Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes. These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.     

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.     

Barramundi (Lates calcarifer) desaturase with Δ6/Δ8 dual activities

Barramundi is a commercially farmed fish in Australia. To examine the potential for barramundi to metabolise dietary α-linolenic acid (ALA, 18:3 n-3), the existence of barramundi desaturase enzymes was examined. A putative fatty acid Δ6 desaturase was cloned from barramundi liver and expressed in yeast. Functional expression revealed Δ6 desaturase activity with both the 18 carbon (C(18)) and C(24) n-3 fatty acids, ALA and 24:5 n-3 as well as the C(18) n-6 fatty, linoleic acid (LA, 18:2 n-6). Metabolism of ALA was favoured over LA. The enzyme also had Δ8 desaturase activity which raises the potential for synthesis in barramundi of omega-3 (n-3) long chain polyunsaturated fatty acids from ALA via a pathway that bypasses the initial Δ6 desaturase step. Our findings not only provide molecular evidence for the fatty acid desaturation pathway in the barramundi but also highlight the importance of taking extracellular fatty acid levels into account when assessing enzyme activity expressed in Saccharomyces cerevisiae.     

Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase

Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase. We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase. For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable to convert exogenously supplied [2-(14)C]-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNA library of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expression in yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids. The enzyme showed strict Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.     

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Delta5-desaturase from Phytophthora megasperma

The biosynthesis of arachidonic acid (20:4(Delta5Z,8Z,11Z,14Z)) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-gamma-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from omega3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Delta5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Delta5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Delta8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.     

Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes

According to Ohba et al. (Ohba, M., Sato, R., Yoshida, Y., Nishino, T. and Katsuki, H. (1978) Biochem. Biophys. Res. Commun. 85, 21-27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of [1,7,15,22,26,30-14C]lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) in the presence of NADPH, NAD+ and molecular oxygen, concomitant with the liberation of 14CO2 derived from C-30 (one of the two methyl groups at the C-4 position). In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system. In this study it was found that the 14CO2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b5 and by palmitoyl-CoA, a substrate of the cytochrome b5-containing fatty acyl-CoA desaturase system of yeast microsomes. However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol). It is concluded that cytochrome b5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14 alpha-demethylation of lanosterol, by yeast microsomes. It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation. The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation.     

Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae

The substrate specificity and regioselectivity of the Brassica napus extraplastidial linoleate desaturase (FAD3) was investigated in vivo in a heterologous expression system. A strain of the yeast Saccharomyces cerevisiae producing the plant enzyme was constructed and cultured in media containing a variety of fatty acids. The products of desaturation of these potential substrates were determined by gas chromatographic and mass spectrometric analysis of the yeast cultures. The results indicate that the enzyme has: (a) omega-3, as opposed to Delta-15 or double-bond-related regioselectivity, (b) the ability to desaturate substrates in the 16 to 22 carbon range, (c) a preference for substrates with omega-6 double bonds, but the ability to desaturate substrates with omega-6 hydroxyl groups or omega-9 or omega-5 double bonds, and (d) a relative insensitivity to double bonds proximal to the carboxyl end of the substrate.     

Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris.

The hypothesis that the Delta9 desaturase of Chlorella vulgaris might operate by a synchronous mechanism has been tested using a kinetic isotope effect (KIE) approach. Thus the intermolecular primary deuterium KIE on the individual C-H bond cleavage steps involved in Delta9 desaturation have been determined by incubating growing cultures of C. vulgaris (strain 211/8K) with mixtures of the appropriate regiospecifically deuterated fatty acid analogues. Our analysis shows that the introduction of a double bond between C-9 and C-10 occurs in two discrete steps as the cleavage of the C9-H bond is very sensitive to isotopic substitution (kH/kD = 6.6 +/- 0.3) whereas a negligible isotope effect (kH/kD = 1.05 +/- 0.05) was observed for the C10-H bond-breaking step. Similar results were obtained for linoleic acid biosynthesis (Delta12 desaturation). These data clearly rule out a synchronous mechanism for these reactions.     

Biosynthesis of 10,12-dienoic fatty acids by a bifunctional Delta11 desaturase in Spodoptera littoralis

In the biosynthetic pathway of Spodoptera littoralis sex pheromone, (E,E)-10,12-tetradecadienoic acid is produced from (Z)-11-tetradecenoic acid by desaturation and concomitant migration of the precursor double bond. With the aim of identifying the enzyme involved in this biotransformation, yeast Deltaelo1/Deltaole mutants, which are both elongase 1 and Delta9 desaturase-deficient, were transformed with the S. littoralis Delta11 desaturase gene using a Cu+2 inducible expression vector. The transformants produced a recombinant polyhistidine-tagged Delta11 desaturase that could be detected by immunoblotting from cell lysates. Lipid analysis revealed that besides producing large quantities of C11-monounsaturated fatty acids, mainly (Z)-11-hexadecenoic acid, (E,E)-10,12-tetradecadienoic acid and minor amounts of (E,Z)-10,12-hexadecadienoic acid were also produced, as well as very low quantities of another tetradecadienoate, which was tentatively identified as the (E,Z)-10,12-tetradecadienoic isomer. None of these dienes was detected with the Delta11 desaturase gene of Trichoplusia ni, which does not produce conjugated dienes as pheromone components. We conclude that the Delta11 desaturase of S. littoralis is a bifunctional enzyme with both Delta11 and Delta10,12 desaturation activities. The relationship between the substrate structure and the stereochemical outcome of the reaction is discussed.     

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.     

delta5 desaturation of fatty acids in L-M cells

L-M cells grown in a lipid-free medium containing ¹⁴C-labeled 9,12-linoleic acid incorporated most of this acid into glycerolipids as linoleic acid. Only a small amount (3%) was elongated to eicosadienoic acid. No Δ6 desaturation occurred. When the cells were incubated with ¹⁴C-labeled 8, 11, 14-eicosatrienoic acid, 22% of the activity was found in 5,8,11,14-eicosatetraenoic acid. Treatment of the cells for 24 hr with N-isopropylethanolamine, a choline analog, depressed this desaturation reaction to about 60% of control values. The identity of the tetraene product was established by two different chromatographic analyses of the fatty acid methyl esters. Location of the double bond at position C-5 was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas-liquid chromatography. These results prove that L-M cells have a Δ5 desaturase and an elongation enzyme converting 18:2 to 20:2, but lack a Δ6 desaturase.     

Desaturation,chain scission,and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase

Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K(M) values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to approximately 3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.     

Role of hydrophobic partitioning in substrate selectivity and turnover of the ricinus communis stearoyl acyl carrier protein delta(9) desaturase.

Stearoyl acyl carrier protein Delta(9) desaturase (Delta9D) uses a diiron center to catalyze the NADPH- and O(2)-dependent desaturation of stearoyl acyl carrier protein (ACP) to form oleoyl-ACP. The reaction of recombinant Ricinus communis Delta9D with natural and nonnatural chain length acyl-ACPs was used to examine the coupling of the reconstituted enzyme complex, the specificity for position of double-bond insertion, the kinetic parameters for the desaturation reaction, and the selectivity for acyl chain length. The coupling of NADPH and O(2) consumption and olefin production was found to be maximal for 18:0-ACP, and the loss of coupling observed for the more slowly desaturated acyl-ACPs was attributed to autoxidation of the electron-transfer chain. Analysis of steady-state kinetic parameters for desaturation of acyl-ACPs having various acyl chain lengths revealed that the K(M) values were similar ( approximately 2.5-fold difference) for 15:0-18:0-ACP, while the k(cat) values increased by approximately 26-fold for the same range of acyl chain lengths. A linear increase in log (k(cat)/K(M)) was observed upon lengthening of the acyl chain from 15:0- to 18:0-ACP, while no further increase was observed for 19:0-ACP. The similarity of the k(cat)/K(M) values for 18:0- and 19:0-ACPs and the retained preference for double-bond insertion at the Delta(9) position with 19:0-ACP (>98% desaturation at the Delta(9) position) suggest that the active-site channel past the diiron center can accommodate at least one more methylene group than is found in the natural substrate. The DeltaDeltaG(binding) estimated from the change in k(cat)/K(M) for increasing substrate acyl-chain length was -3 kJ/mol per methylene group, similar to the value of -3.5 kJ/mol estimated for the hydrophobic partition of long-chain fatty acids (C-7 to C-21) from water to heptane [Smith, R. , and Tanford, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 289-293]. Since the K(M) values are overall similar for all acyl-ACPs tested, the progressive increase in hydrophobic binding energy available from increased chain length is apparently utilized to enhance catalytic steps, which thus provides the underlying physical mechanism for acyl chain selectivity observed with Delta9D.     

Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae

The unsaturated fatty acid (ufa) requiring ole1 mutant of Saccharomyces cerevisiae appears to produce a defective delta-9 fatty acid desaturase. This enzyme catalyzes double bond formation between carbons 9 and 10 of palmitoyl and stearoyl coenzyme A. A DNA fragment isolated by complementation of an ole1 strain repairs the ufa requirement in mutant cells. Genetic analysis of the cloned DNA fragment indicates that it is allelic to the OLE1 gene. Disruption of a single copy of the wild type gene in a diploid strain produces both wild type and nonreverting ufa-requiring haploid progeny upon sporulation. Membrane lipids of the disrupted haploid strains contain only ufas supplied in the growth medium. The recovery of activity in both wild type and disrupted segregants was examined after removal of ufas from the growth medium. Following ufa deprivation disruptant cells grew normally for about three generations and then at a slower rate for at least 0.6 generations. During that time cellular ufas dropped from 63 to 7.3 mol % of the total fatty acids. No production of the 16:1 and 18:1 products of the desaturase was observed in disruptant cells, whereas desaturation in wild type control cells was evident 2 h after deprivation. These results indicate that 1) the OLE1 gene is essential for production of monounsaturated fatty acids and is probably the structural gene for the delta-9 desaturase enzyme. 2) A large part of membrane ufas present under normal culture conditions are not essential for growth and cell division.     

A palmitoyl-CoA-specific delta9 fatty acid desaturase from Caenorhabditis elegans

Biosynthesis of polyunsaturated fatty acids in C. elegans is initiated by the introduction of a double bond at the delta9 position of a saturated fatty acid. We identified three C. elegans fatty acid desaturase genes related to the yeast delta9 desaturase OLE1 and the rat stearoyl-CoA desaturase SCD1. Heterologous expression of all three genes rescues the fatty acid auxotrophy of the yeast delta9 desaturase mutant ole1. Examination of the fatty acid composition of the transgenic yeast reveals striking differences in the substrate specificities of these desaturases. Two desaturases, FAT-6 and FAT-7, readily desaturate stearic acid (18:0) and show less activity on palmitic acid (16:0). In contrast, the other desaturase, FAT-5, readily desaturates palmitic acid (16:0), but shows nearly undetectable activity on the common delta9 substrate stearic acid. This is the first report of a palmitoyl-CoA-specific membrane fatty acid desaturase.     

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum

Enzymatic activation of PAA (phenylacetic acid) to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (phenylacetate-CoA ligase), was cloned in Escherichia coli as a maltose-binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA into phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k(cat)/K(m) values of 0.23+/-0.06 and 7.8+/-1.2 mM(-1).s(-1) for PAA and POA respectively. Surprisingly, PCL was very efficient in catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [k(cat)/K(m)=(3.1+/-0.4)x10(2) mM(-1).s(-1) for trans-cinnamic acid]. Of all the substrates screened, medium-chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on the basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty-acid-activating ancestral enzyme that may have been involved in the beta-oxidation of fatty acids.     

Mechanism of fatty acid desaturation: a bioorganic perspective

The desaturation of long chain fatty acids is a ubiquitous transformation which plays a critical role in the biosynthesis of lipids. Of particular interest to the bioorganic chemist is the unique ability of desaturases to oxidize unactivated hydrocarbon chains in a chemo-, regio- and stereoselective manner. The mechanism of membrane-bound desaturases has been examined using regiospecifically labelled analogues bearing deuterium, sulfur or fluorine-substituted methylene isosteres. These probes have been applied in the study of several biomedically important desaturase systems including a prototypical yeast stearoyl CoA delta(9) desaturase. In all cases, it has been found that the dehydrogenation (desaturation) process is initiated by a kinetically important hydrogen activation step at the carbon of the incipient double bond which is closest to the acyl terminus of the fatty acid chain. These results point to a common active site architecture which is highly conserved among a wide range of membranous desaturases.     

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.     

少根根霉Δ^6-脂肪酸脱氢酶基因在毕赤酵母中的表达

Δ^6-脂肪酸脱氢酶是一种膜整合蛋白,也是多不饱和脂肪酸合成途径中的限速酶。在前期工作中,通过RT-PCR和RACE技术,从少根根霉NK300037中克隆到一个潜在编码Δ^6-脂肪酸脱氢酶的序列,序列和功能分析结果表明该序列具有一个长度为1377bp、编码由458个氨基酸组成、大小为52kD的新的Δ^6-肪酸脱氢酶基因。把少根根霉Δ^6-脂肪酸脱氢酶基因（RAD6）亚克隆到表达载体pPIC3．5K,构建重组表达载体pPICRAD6,并转化到毕赤酵母菌株GS115进行表达。提取酵母细胞总脂肪酸和进行甲酯化,经气相色谱和气相色谱-质谱连用分析表明,目的基因的编码产物能将C16：1、C17：1、C18：1、亚油酸和α-亚麻酸在△6和7位间特异性脱氢而引入一个新的双键,生成更高不饱和的脂肪酸,该催化反应没有链长特异性,只有键位特异性。此外,按Kozak序列特点,改变目的基因转译起始密码子周边序列结构,并把改变后序列导入毕赤酵母GS115中进行功能表达分析,结果表明在毕赤酵母中这种改变同样能提高目的基因的表达水平。综合所有分析结果表明,巴斯德毕赤酵母更适合用来综合分析Δ^6-脂肪酸脱氢酶基因的功能。