hydrophobic environment. As the strains enter stationaryphase, lipid content decreases, indicating that the amount of FApresent in the media is no longer in excess and can be efficientlyutilized. Interestingly, although the �acs-3 mutant accumulatedsignificantly more lipid under glucose conditions (Fig. 2C), itshowed a lipid profile similar to that of the wild type when grownin media with exogenously supplied FAs (Fig. 3). These data indi-cate that exogenously supplied FA is sufficient to repress FA bio-synthesis and overaccumulation of lipid in this strain.

Subcellular localization of ACSs. To investigate the subcellu-lar localization of the N. crassa ACSs, C-terminal sGFP fusionconstructs, driven by the constitutive Myceliophthora thermophilagpdA promoter, were prepared and transformed into the csr-1locus (35) of �acs-2, �acs-3, �acs-4, and �acs-6 mutants (see Ma-terials and Methods). Three independent clones were evaluatedfor each construct by spinning disc confocal microscopy. Trunkand branch hyphae, as well as apical and distal hyphal regions,were analyzed for marker fluorescence. ACS-1 and ACS-7 werenot tagged, as they were previously localized to the proteome ofthe peroxisome/glyoxysome (43). Similarly, a GFP-tagged ACS-5was shown to localize to peroxisomes when introduced into A.nidulans (46).

Confocal fluorescence microscopy revealed that ACS-2 andACS-3 have similar localization patterns (Fig. 4A and B) and lo-calized to membrane-bound structures that resemble the endo-plasmic reticulum (ER). Colocalization of H1::dsRed (i.e., dsRed-labeled histone 1, nuclear marker) with either ACS-2–GFP orACS-3–GFP verified ER localization of ACS-2 and ACS-3 (Fig. 5Aand B). Additionally, ACS-3 localized to the plasma membrane(PM) and septa, consistent with our finding that ACS-3 is themajor activator of exogenous FAs. The ACS-4 –GFP fusion local-ized to mitochondria (Fig. 4C), which in the apical tip regionappear as long tubular structures, while in subapical regions, thestructures were shorter and more diffuse; ACS-4 –GFP colocalizedwith the MitoTracker Red FM stain (Fig. 5C). Finally, the �acs-6strain expressing ACS-6 –GFP showed localization to the plasmamembrane in distal hyphal regions, as well as to puncta through-out the hypha, but did not colocalize with the glyoxysome (datanot shown). While localization to the plasma membrane was notobserved in all hyphae (Fig. 4D), this localization pattern is ex-pected for ACS-6, which is the homolog to the S. cerevisiae long-chain FA transporter, Fat1p.

Subcellular localization of each ACS gives an indication of thepathways for which they provide activated FAs. For example, theglyoxysomal localization of ACS-1 and ACS-7 and the mitochon-drial localization of ACS-4 support their role in providing acti-vated FA for the glyoxysomal and mitochondrial �-oxidationpathways, respectively. ACS-2 and ACS-3, which exhibited ERlocalization, are likely involved in activating FAs for anabolicpathways such as FA elongation, desaturation, and complex lipidassembly, as the ER houses other enzymes involved in these path-ways. Since most of the �acs mutant strains did not exhibit phe-notypes different from that of the wild-type strain, we were unableto determine functional complementation. However, the wild-

FIG 3 Phenotypic analysis of N. crassa �acs-3 mutant strain compared to thewild type with FA-supplemented medium. Open and solid diamonds repre-sent data for the wild type, while open and solid circles represent data for the�acs-3 mutant. Solid diamonds and circles represent biomass accumulation insubmerged liquid culture growth in VM–2% Tween 80. Open diamonds andcircles represent GC-FID quantification of FAMEs derived from total lipidsover the growth time course. Lipid was quantified per mg of lyophilized bio-mass. All values are representative of triplicate biological samples. Error barsindicate standard deviations.

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type FA phenotype was restored in the �acs-3 mutant by the in-troduction of the ACS-3–GFP fusion construct (see Fig. S5 in thesupplemental material).

Double mutant analysis. To gain further insight into the phys-iological role of specific ACSs and to elucidate possible functionalredundancy, double deletion mutants of the long-chain clade acsgenes were constructed and evaluated for FA composition: �acs-1;�acs-2, �acs-1; �acs-3, and �acs-3; �acs-2 mutants. We were un-able to produce the triple deletion strain (�acs-1; �acs-3; �acs-2),suggesting that a strain bearing all three of these lesions is not

viable. The �acs-1; �acs-2 and �acs-1; �acs-3 mutants exhibited awild-type growth phenotype on solids and in liquid medium,while the �acs-3; �acs-2 mutant strain lacked conidia and dis-played a 58% reduction in linear growth rate compared to the wildtype. The total FA contents and compositional profiles are given inTable 2.

Combinatorial deletions of the acs genes led to a spectrum offatty acid phenotypes. The �acs-1; �acs-2 mutant strain exhibitednearly wild-type levels and composition of fatty acids, while the�acs-3; �acs-2 mutant accumulated �acs-3 levels of fatty acids,

FIG 4 Localization of ACS-GFP fusion proteins. ACS-GFP fusions were expressed in the background strain of the respective �acs strain. Bright-field and GPFfluorescence images were obtaining by spinning disc confocal microscopy (see Materials and Methods). The two columns of panels on the left showlocalization in the tip region of a hypha, while the two right columns show localization in subapical regions. Arrows indicate putative endoplasmicreticulum (ER) localization for ACS-2–GFP and ER, plasma membrane, and septal localization for ACS-3–GFP. For ACS-6 –GFP, arrows show plasmamembrane localization. Bars � 10 �m.

GFPdsRed or

MitoTracker Merged

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itoTr

acke

r R

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FIG 5 Colocalization of ACS proteins with fluorescent markers for different cellular structures. (A) Forced heterokaryon of �acs-2 ACS-2–sGFP plus �ridH1-dsRed strains. (B) Forced heterokaryon of �acs-3 ACS-3–sGFP plus �rid H1-dsRed strains. ACS-2–GFP and ACS-3–GFP localizes to the membranesurrounding the nuclei, as indicated by the H1-dsRed fluorescence, indicating endoplasmic reticulum localization. (C) �acs-4 strain ACS-4 –GFP colocalizeswith the mitochondria, as indicated by MitoTracker Red FM dye. Fluorescence images were obtaining by spinning disc confocal microscopy (see Materials andMethods). Bars � 10 �m.

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with an even greater increase in 16:0 and concomitant decrease inpolyunsaturated 18:2 and 18:3 FAs (Table 2). These data supportthe hypothesis of functional redundancy of ACS-2 and ACS-3 inactivating FAs for desaturation and elongation. Surprisingly, the�acs-1; �acs-3 mutant strain gave an intermediate fatty acid phe-notype compared to the �acs-3 single deletion strain (compareFig. 2C and D with Table 2). Thus, contrary to expectations, thedeletion of the major peroxisomal ACS, presumably activating FAfor �-oxidation, in the �acs-3 background resulted in a decreasein FA accumulation. No evidence of FA secretion was detected inthe extracellular medium for any of the double deletion strains.

As a means to determine how further perturbations in ACSactivity affected the �acs-5 strain, double deletion strains with�acs-2 and �acs-3 were constructed and evaluated for growth lagin liquid culture growth. As shown in Fig. 6, the �acs-2; �acs-5mutant exhibited a prolonged lag phase similar to the single�acs-5 deletion strain. However, the �acs-3; �acs-5 mutantshowed partial recovery of the lag in growth phenotype (Fig. 6)and exhibited nearly the same FA levels as the �acs-3 mutant (datanot shown). These data are consistent with the reduction in lagphase when the �acs-5 mutant was grown in fatty acid-supple-mented media (see Fig. S6 in the supplemental material).

DISCUSSION

Fatty ACSs play an important role in lipid metabolism by catalyz-ing the formation of acyl-CoA esters. In addition to activating FAsfor degradation via �-oxidation or for biosynthesis of glycerolip-

ids, acyl-CoA esters have been reported to act as regulators ofvarious intracellular functions and in cell signaling (52). In thiswork, we identified seven putative fatty ACSs encoded in the ge-nome of N. crassa; these enzymes are predicted to activate specificFA pools for various intracellular processes. We took a global per-spective on the role of ACS in lipid metabolism by characterizingthe FA content and composition in acs deletion strains in N. crassaand assessed cellular localization, thus providing further insightsfor FA elongation and desaturation pathways (Table 3). Studies inother fungi have taken a more focused approach to analyze ACSinvolvement in N-myristoylation (44, 53), FA import (54), and�-oxidation (21).

Subcellular localization provides an excellent starting point fordeciphering the physiological role of ACSs, as it reflects the loca-tion of the pathway for which it is providing activated FAs. Tofurther support our finding for the physiological role of the N.crassa ACSs and broadly define the role of ACSs in fungal metab-olism, we compared our results with S. cerevisiae and A. nidulansACS homologs (Fig. 7). ACS homologs were identified from thesethree species in all clades, with the exception of the absence of arepresentative member from S. cerevisiae in family 1 (Fig. 1A and7). While originally these clades were named based on known orpredicted FA specificity of the ACS, we find a high conservation ofsubcellular localization (Fig. 7).

Three of the four clades contain members that localize to theperoxisome or glyoxysome, where the glyoxysome contains only asubset of the peroxisomal proteins. However, both of these organ-elles house the machinery required for FA �-oxidation. N. crassa,A. nidulans, and S. cerevisiae each have one ACS within the LCclade that localizes to the peroxisome/glyoxysome (Fig. 7 and 8).Sc-Faa2p activates MC FAs for degradation within the peroxisome(55), while an A. nidulans �faaB strain exhibits a growth defect onshort-, medium-, and long-chain FAs (21). The N. crassa �acs-1strain exhibited a growth defect on lauric acid (12:0) as the solecarbon source, even when supplemented with lactose, both withand without FA biosynthesis inhibition. These results indicate thatACS-1 mediates toxicity resistance to lauric acid by activating itfor degradation, similar to FaaB (21).

S. cerevisiae and N. crassa contain several additional ACSswithin the LC clade (Sc-Faa1p, Sc-Faa3p, Sc-Faa4p, Nc-ACS-2,and Nc-ACS-3), while A. nidulans only has one (An-FaaA). ACSsin this subclade exhibit broad cellular localization (Fig. 7). S.cerevisiae faa1� and faa4� strains exhibit reduced �-oxidation ofoleic acid (18:1) compared to the wild-type strain, indicating arole for Faa1p and Faa4p in FA catabolism (54). Although Sc-Faa1p and -Faa4p provide FA-CoA for �-oxidation, the more cen-tral physiological roles of these proteins is in FA uptake via vecto-

FIG 6 Biomass accumulation of �acs-5 single- and multiple-gene deletionstrains in VM–2% glucose. All values are representative of triplicate biologicalsamples. Error bars indicate standard deviations.

TABLE 2 Fatty acid composition at 60 h growth on VM–2% glucose

StrainFAME amt(�g/mg biomass)

% (of total FAs) of FA:

16:0 16:1 18:0 18:1 18:2 18:3

WT 51.04 � 2.7 17.26 � 0.57 2.27 � 0.09 2.79 � 0.72 12.3 � 0.63 56.41 � 0.82 6.33 � 0.49�acs-1 58.22 � 3.39 17.34 � 0.78 1.91 � 0.14 3.12 � 0.30 11.76 � 1.15 57.67 � 1.41 5.38 � 0.30�acs-2 55.58 � 1.54 17.14 � 0.42 2.16 � 0.05 3.44 � 0.34 12.83 � 0.94 56.11 � 0.83 5.66 � 0.25�acs-3 86.81 � 4.42 24.28 � 0.38 1.06 � 0.08 7.12 � 0.36 21.26 � 0.78 41.88 � 0.89 1.52 � 0.19�acs-1 �acs-2 57.55 � 1.71 16.67 � 0.57 2.01 � 0.04 2.91 � 0.19 13.57 � 0.53 57.74 � 0.69 5.68 � 0.28�acs-1 �acs-3 68.13 � 1.56 19.63 � 0.49 1.02 � 0.03 4.01 � 0.17 17.18 � 1.07 54.66 � 0.84 2.35 � 0.3�acs-3 �acs-2 84.83 � 21.05 33.52 � 0.31 1.59 � 0.03 5.61 � 0.36 18.05 � 0.20 38.53 � 0.30 0.5 � 0.04

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rial acylation and activation of exogenous FA for anabolic lipidmetabolic processes such as N-myristoylation (53, 54, 56, 57).Here, we showed that ACS-2 and ACS-3 were broadly distributedthroughout the fungal cell in the cytoplasm and membrane-bound compartments, particularly the ER, as well as the plasmamembrane and septa. The ER localization pattern indicates thatACS-2 and ACS-3 are FA activators for anabolic lipid metabolicpathways, since the ER is the site of complex lipid assembly, FAdesaturation, and FA elongation (Fig. 8). The N. crassa �acs-2strain did not exhibit any growth or lipid phenotype comparedwith the wild-type strain, while the N. crassa �acs-3 strain dis-played the most pronounced lipid phenotype of all of the N. crassa�acs mutants. Like Sc-Faa1p, Nc-ACS-3 provides the prominentACS activity for exogenous FA fed into anabolic pathways, whichis also consistent with its plasma membrane localization. The A.nidulans member of this clade, encoded by faaA, was not evaluated

for a role in anabolic activation; we predict that the A. nidulans�faaA strain would exhibit a phenotype similar to that of the N.crassa �acs-3 mutant.

One of the most prominent phenotypes observed in the N.crassa �acs-3 strain was the overaccumulation of FAs. Because acatabolism phenotype was not observed for the �acs-3 mutant onexogenous FA, the cause for increased lipid accumulation in the�acs-3 strain must be increased FA synthesis. The increased con-tent of saturated and shorter FAs of the �acs-3 mutant and evenmore so of the �acs-3; �acs-2 double mutant suggests the roles ofACS-2 and ACS-3 in FA activation for desaturation and elonga-tion, consistent with a findings on a homologous protein in theoleaginous yeast Yarrowia lipolytica (58). Increased FA biosynthe-sis may result from relief of feedback inhibition by acyl-CoA estersor possibly an attempt by the cell to fill an unmet need for unsat-urated FAs. However, when the �acs-3 strain was grown in media

TABLE 3 Overview of putative acyl-CoA synthetases

Protein Gene LocalizationAnabolic/catabolicpathway

Growth phenotype of �acs strain on: Lipid phenotype of �acs strain on:

Glucose FA Glucose FA

ACS-1 NCU01654 Glyoxysomea Catabolicc WT No growth on 12:0 WT NDg

ACS-2 NCU03929 ERe Anabolicd WT WT WT NDACS-3 NCU04380 ER, PM,f septa Anabolicc WT No growth on 12:0 175%;2polyunsat FAh WTACS-4 NCU00608 Mitochondria Catabolicd WT WT WT NDACS-5 NCU06063 Peroxisomeb Anabolic (siderophore) Prolonged lag WT Different early time 18:3 NDACS-6 NCU06032 PM, puncta Uncertain WT WT WT WTACS-7 NCU08935 Glyoxysomea Catabolicd WT WT WT NDa See reference 43.b Localized in A. nidulans (46).c Determined by assay.d Predicted based on localization.e ER, endoplasmic reticulum.f PM, plasma membrane.g ND, not determined.h Total FA increased by 75%; polyunsaturated FAs decreased compared to the wild-type strain.

FIG 7 Comparison of localization patterns and physiological roles of N. crassa, A. nidulans, and S. cerevisiae ACSs that clustered by phylogeny. See Materials andMethods for tree construction. Black circles, �95% bootstrap support; gray circles, 80 to 95%; white circles, 50 to 80%.

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supplemented with FA, the mutant no longer accumulated lipidabove wild-type levels, supporting the hypothesis of feedback in-hibition relief. Our results differ from the FA secretion phenotypeobserved in the S. cerevisiae �faa1 and �faa1 �faa4 strains (27,28). This indicates a different capacity of N. crassa to store excesslipid, which is more consistent with oleaginous microorganisms(58).

The partial compensation of activation of VLC FAs for ana-bolic pathways is observed in the N. crassa �acs-3 strain, and wepredict that ACS-6 provides this activity. The most well-charac-terized member of the VLC clade of ACS is S. cerevisiae Fat1p. In S.cerevisiae, Fat1p was shown both to activate VLC FAs for �-oxi-dation and to be required for uptake of LC FAs from the environ-ment; both functions are supported by subcellular localization ofFat1p to the plasma membrane and lipid bodies (59, 60). How-ever, N. crassa ACS-6 was not required for growth on exogenouslysupplied FA, similar to A. nidulans �fatA, �fatB, and �fatC mu-tants (21). ACS-6 –GFP localized to the plasma membrane insome hyphae but was more frequently observed in puncta. Whilethe plasma membrane localization is consistent with that of Fat1p,the puncta did not colocalize with the glyoxysome. These resultsindicate either that ACS-6 is nonfunctional for FA transport orthat another FA transport protein(s) exists.

The family 2 clade, which contains S. cerevisiae Fat2p, A. nidu-lans FatD, and N. crassa ACS-7, is poorly characterized. Sc-Fat2p,An-FatD and Nc-ACS-7 all localize to the peroxisome/glyoxy-some. However, the natural substrate of Sc-Fat2p has yet to beidentified (61). The A. nidulans fatD� (21) and N. crassa �acs-7mutants also do not exhibit a growth phenotype in any of theconditions evaluated, nor did we observe a lipid phenotype of the�acs-7 mutant when grown on either glucose or FA. Further workis needed to define the physiological role and biochemical speci-ficity of these ACSs.

In many eukaryotic cells, FA �-oxidation occurs in both theperoxisome (or glyoxysome) and the mitochondria. S. cerevisiaelacks mitochondrial �-oxidation, and until recently this was as-sumed to be true across the entire fungal kingdom. The character-

ization of both peroxisomal and mitochondrial �-oxidation path-ways in A. nidulans (11) led to a comparative genomic study of�-oxidation pathways across the fungal kingdom, which revealedthat both pathways exist in the majority of fungi (9, 62). Themitochondrial �-oxidation pathway is specific for short-chainFAs and has also been implicated in breakdown of the amino acidsisoleucine and valine (63, 64). Although enzyme activities for�-oxidation were not identified in the mitochondrial fraction ofN. crassa cell lysates (10), our data support the mitochondrial�-oxidation hypothesis. Subcellular localization of ACS-4 to mi-tochondria indicates an involvement of this protein in mitochon-drial �-oxidation and is consistent with previous data of oleate-induced mitochondrial ACS activity in N. crassa (65). In A.nidulans, FA degradation in the mitochondria is limited to short-,medium-, and long-chain FAs and is essential for growth onshort-chain FAs. The A. nidulans homolog of ACS-4 (AN4659)was suggested by in silico analyses to be a putative short-chain ACSthat localizes to mitochondria (21). N. crassa was unable to growon most short- and medium-chain FAs, and for those that Neuros-pora is able to utilize, the �acs-4 strain did not exhibit a growthdefect. One possible explanation for these observations is ACS-4 isactive against endogenous pools of short- and/or medium-chainFAs resulting from partial �-oxidation of long-chain FAs in theglyoxysome.

Within the family 1 ACS subclade, ACS-5 clusters with A. ni-dulans AN0609, whose gene is an ortholog of A. fumigatus sidl,which is required for the TAFC siderophore biosynthesis (45).Growth phenotypes observed for the �acs-5 mutant strain areconsistent with ACS-5 involvement in siderophore biosynthesis inN. crassa. Supplementation experiments further supported thishypothesis and specifically the role in biosynthesis of the N. crassaextracellular iron scavenger coprogen (47). Thus, members ofACS family 1 exhibit divergent functions, and some do not func-tion primarily to activate FAs.

Cellular localization through proteomics (43) and microscopyreveal that, in N. crassa, ACSs are found in glyoxysomes, micro-somes, PM, ER, and mitochondria, which reflects the pathway for

FIG 8 Cellular localization and schematic representation of fatty acid activation by ACSs in N. crassa for anabolic and catabolic lipid metabolic pathways. ACS-5likely activates mevalonate for siderophore biosynthesis.

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which the ACSs are providing activated substrate: complex lipidbiosynthesis and FA modification in the ER and FA �-oxidation inthe glyoxysome and mitochondria (Fig. 8). It is clear that there isfunctional redundancy among the seven ACSs in N. crassa foractivation of exogenous FA for degradation; however, one, ACS-3,is the predominant FA activator for anabolic pathways. In addi-tion to elucidating lipid metabolic pathways specific for ACS func-tion, we identified candidate �acs strains that exhibited a disrup-tion in FA equilibrium, altered FA profile, and overaccumulationof FAs. These results can inform future work toward tailoring FAhyperaccumulation for applications such as biofuel and biochem-ical production.

ACKNOWLEDGMENTS

This work was supported by a grant from the Energy Biosciences toD.S.C., H.W.B., and N.L.G. C.M.R. is supported in part by the Depart-ment of Energy Office of Science Graduate Fellowship Program, madepossible in part by the American Recovery and Reinvestment Act of2009, administered by ORISE-ORAU under contract no. DE-AC05-06OR23100. We acknowledge use of materials generated by P01GM068087 (Functional Analysis of a Model Filamentous Fungus).

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