Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae.

The functional expression in yeast of the Arabidopsis thaliana FAD2 gene, encoding the extraplastidial oleate desaturase (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase) is reported. Dienoic fatty acids constituted up to 11% (w/w) of the total fatty acids in transformed Saccharomyces cerevisiae cells and were confirmed to be linoleic acid and delta 9, delta 12-hexadecadienoic acid by gas chromatography-mass spectrometry.     

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 Delta(9) position. We expressed two sunflower (Helianthus annuus) oleate Delta(12) 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 18:2Delta(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 degrees 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 degrees C or 15 degrees 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.     

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.     

玉米△12脂肪酸脱氢酶基因（FAD2）在酿酒酵母中的表达

玉米△12脂肪酸脱氢酶是催化油酸形成亚油酸的关键酶。将其编码基因FAD2（GenBank登陆号：DQ496227）克隆到酿酒酵母表达载体pYES2．0中,构建成重组质粒pYE／FAD2,转化到酿酒酵母进行诱导表达,同时以pYES2．0转化子为对照。气相色谱（Gc）分析表明,重组转化子亚油酸的含量占酵母总脂肪酸的1．54％,而对照未检测到亚油酸。表明FAD2基因具有编码△12脂肪酸脱氢酶的功能。为探索转译起始密码子周边序列的改变对FAD2基因表达产生的影响,将该基因的起始密码子上游序列进行修改,构建重组表达载体pYE／FAD2—1,转化酿酒酵母进行表达。GC分析表明,pYE／FAD2—1转化子的亚油酸含量占总脂肪酸含量的8．81％,是对照pYE／FAD2转化子的近5倍。     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

Hormonal regulation of tissue-specific ectopic expression of an Arabidopsis endoplasmic reticulum-type ω-3 fatty acid desaturase (FAD3) gene

A common feature of the membrane lipids of higher plants is a large content of polyunsaturated fatty acids, which typically consist of dienoic and trienoic fatty acids. Two types of omega-3 fatty acid desaturase. which are present in the plastids and in the endoplasmic reticulum (ER), respectively, are responsible for the conversion of dienoic to trienoic fatty acids. To establish a system for investigating the tissue-specific, and hor-mone-regulated expression of the ER-type desaturase gene (FAD3), transgenic plants of Arabidopsis thaliana (L.) Heynh. containing the firefly luciferase gene (LUC) fused to the FAD3 promoter (FAD3::LUC) were constructed. At different times during plant development, FAD3::LUC was actively expressed at two major sites, the vegetative shoot meristem and the floral organs. Transgenic plants with LUC fused to the promoter of FAD7 (FAD7::LUC) which encodes plastid-type desaturase, were also constructed. FAD3::LUC and FAD7::LUC were expressed in the same organs during reproductive growth, but not during vegetative growth. In plants exposed to both auxin and cytokinin, FAD3::LUC expression was ectopically induced in the root tissues. However, this induction by auxin and cytokinin was inhibited when abscisic acid was also present. FAD3::LUC expression could be induced in the roots by auxin and cytokinin if the hormones were applied during vegetative growth, but not if they were applied during germination or reproductive growth. Analysis of the fatty acid composition in the roots of Arabidopsis fad mutant and wild-type plants confirmed that the response of FAD3::LUC expression to various hormones reflected the response of endogenous FAD3 gene expression. These results suggest that the expression of ER-type desaturase is regulated through synergistic and antagonistic hormonal interactions, and that such hormonal regulation and the tissue specificity of the expression of this gene are further modified in accordance with the growth phase in plant development.     

Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds

The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid beta-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 ( FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.     

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.     

Polyunsaturated fatty acid biosynthesis in Saccharomyces cerevisiae: expression of ethanol tolerance and the FAD2 gene from Arabidopsis thaliana.

The Arabidopsis thaliana delta-12 fatty acid desaturase gene (FAD2) was overexpressed in Saccharomyces cerevisiae by using the GAL1 promoter. S. cerevisiae harboring the FAD2 gene was capable of forming hexadecadienoyl (16:2) and linoleoyl (18:2) residues in the membrane lipid when cultured in medium containing galactose. Gas-liquid chromatography analysis of total lipids indicated that the transformed S. cerevisiae accumulated these dienoic fatty acyl residues and that they accounted for approximately 50% of the total fatty acyl residues. Phospholipid analysis of this strain indicated that the oleoyl (18:1) residue binding phosphatidylcholine (PC) was mostly converted to the 18:2 residue binding PC, whereas 50% of the palmitoleoyl (16:1) residue binding PC was converted to the 16:2 residue binding PC. A marked effect on the unsaturation of 16:1 and 18:1 was observed when S. cerevisiae harboring the FAD2 gene was cultured at 8 degrees C. To assess the ethanol tolerance of S. cerevisiae producing polyunsaturated fatty acids, the cell viability of this strain in the presence of ethanol was examined. The results indicated that S. cerevisiae cells overexpressing the FAD2 gene had greater resistance to 15% (vol/vol) ethanol than did the control cells.     

植物脂肪酸去饱和酶及其编码基因研究进展

脂肪酸去饱和酶是催化脂肪酸链特定位置形成双键和产生不饱和脂肪酸的酶类。植物脂肪酸去饱和酶主要有5种(FAD2、FAD3、FAD6、FAD7和FAD8), 可分为ω-3型 (FAD2、FAD6)和ω-6型(FAD3、FAD7、FAD8)两大类。其编码基因(FAD2、FAD3、FAD6、FAD7和FAD8)在植物中一般有多个拷贝。同种基因在不同植物中拷贝数不同, 同一植物中相同基因的不同拷贝间在序列特征、表达调控和功能等方面也存在显著差异。本文根据国内外对脂肪酸去饱和酶基因及编码产物的研究现状, 分别从它们的分类、拷贝数、结构、作用机制、表达调控等方面的研究进展进行了详细的分类阐述。     

Ozone-induced expression of the Arabidopsis FAD7 gene requires salicylic acid, but not NPR1 and SID2

The Arabidopsis FAD7 gene encodes a plastid omega-3 fatty acid desaturase that catalyzes the desaturation of dienoic fatty acids to trienoic fatty acids in chloroplast membrane lipids. The expression of FAD7 was rapidly and locally induced by ozone exposure, which causes oxidative responses equivalent to pathogen-induced hypersensitive responses and subsequently activates various defense-related genes. This induction was reduced in salicylic acid (SA)-deficient NahG plants expressing SA hydroxylase, but was unaffected in etr1 and jar1 mutants, which are insensitive to ethylene and jasmonic acid (JA), respectively. The SA dependence of the FAD7 induction was confirmed by the exogenous application of SA. SA-induced expression of FAD7 in the npr1 mutant which is defective in an SA signaling pathway occurred to the same extent as in the wild type. Furthermore, in the sid2 mutant which lacks an enzyme required for SA biosynthesis, the expression of FAD7 was induced by ozone exposure. These results suggest that the ozone-induced expression of FAD7 gene requires SA, but not ethylene, JA, NPR1 and SID2.     

Molecular cloning and functional expression of the gene for a cotton Delta-12 fatty acid desaturase (FAD2).

Two overlapping genomic clones spanning 16.5 kb of cotton DNA were found to encompass a Delta-12 fatty acid desaturase (FAD2-3) gene. A partial FAD2-3 cDNA clone was also analyzed. The FAD2-3 gene has one large intron of 2967 bp entirely within its 5'-untranslated region, only 12 bp upstream from the ATG initiation codon. Several potential promoter elements, including several light-responsive motifs, occur in the 5'-flanking region. The continuous FAD2-3 coding region is 1155 bp and would encode a protein of 384 amino acids. The polypeptide has four putative membrane-spanning helices, indicative of an integral membrane protein, and is most likely localized in the endoplasmic reticulum. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.     

Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared toward the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically expressed enzymes. Here, we show that the addition of an N-terminal epitope tag sequence (either Myc or hemagglutinin) to oleate desaturase (FAD2) or omega-3 linoleate desaturase (FAD3) enzymes from plants, which catalyze consecutive reactions in the production of long chain omega-3 fatty acids, significantly increases their activity up to fourfold when expressed in yeast cells. Quantitative protein blotting using an antibody specific for native FAD2 revealed that the steady-state amount of the epitope-tagged FAD2 protein was also approximately fourfold higher than that of its untagged counterpart, demonstrating a direct relationship between the epitope tag-induced increase in enzyme amount and fatty acid product formation. Protein half-life and RNA blotting experiments indicated that the half-lives and mRNA content of the tagged and untagged FAD2 proteins were essentially the same, suggesting that the epitope tags increased protein abundance by improving translational efficiency. Taken together, these results indicate that the addition of an epitope tag sequence to a plant fatty acid desaturase (FAD) not only provides a useful means for protein immunodetection using highly specific, commercially available antibodies, but that it also significantly increases FAD activity and the production of polyunsaturated fatty acids in yeast cells.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.     

Direct Evaluation of Effects of Fatty-Acid Unsaturation on the Thermal Properties of Photosynthetic Activities, as Studied by Mutation and Transformation of Synechocystis PCC6803

Glycerolipids of thylakoid membranes isolated from the cyanobacteriumSynechocystis PCC6803 contained high levels of dienoic and trienoicC₁₈ fatty acids, in addition to saturated C₁₆ and monoenoicC₁₈ fatty acids. A mutant (Fadl2) of this cyanobacterium wasdefective in the desaturation of C₁₈ fatty acids at the ¹² position,and its thylakoid membranes lacked trienoic acids and containeda very reduced level of dienoic acids. A derivative strain ofFadl2 (Fadl2/desA), which had been transformed with a gene fordesaturation at the ¹² position, fully recovered the abilityto desaturate the fatty acids in the glycerolipids of thylakoidmembranes. The thermal properties of the photosynthetic activitiesof the mutant and the transformant were compared with thoseof the wild-type strain. Despite great diversity in the extentof unsaturation of fatty acids between the wild-type, Fadl2,and Fad12/desA strains, no significant differences were foundeither in the temperature dependence of photosynthesis or inthe heat stability of photosynthetic, photosystem II and photosystemI activities. These results demonstrate that the trienoic fattyacids and, probably, the dienoic acids of the lipids in thethylakoid membrane do not affect the thermal properties of theabove-mentioned activities of photosynthesis. ³Permanent address: Institute of Plant Physiology, BiologicalResearch Center of Hungarian Academy of Sciences, H-6701 Szeged,P.O. Box 521, Hungary (Received August 9, 1990; Accepted December 7, 1990)     

Delta6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Gene cloning and its heterologous expression in a fungus, Aspergillus

A DNA fragment was cloned from the fungal strain, Mortierella alpina 1S-4 (which is used industrially to produce arachidonic acid), after PCR amplification with oligonucleotide primers designed based on the sequence information for delta6-desaturase genes (from borage and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (delta9, delta12-18:2) to gamma-linolenic acid (delta6, delta9, delta12-18:3). This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 457 amino acids from a M. calpina 1S-4 library. The predicted amino-acid sequence showed similarity to those of the above delta6-desaturases, and contained a cytochrome b5-like domain at the N-terminus, being different from the yeast delta9-desaturase which has the corresponding domain at the C-terminus. The full-length cDNA clone was expressed under the control of the amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) to the level of 25.2% of the total fatty acids. These findings revealed that the recombinant product has delta6-desaturase activity. The Mortierella delta6-desaturase is the first to be reported in fungi.     

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum)

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.