Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Delta(9)Z-C(16:1)) and oleic acid (Delta(9)Z-C(18:1)), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C(16:0)) and stearic acid (C(18:0)), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Delta(9) and Delta(11)) and substrate chain-length preferences (i.e., C(16:0) and C(18:0)); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Delta(11)Z-C(18:1)), whereas neither Delta(11)Z-C(16:1) nor palmitoleic acid (Delta(9)Z-C(16:1)) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Effects of sodium chloride concentration on phospholipid fatty acid composition of yeasts differing in osmotolerance

The effect of growth medium NaCl concentration on the fatty acid composition of phospholipids of 3 strains of Saccharomyces cerevisiae and 6 osmotolerant yeast strains was examined. The S. cerevisiae strains were characterized by a high content of palmitoleic (C_16:1) acid and by having no polyunsaturated C₁₈ acids, whereas the osmotolerant strains had a low content of C_16:1 and a high proportion of polyenoic C₁₈ acids. An increase of the NaCl concentration from 0% to 8% resulted in a decrease of the cellular phospholipid content on a dry-weight basis, for all strains but one of the osmotolerant strains. For the S. cerevisiae strains increased salinity produced a slight decrease of the proportion of C₁₆ fatty acids with a concomitant increase of C₁₈ acids, whereas the osmotolerant strains showed an increase of the relative content of oleic acid (C_18:1) at the expense of the proportion of polyenoic C₁₈ acids.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C₁₆ to C₂₀ fatty acids preferentially. It converted oleic acid (C_18:1) more efficiently than stearic acid (C_18:0) and linoleic acid (C_18:2) and much more effectively than α-linolenic acid (C_18:3). No products were detected when C₁₀ to C₁₂ fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C₁₄ to C₂₀ saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps.     

Biogenesis of mitochondria 19 the effects of unsaturated fatty acid depletion on the lipid composition and energy metabolism of a fatty acid desaturase mutant of

1. The lipid composition of a mutant ofSaccharomyces cerevisiae which cannot synthesize unsaturated fatty acid (UFA) can be extensively manipulated by growing the organism in the presence of added fatty acids.
2. Growth of the mutant is supported by a wide range of unsaturated fatty acids including oleic, palmitoleic, petroselenic, 11-eicosaenoic, ricinoleic, arachidonic, clupanodonic, linoleic and linolenic acids; 9- and 10-hydroxystearic acids support growth less effectively, but erucic, nervonic, elaidic and saturated fatty acids (C_8∶0?C_20∶0)^* are ineffective. All the fatty acids which support growth are incorporated into cell lipids, apparently without further metabolism.
3. The effects of altered lipid composition on the energy metabolism of yeast cells were investigated. Cells containing less than approximately 20% of their fatty acids as UFA cannot grow on non-fermentable substrates, and their growth on glucose is restricted to that which can be supported by fermentation alone.
4. UFA-depleted cells contain mitochondria which are apparently normal in morphology, furthermore they have normal levels of cytochromesa+a ₃,b,c ₁ andc and respire at normal rates. This suggests that the lesion in energy metabolism produced by UFA-depletion may be the loss of the ability of the mitochondria to couple respiration to phosphorylation.
5. UFA-depleted cells incorporate added UFA into their cell lipids and subsequently regain the ability to grow on non-fermentable substrates, showing that the lesion in energy metabolism is fully reversible.

     

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.     

Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth

The inositol 1,3,4,5,6-pentakisphosphate (IP₅) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP₆) in eukaryotic cells. Previous studies have shown that IP₆ is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1⁺ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1⁺ deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP₆ production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1⁺ function, encoding an IP₆ kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1⁺ upstream of ipk1⁺. We propose that IP₆ production is a key signaling linchpin for regulating multiple essential cellular processes.     

Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺), was expressed in the gpd1Δ gpd2Δ strain, anaerobic growth was restored by supplementation with 2.0 g liter⁻¹ acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).Bioethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology. A global research effort is under way to expand the substrate range of S. cerevisiae to include lignocellulosic hydrolysates of nonfood feedstocks (e.g., energy crops and agricultural residues) and to increase productivity, robustness, and product yield (for reviews see references 20 and 35). A major challenge relating to the stoichiometry of yeast-based ethanol production is that substantial amounts of glycerol are invariably formed as a by-product (24). It has been estimated that, in typical industrial ethanol processes, up to 4% of the sugar feedstock is converted into glycerol (24). Although glycerol also serves as a compatible solute at high extracellular osmolarity (10), glycerol production under anaerobic conditions is primarily linked to redox metabolism (34).During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD⁺-dependent alcohol dehydrogenase. The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD⁺ to NADH occurs elsewhere in the metabolism. Such a net production of NADH occurs in assimilation when yeast biomass is synthesized from glucose and ammonia (34). Under anaerobic conditions, NADH reoxidation in S. cerevisiae is strictly dependent on reduction of sugar to glycerol (34). Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol-3-phosphate, a reaction catalyzed by NAD⁺-dependent glycerol-3-phosphate dehydrogenase. Subsequently, the glycerol-3-phosphate formed in this reaction is hydrolyzed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate.The importance of glycerol production for fermentative growth of yeasts was already observed in the 1960s during studies of non-Saccharomyces yeasts that exhibit a so-called “Custers effect.” In such yeast species, which are naturally unable to produce glycerol, fermentative growth on glucose is possible only in the presence of an external electron acceptor that can be reduced via an NADH-dependent reaction (e.g., the reduction of acetoin to butanediol via NAD⁺-dependent butanediol dehydrogenase) (29). It was later shown that gpd1Δ gpd2Δ strains of S. cerevisiae, which are also unable to produce glycerol, are similarly unable to grow under anaerobic conditions unless provided with acetoin as an external electron acceptor (8).In view of its large economic significance, several metabolic engineering strategies have been explored to reduce or eliminate glycerol production in anaerobic cultures of S. cerevisiae. Nissen et al. (25) changed the cofactor specificity of glutamate dehydrogenase, the major ammonia-fixing enzyme of S. cerevisiae, thereby increasing NADH consumption in biosynthesis. This approach significantly reduced glycerol production in anaerobic cultures grown with ammonia as the nitrogen source. Attempts to further reduce glycerol production by expression of a heterologous transhydrogenase, with the aim to convert NADH and NADP⁺ into NAD⁺ and NADPH, were unsuccessful (24) because intracellular concentrations of these pyridine nucleotide cofactor couples favor the reverse reaction (23).The goal of the present study was to investigate whether the engineering of a linear pathway for the NADH-dependent reduction of acetic acid to ethanol can replace glycerol formation as a redox sink in anaerobic, glucose-grown cultures of S. cerevisiae and thus provide a stoichiometric basis for elimination of glycerol production during industrial ethanol production. Significant amounts of acetic acid are released upon hydrolysis of lignocellulosic biomass, and, in fact, acetic acid is studied as an inhibitor of yeast metabolism in lignocellulosic hydrolysates (5, 7, 26). The S. cerevisiae genome already contains genes encoding acetyl coenzyme A (acetyl-CoA) synthetase (32) and NAD⁺-dependent alcohol dehydrogenases (ADH1-5 [12]). To complete the linear pathway for acetic acid reduction, we expressed an NAD⁺-dependent, acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) from Escherichia coli into a gpd1Δ gpd2Δ strain of S. cerevisiae. This enzyme, encoded by the E. coli mhpF gene (15), catalyzes the reaction acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺. Growth and product formation of the engineered strain were then compared in the presence and absence of acetic acid and compared to those of a congenic reference strain.     

Efficient accumulation of oleic acid in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> caused by expression of rat elongase 2 gene (<Emphasis Type="Italic">rELO2</Emphasis>) and its contribution to tolerance to alcohols

When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.     

Expression of the Yeast Delta-9 Fatty Acid Desaturase in Nicotiana tabacum

To examine the processes of plant cytoplasmic fatty acid desaturation and glycerolipid biosynthesis, the protein coding sequence of the endoplasmic reticulum cytochrome b₅-dependent, Δ-9 fatty acid desaturase gene from Saccharomyces cerevisiae was introduced into Nicotiana tabacum via Agrobacterium transformation. All transformed plants expressing the yeast gene at the mRNA level exhibited an approximately 10-fold increase in the levels of palmitoleic acid (16:1) in leaf tissue. This fatty acid species is found in very low levels (less than 2%) in wild-type plants. These results indicate that the yeast desaturase can function in plants, presumably by using a leaf microsomal cytochrome b₅-mediated electron transport system. Lipid analysis demonstrated that the overproduced 16:1 is incorporated into most of the major polar lipid classes, including the cytoplasmically produced “eukaryotic” fraction of the chloroplast galactolipids. 16:1 was not found, however, in phosphatidyl glycerol, which is considered to be produced almost exclusively in the chloroplast. Despite these changes in membrane lipid composition, no obvious phenotypic differences were apparent in the transformed plants. Positional analysis shows that the cytoplasmically produced 16:1 is found primarily in the sn-2 position of phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol. The positional data suggest that the sn-2 acyltransferases responsible for the “eukaryotic” arrangement of 16- and 18- carbon fatty acids in glycerolipids are selective for unsaturated fatty acids rather than chain length.     

Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.     

Genome-Wide Screen for Oxalate-Sensitive Mutants of Saccharomyces cerevisiae

Oxalic acid is an important virulence factor produced by phytopathogenic filamentous fungi. In order to discover yeast genes whose orthologs in the pathogen may confer self-tolerance and whose plant orthologs may protect the host, a Saccharomyces cerevisiae deletion library consisting of 4,827 haploid mutants harboring deletions in nonessential genes was screened for growth inhibition and survival in a rich medium containing 30 mM oxalic acid at pH 3. A total of 31 mutants were identified that had significantly lower cell yields in oxalate medium than in an oxalate-free medium. About 35% of these mutants had not previously been detected in published screens for sensitivity to sorbic or citric acid. Mutants impaired in endosomal transport, the rgp1Δ, ric1Δ, snf7Δ, vps16Δ, vps20Δ, and vps51Δ mutants, were significantly overrepresented relative to their frequency among all verified yeast open reading frames. Oxalate exposure to a subset of five mutants, the drs2Δ, vps16Δ, vps51Δ, ric1Δ, and rib4Δ mutants, was lethal. With the exception of the rib4Δ mutant, all of these mutants are impaired in vesicle-mediated transport. Indirect evidence is provided suggesting that the sensitivity of the rib4Δ mutant, a riboflavin auxotroph, is due to oxalate-mediated interference with riboflavin uptake by the putative monocarboxylate transporter Mch5.     

Quality Control of Plasma Membrane Proteins by Saccharomyces cerevisiae Nedd4-Like Ubiquitin Ligase Rsp5p under Environmental Stress Conditions

In Saccharomyces cerevisiae, when a rich nitrogen source such as ammonium is added to the culture medium, the general amino acid permease Gap1p is ubiquitinated by the yeast Nedd4-like ubiquitin ligase Rsp5p, followed by its endocytosis to the vacuole. The arrestin-like Bul1/2p adaptors for Rsp5p specifically mediate this process. In this study, to investigate the downregulation of Gap1p in response to environmental stresses, we determined the intracellular trafficking of Gap1p under various stress conditions. An increase in the extracellular ethanol concentration induced ubiquitination and trafficking of Gap1p from the plasma membrane to the vacuole in wild-type cells, whereas Gap1p remained stable on the plasma membrane under the same conditions in rsp5^A401E and Δend3 cells. A ¹⁴C-labeled citrulline uptake assay using a nonubiquitinated form of Gap1p (Gap1p^K9R/K16R) revealed that ethanol stress caused a dramatic decrease of Gap1p activity. These results suggest that Gap1p is inactivated and ubiquitinated by Rsp5p for endocytosis when S. cerevisiae cells are exposed to a high concentration of ethanol. It is noteworthy that this endocytosis occurs in a Bul1/2p-independent manner, whereas ammonium-triggered downregulation of Gap1p was almost completely inhibited in Δbul1/2 cells. We also found that other environmental stresses, such as high temperature, H₂O₂, and LiCl, also promoted endocytosis of Gap1p. Similar intracellular trafficking caused by ethanol occurred in other plasma membrane proteins (Agp1p, Tat2p, and Gnp1p). Our findings suggest that stress-induced quality control is a common process requiring Rsp5p for plasma membrane proteins in yeast.     

Deletion of ELOVL6 blocks the synthesis of oleic acid but does not prevent the development of fatty liver or insulin resistance

Elongation of very long chain fatty acid-like family member 6 (ELOVL6) is a fatty acyl elongase that performs the initial and rate-limiting condensing reaction required for microsomal elongation of long-chain fatty acids. Our previous in vitro studies suggested that ELOVL6 elongated long-chain saturated fatty acids and monounsaturated fatty acids with chain lengths of 12 to 16 carbons. Here, we describe the generation and phenotypic characterization of Elovl6^−/− mice. As predicted from the in vitro studies, livers from Elovl6^−/− mice accumulated palmitic (C16:0) and palmitoleic (C16:1, n-7) fatty acids and contained significantly less stearic (C18:0) and oleic (C18:1, n-9) acids, confirming that ELOVL6 is the only enzyme capable of elongating palmitate (C16:0). Unexpectedly, Elovl6^−/− mice produced vaccenic acid (C18:1, n-7), the elongated product of palmitoleate (C16:1, n-7), suggesting that palmitoleate (C16:1, n-7) to vaccenate (C18:1, n-7) elongation was not specific to ELOVL6. The only detected consequence of deleting Elovl6^−/− in mice was that their livers accumulated significantly more triglycerides than wild-type mice when fed a fat-free/high-carbohydrate diet. When mice were fed a high-fat diet or ELOVL6 was deleted in ob/ob mice, the absence of ELOVL6 did not alter the development of obesity, fatty liver, hyperglycemia, or hyperinsulinemia. Combined, these results suggest that palmitoleic (C16:1, n-7) and vaccenic (C18:1, n-7) acids can largely replace the roles of oleic acid (C18:1, n-9) in vivo and that the deletion of ELOVL6 does not protect mice from the development of hepatic steatosis or insulin resistance.     

The Effects of Cutaneous Fatty Acids on the Growth of Pseudogymnoascus destructans,the Etiological Agent of White-Nose Syndrome (WNS)

White Nose Syndrome (WNS) greatly increases the over-winter mortality of little brown (Myotis lucifugus), Indiana (Myotis sodalis), northern (Myotis septentrionalis), and tricolored (Perimyotis subflavus) bats. It is caused by a cutaneous infection with the fungus Pseudogymnoascus destructans (Pd). Big brown bats (Eptesicus fuscus) are much more resistant to cutaneous infection with Pd, however. We thus conducted analyses of wing epidermis from hibernating E. fuscus and M. lucifugus to determine their fatty acid compositions, and laboratory Pd culture experiments at 4.0–13.4°C to determine the effects of these fatty acids on Pd growth. Our analyses revealed that the epidermis of both bat species contain the same 7 fatty acid types (14:0, 15:0, 16:0. 16:1, 18:0, 18:1, & 18:2), but the epidermis of M. lucifugus contains: a) more stearic (18:0) acid, b) less palmitoleic (16:1) acid, c) less myristic (14:0) acid, and, d) less oleic (18:1) acid than that of E. fuscus. The growth of Pd was inhibited by: a) myristic and stearic acids at 10.5–13.4°C, but not at 4.0–5.0°C, b) oleic acid at 5.0–10.6°C, c) palmitoleic acid, and, d) linoleic (18:2) acid at 5.0–10.6°C. One set of factors that enables E. fuscus to better resist cutaneous P. destructans infections (and thus WNS) therefore appears to be the relatively higher myristic, palmitoleic, and oleic acid contents of the epidermis.     

Oleaginous yeast Meyerozyma guilliermondii shows fermentative metabolism of sugars in the biosynthesis of ethanol and converts raw glycerol and cheese whey permeate into polyunsaturated fatty acids

We studied the biotechnological potential of the recently isolated yeast Meyerozyma guilliermondii BI281A to produce polyunsaturated fatty acids and ethanol, comparing products yields using glucose, raw glycerol from biodiesel synthesis, or whey permeate as substrates. The yeast metabolism was evaluated for different C/N ratios (100:1 and 50:1). Results found that M. guilliermondii BI281A was able to assimilate all tested substrates, and the most efficient conversion obtained was observed using raw glycerol as carbon source (C/N ratio 50:1), concerning biomass formation (5.67 g·L⁻¹) and lipid production (1.04 g·L⁻¹), representing 18% of dry cell weight. Bioreactors experiments under pH and aeration-controlled conditions were conducted. Obtained fatty acids were composed of ~67% of unsaturated fatty acids, distributed as palmitoleic acid (C_16:1, 9.4%), oleic acid (C_18:1, 47.2%), linoleic acid (C_{18:2 n−6}, 9.6%), and linolenic acid (C_{18:3 n−3}, 1.3%). Showing fermentative metabolism, which is unusual for oleaginous yeasts, M. guilliermondii produced 13.7 g·L⁻¹ of ethanol (yields of 0.27) when growing on glucose medium. These results suggest the promising use of this uncommonly studied yeast to produce unsaturated fatty acids and ethanol using cheap agro-industrial residues as substrates in bioprocess.     

Yeast engineering to express sex pheromone gland genes of the oriental fruit moth,Grapholita molesta

Mating disruption by using sex pheromone is an ecofriendly alternative way to control insect pests. To be effective, large amounts of sex pheromone are needed, leading to a relatively high production cost. To reduce the cost for chemical synthesis of sex pheromone, yeast engineering technology has been devised. This study used a baker's yeast, Saccharomyces cerevisiae, to express genes associated with sex pheromone biosynthesis of the Oriental fruit moth, Grapholita molesta. Compared to other fatty acid biosynthetic pathways, two steps that are unique to pheromone gland of G. molesta are proposed: desaturation at even number catalyzed by desaturase (Gm-DES) and terminal reduction catalyzed by fatty acyl reductase (Gm-FAR). Gm-DES and Gm-FAR were cloned into a yeast expression vector, pYES2.1. They were used to transform S. cerevisiae by a double transfection method. The transformed yeast was induced with 2% galactose to over-express these two exogenous genes. Their expression was confirmed by RT-PCR and western blotting. To facilitate pheromone production, transformed yeasts were supplied with myristic acid during over-expression. Resulting fatty acid composition was analyzed by GC-MS after fatty acid methyl ester derivatization. Control yeast produced mostly saturated fatty acids. However, a single gene (Gm-DES)-transformed yeast produced unsaturated fatty acids at ∆9 such as Z9-tetradecenoic acid (Z9-14:1), palmitoleic acid (Z9-16:1), and oleic acid (Z9-18:1) in addition to saturated fatty acids. The double-transformed yeast produced an additional component, alcohol form of oleic acid (Z9-18:OH). These results suggest that Gm-DES can catalyze desaturation of fatty acids at ∆9 and Gm-FAR can reduce terminal carboxylic acid into alcohol.     

Biosynthesis and Characterization of Polyhydroxyalkanoates Copolymers Produced by Pseudomonas putida Bet001 Isolated from Palm Oil Mill Effluent

The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C_8∶0) to oleic acid (C_18∶1) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the ¹H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T _d) of 264.6 to 318.8 (±0.2) ^oC, melting temperature (T _m) of 43. (±0.2) ^oC, glass transition temperature (T _g) of −1.0 (±0.2) ^oC and apparent melting enthalpy of fusion (ΔH _f) of 100.9 (±0.1) J g⁻¹.