Atypical Biosynthetic Properties of a Δ12/ν+3 Desaturase from the Model Basidiomycete Phanerochaete chrysosporium

The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Δ¹²-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structural domains. In vivo activity of the heterologously expressed enzyme favors C₁₈ substrates with ν+3 regioselectivity, where the site of desaturation is three carbons carboxy-distal to the reference position of a preexisting double bond (ν). Linoleate accumulated to levels in excess of 12% of the total fatty acids upon heterologous expression of P. chrysosporium FAD2 in Saccharomyces cerevisiae. In contrast to the behavior of the plant FAD2 enzymes, this oleate desaturase does not 12-hydroxylate lipids and is the first example whose activity increases at higher temperatures (30°C versus 15°C). Thus, while maintaining the hallmark activity of the fatty acyl Δ¹²-desaturase family, the basidiomycete fad2 genes appear to have evolved substantially from an ancestral desaturase.Desaturases, the enzymes responsible for unsaturated fatty acid biosynthesis, are found throughout the eukaryotic taxa. Critical cellular processes dependent on the modification of acyl lipids by desaturases include the regulation of membrane structure and fluidity, proper function of ion channels and other membrane proteins, and the biosynthesis of signaling molecules, such as jasmonic acid and arachidonic acid-derived second messengers (53, 71). Polyunsaturated fatty acids (PUFAs) with double bonds at carbon-12, such as linoleic acid (18:2Δ9c,12c), are not synthesized by animals, who therefore depend upon the activities of the stepwise action of the Δ⁹- and Δ¹²-desaturases from plants and lower eukaryotes to generate these essential lipids.Supplementation of our diet with PUFAs derived from transgenic organisms has been targeted in recent years. Expression of fungal (37) and plant (56) desaturase genes in mammalian cells has been explored as a means to enhance the nutritional quality of meat products. Oleate and PUFA desaturases and elongases are gene targets sought after for transgenic production of the C₂₀ and C₂₂ polyunsaturated food supplements docosahexenoic and eicosapentenoic acids in alga, plants, and yeast (35, 51). The practical success of lipid metabolic engineering studies is dependent upon the expression of enzymes with high chemo- and regioselectivity within the transgenic organism, coupled with the manipulation of lipid biochemical flux to result in high, economically viable levels of unsaturated storage oil accumulation.Two evolutionarily distinct desaturase types exist: the soluble plastidal and the membrane-bound endoplasmic reticulum (ER)-localized enzymes, both of which use NAD(P)H and O₂ to sequentially abstract two hydrogens from vicinal sp³-hybridized carbons leading to a cis-alkene (15, 61). Current models for all fatty acyl desaturases postulate the activation of molecular oxygen at a nonheme diferrous active site that culminates with two C-H bond scissions and the formation of water (20, 29, 39, 46). In the case of the microsomal desaturases, conserved iron ligands appear to be located in three distinctive histidine box motifs (61). Microsomal Δ¹²-desaturases (FAD2s) are best known from plants, where they exhibit substantial (60 to 90%) sequence identity and have focused regioselectivity yet have evolved into the diverged desaturases that catalyze distinct oxidative processes, resulting in natural products with hydroxy, conjugated polyalkenyl, epoxy, and acetylenic functionalities (65). Studies of the FAD2 superfamily have been propelled by commercial interest in the modification of standard oilseed crops with “diverged” fad2 genes that show atypical regio- and/or chemoselectivity (32, 38, 50). Despite the sheer number of plant Δ¹²-desaturases, the refractory nature of these membrane-bound enzymes to purification has left structure/function relationships ill defined (33). Consequently, only the rough classification of the FAD2 enzymes into distinct functional or evolutionary classes has occurred, largely using genetic and in vivo functional characterization (12, 38, 42).While the Δ¹²- or FAD2 desaturases, which form a 12,13-double bond, are best known from plants, fungal fad2 homologs have been found in zygomycetes and ascomycetes (8, 52, 59). As suggested by molecular clock data, Basidiomycota and Ascomycotina diverged approximately 550 million years ago (3), indicating that metabolic basidiomycete genes may differ significantly from those of other fungal subtypes. With their high linoleic acid content, typically 60 to 80% of the lipid in basidiomycete fruiting bodies (63), and their ability to grow under varied temperature regimes, macrofungi provide an untapped genetic resource for desaturases that may be well suited for biotechnological applications. Indeed, two homobasidiomycete Δ¹²-desaturases have been recently reported (57, 77).In fungi, variations in membrane lipid composition caused by temperature cycling may be integral to the morphological changes of fruit body formation (58). Linoleate-derived hydroxy fatty acids and lactones have been shown to provide molecular signals, called Psi factors, involved in ascomycete sporulation (8, 9). Disruption of the oleoyl-phosphatidycholine desaturase odeA in Aspergillus parasiticus results in diminished growth; delayed germination has been proposed as a countermeasure for controlling this aflatoxigenic species (74). Additionally, volatile organic species emitted by fungi (e.g., (−)-1-octen-3-ol and 10-oxodecanoic acid) play a role in the palatability of mushrooms and may also mediate sporulation and the transition from vegetative to reproductive tissues (10). Separately, targeting Δ¹²-desaturases, which have no known homologs in humans, in pathogenic basidiomycetes has real potential as selective fungicidal targets. Cryptococcus neoformans infections in AIDS and immunosuppressed patients are frequently observed in the clinic; consequently, developing antimicrobial agents targeting C. neoformans will markedly improve the health of these patients (55).Phanerochaete chrysosporium is a widely distributed wood decay homobasidiomycete that has become a model system for studying lignocellulose degradation (41). It harbors an array of peroxidases and degrading lignocellulose as well as aromatic pollutants (14, 26). A role for linoleate (18:2), which may be supplied from endogenous wood lipids or through fungal Δ¹²-desaturation, in the mediation of lignin degradation has been suggested whereby diffusible lipid-derived peroxyl or alkoxy radicals aid in the initial decay of sound wood, particularly in white-rot fungi lacking lignin peroxidase (36, 73). The production of free 18:2 during early colonization of wood meal, followed by extracellular lipid peroxidation and in vitro degradation of nonphenolic lignin, has been shown for the white-rot fungus Ceriporiopsis subvermispora (16).As part of our program to elucidate the biosynthetic networks leading to highly unsaturated natural products in basidiomycetes (e.g., the polyacetylenes) (45), we carried out the cloning and sequence analysis of the gene encoding the sole Δ¹²-desaturase from P. chrysosporium. In this paper, we demonstrate its function through heterologous expression in Saccharomyces cerevisiae and show that this enzyme has features distinct from other fungal and plant FAD2 desaturases, which should facilitate future isolation and structure-function analysis of diverged macrofungal desaturases.     

Regioselectivity mechanism of the Thunbergia alata Δ6-16:0-acyl carrier protein desaturase

Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ⁹cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ⁶-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ⁶ desaturase to Δ⁹ regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ⁶ to Δ⁹ selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ⁹ to Δ⁶ selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ⁶ position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

Predictions regarding the mechanism of Δ⁶ regioselectivity of the Thunbergia alata desaturase based on X-ray crystallography and molecular dynamics simulations are confirmed by experiment.     

Functional Diversity in Fungal Fatty Acid Synthesis: THE FIRST ACETYLENASE FROM THE PACIFIC GOLDEN CHANTERELLE,CANTHARELLUS FORMOSUS*

Acetylenic specialized metabolites containing one or more carbon-carbon triple bonds are widespread, being found in fungi, vascular and lower plants, marine sponges and algae, and insects. Plants, moss, and most recently, insects, have been shown to employ an energetically difficult, sequential dehydrogenation mechanism for acetylenic bond formation. Here, we describe the cloning and heterologous expression in yeast of a linoleoyl 12-desaturase (acetylenase) and a bifunctional desaturase with Δ¹²-/Δ¹⁴-regiospecificity from the Pacific golden chanterelle. The acetylenase gene, which is the first identified from a fungus, is phylogenetically distinct from known plant and fungal desaturases. Together, the bifunctional desaturase and the acetylenase provide the enzymatic activities required to drive oleate through linoleate to crepenynate and the conjugated enyne (14Z)-dehydrocrepenynate, the branchpoint precursors to a major class of acetylenic natural products.     

A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat''s Claw Seed

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase.     

Molecular cloning and functional analysis of two FAD2 genes from American grape (Vitis labrusca L.)

The synthesis of polyunsaturated fatty acids (PUFAs), the most abundant fatty acids in plants, begins with a reaction catalyzed by fatty acid desaturase 2 (FAD2; EC 1.3.1.35), also called microsomal oleate Δ12-desaturase. Since the FAD2 gene was first identified in Arabidopsis thaliana, FAD2 research has gained wide interest as the essential enzyme for synthesizing PUFA. Grapes are one of the most frequently cultivated fruits in the world, with most commercial growers cultivating Vitis vinifera and V. labrusca. Grapeseed oil contains a high proportion, 60–70% of linoleic acid (18:2). We cloned two putative FAD2 genes from V. labrusca cv. Campbell Early based on V. vinifera genome sequences. Deduced amino acid sequences of two putative genes showed that VlFAD2s show high similarity to Arabidopsis FAD2 and commonly contain six transmembrane domain, three histidine boxes and endoplasmic reticulum (ER) retrieval motif representing the characteristics of fatty acid desaturase. Phylogenetic analyses of various plant FAD2s showed that VlFAD2-1 and VlFAD2-2 are separately grouped with constitutive and seed-type FAD2s, respectively. Southern blot showed that one or two bands are found in each lane. Because Campbell Early is a hybrid cultivar, FAD2-1 and FAD2-2 genes may exist as one copy in V. labrusca. Expression analysis in different tissues indicated that VlFAD2-1 is a constitutive gene but VlFAD2-2 is a seed-type gene. Complementation experiments of fad2-1 mutant Arabidopsis with VlFAD2-1 or VlFAD2-2 demonstrated that VlFAD2-1 and VlFAD2-2 can restore low PUFA proportion of fad2 to normal PUFA proportion.     

FAD2 and FAD3 Desaturases Form Heterodimers That Facilitate Metabolic Channeling in Vivo

Plant desaturases comprise two independently evolved classes, a structurally well characterized soluble class responsible for the production of monoenes in the plastids of higher plants and the poorly structurally characterized integral membrane class that has members in the plastid and endoplasmic reticulum that are responsible for producing mono- and polyunsaturated fatty acids. Both require iron and oxygen for activity and are inhibited by azide and cyanide underscoring their common chemical imperatives. We previously showed that the Δ⁹ acyl-CoA integral membrane desaturase Ole1p from Saccharomyces cerevisiae exhibits dimeric organization, like the soluble plastidial acyl-ACP desaturases. Here we use two independent bimolecular complementation assays, i.e. yeast two-hybrid analysis and Arabidopsis leaf protoplast split luciferase assay, to demonstrate that members of the plant integral membrane fatty acid desaturase (FAD) family, FAD2, FAD3, FAD6, FAD7, and FAD8, self-associate. Further, the endoplasmic reticulum-localized desaturase FAD2 can associate with FAD3, as can the plastid-localized FAD6 desaturase with either FAD7 or FAD8. These pairings appear to be specific because pairs such as FAD3 and FAD7 (or FAD8) and FAD2 and FAD6 do not interact despite their high amino acid similarity. These results are consistent also with their known endoplasmic reticulum and plastid subcellular localizations. Chemical cross-linking experiments confirm that FAD2 and FAD3 can form dimers like the yeast Ole1p and, when coexpressed, can form FAD2-FAD3 heterodimers. Metabolic flux analysis of yeast coexpressing FAD2 and FAD3 indicates that heterodimers can form a metabolic channel in which 18:1-PC is converted to 18:3-PC without releasing a free 18:2-PC intermediate.     

Abnormal n-6 fatty acid metabolism in cystic fibrosis is caused by activation of AMP-activated protein kinase

Cystic fibrosis (CF) patients and model systems exhibit consistent abnormalities in PUFA metabolism, including increased metabolism of linoleate to arachidonate. Recent studies have connected these abnormalities to increased expression and activity of the Δ6- and Δ5-desaturase enzymes. However, the mechanism connecting these changes to the CF transmembrane conductance regulator (CFTR) mutations responsible for CF is unknown. This study tests the hypothesis that increased activity of AMP-activated protein kinase (AMPK), previously described in CF bronchial epithelial cells, causes these changes in fatty acid metabolism by driving desaturase expression. Using CF bronchial epithelial cell culture models, we confirm elevated activity of AMPK in CF cells and show that it is due to increased phosphorylation of AMPK by Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ). We also show that inhibition of AMPK or CaMKKβ reduces desaturase expression and reverses the metabolic alterations seen in CF cells. These results signify a novel AMPK-dependent mechanism linking the genetic defect in CF to alterations in PUFA metabolism.     

Degradation of the γ-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4′-Ethoxy-3′-Methoxyphenyl)-2-(4″-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4′-ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [¹⁴C]lignin to ¹⁴CO₂. 1-(4′-Ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by α, β cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under ¹⁸O₂, ¹⁸O was incorporated into the β position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.     

Heterologous Production of Dihomo-γ-Linolenic Acid in Saccharomyces cerevisiae

To make dihomo-γ-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Δ12 fatty acid desaturase, rat Δ6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15°C than in those grown at 20°C, and no DGLA production was observed in the cells grown at 30°C. In NSD at 15°C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15°C, the yield of DGLA reached 2.19 μg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.     

Role of ςB in Heat, Ethanol, Acid, and Oxidative Stress Resistance and during Carbon Starvation in Listeria monocytogenes

To determine the contribution of sigma B (ς^B) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ΔsigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the ΔsigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ΔsigB strain. These results suggest the existence in L. monocytogenes of both a ς^B-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. ς^B contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The ΔsigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ΔsigB strain lost viability more rapidly than the parent strain. ς^B contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that ς^B plays a role in protecting L. monocytogenes against environmental adversities.     

A More Desirable Balanced Polyunsaturated Fatty Acid Composition Achieved by Heterologous Expression of Δ15/Δ4 Desaturases in Mammalian Cells

Arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway–a further elongation step from docosapentaenoic acid (DPA), a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage), coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line). Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs) in transgenic HEK293 cells made a more desirable fatty acid composition – a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health.     

Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

The replication of double-stranded plasmids containing a single N-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae. The strains used were proficient or deficient for the activity of DNA polymerase ζ (REV3 and rev3Δ, respectively) in a mismatch and nucleotide excision repair-defective background (msh2Δ rad10Δ). The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication. To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, ³²P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3Δ strains, the two strands of an unmodified heteroduplex plasmid were replicated in ~80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair. However, in the presence of the AAF adduct, TLS occurred in only 8% of the REV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in the REV3 strain was abolished in the rev3Δ mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.     

Fluidization of Membrane Lipids Enhances the Tolerance of Saccharomyces cerevisiae to Freezing and Salt Stress

Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the Δ⁹ position. We expressed two sunflower (Helianthus annuus) oleate Δ¹² desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2Δ^9,12, the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp⁺ or Trp⁻ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.     

The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.     

An alternate pathway to long-chain polyunsaturates: the FADS2 gene product ��8-desaturates 20:2n-6 and 20:3n-3

The mammalian Δ6-desaturase coded by fatty acid desaturase 2 (FADS2; HSA11q12-q13.1) catalyzes the first and rate-limiting step for the biosynthesis of long-chain polyunsaturated fatty acids. FADS2 is known to act on at least five substrates, and we hypothesized that the FADS2 gene product would have Δ8-desaturase activity. Saccharomyces cerevisiae transformed with a FADS2 construct from baboon neonate liver cDNA gained the function to desaturate 11,14-eicosadienoic acid (20:2n-6) and 11,14,17-eicosatrienoic acid (20:3n-3) to yield 20:3n-6 and 20:4n-3, respectively. Competition experiments indicate that Δ8-desaturation favors activity toward 20:3n-3 over 20:2n-6 by 3-fold. Similar experiments show that Δ6-desaturase activity is favored over Δ8-desaturase activity by 7-fold and 23-fold for n-6 (18:2n-6 vs 20:2n-6) and n-3 (18:3n-3 vs 20:3n-3), respectively. In mammals, 20:3n-6 is the immediate precursor of prostaglandin E1 and thromboxane B1. 20:3n-6 and 20:4n-3 are also immediate precursors of long-chain polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid, respectively. These findings provide unequivocal molecular evidence for a novel alternative biosynthetic route to long-chain polyunsaturated fatty acids in mammals from substrates previously considered to be dead-end products.     

Cytochrome b5 Coexpression Increases Tetrahymena thermophila Δ6 Fatty Acid Desaturase Activity in Saccharomyces cerevisiae

Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2^Δ9,12 substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b₅ protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b₅ domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b₅s were coexpressed with the desaturase. Coexpression of a cytochrome b₅ from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b₅ proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.