Identification of a Caenorhabditis elegans Delta6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Napier JA
PROVIDER: S-EPMC1219180 | biostudies-other | 1998 Mar
REPOSITORIES: biostudies-other
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Project description:In animals, fatty acid desaturases catalyze key reactions in the synthesis of arachidonic acid and other polyunsaturated fatty acids. A search of the Caenorhabditis elegans DNA databases, using the sequences of Arabidopsis genes, identified several putative desaturases. Here we describe the characterization of the first of these genes, fat-1. The predicted protein encoded by a fat-1 cDNA showed 32-35% identity with both FAD2 and FAD3 of Arabidopsis. When expressed in transgenic plants, fat-1 resulted in a 90% increase in the proportion of alpha-linolenic acid in root lipids. Wild-type Arabidopsis incorporated omega-6 fatty acids (delta8,11,14-20:3 and delta5,8,11,14-20:4) into membrane lipids but did not desaturate them. By contrast, fat-1 transgenic plants efficiently desaturated both of these fatty acids to the corresponding omega-3 products. These findings indicate that the C. elegans fat-1 gene encodes the first animal representative of a class of glycerolipid desaturases that have previously been characterized in plants and cyanobacteria. The FAT-1 protein is an omega-3 fatty acyl desaturase that recognizes a range of 18- and 20-carbon omega-6 substrates.
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Project description:BackgroundIn the nematode Caenorhabditis elegans, longevity in response to germline ablation, but not in response to reduced insulin/IGF1-like signaling, is strongly dependent on the conserved protein kinase minibrain-related kinase 1 (MBK-1). In humans, the MBK-1 ortholog DYRK1A is associated with a variety of disorders, most prominently with neurological defects observed in Down syndrome. To better understand mbk-1's physiological roles and their dependence on genetic background, we analyzed the influence of mbk-1 loss on the transcriptomes of wildtype and long-lived, germline-deficient or insulin-receptor defective, C. elegans strains by RNA-sequencing.Resultsmbk-1 loss elicited global changes in transcription that were less pronounced in insulin-receptor mutant than in germline-deficient or wildtype C. elegans. Irrespective of genetic background, mbk-1 regulated genes were enriched for functions in biological processes related to organic acid metabolism and pathogen defense. qPCR-studies confirmed mbk-1 dependent induction of all three C. elegans Δ9-fatty acid desaturases, fat-5, fat-6 and fat-7, in wildtype, germline-deficient and insulin-receptor mutant strains. Conversely, mbk-1 dependent expression patterns of selected pathogen resistance genes, including asp-12, dod-24 and drd-50, differed across the genetic backgrounds examined. Finally, cth-1 and cysl-2, two genes which connect pathogen resistance to the metabolism of the gaseous messenger and lifespan regulator hydrogen sulfide (H2S), were commonly suppressed by mbk-1 loss only in wildtype and germline-deficient, but not in insulin-receptor mutant C. elegans.ConclusionOur work reveals previously unknown roles of C. elegans mbk-1 in the regulation of fatty acid desaturase- and H2S metabolic-genes. These roles are only partially dependent on genetic background. Considering the particular importance of fatty acid desaturation and H2S for longevity of germline-deficient C. elegans, we propose that these processes at least in part account for the previous observation that mbk-1 preferentially regulates lifespan in these worms.
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Project description:Omega--3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n--3 PUFAs is insufficient in most Western people, whereas the level of n--6 PUFAs is relatively too high, with an n--6/n--3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n--3 PUFAs in mammals relies on chronic dietary intake of fat rich in n--3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n--3 PUFAs or to convert n--6 to n--3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n--3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n--3 PUFA contents and dramatically balance the ratio of n--6/n--3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n--6 PUFAs to the corresponding n--3 PUFAs, and changed the n--6/n--3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n--6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.
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Project description: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.
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Project description:BackgroundLevopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. This work aimed to apply synthetic biology to produce LA in yeast strains from a simple carbon source.ResultsLevopimaradiene (LP), the precursor of LA, was produced via LP synthase (LPS) expression in yeast. LPS was then modified by N-terminal truncating and site-directed mutagenesis. The strain containing t79LPSMM (79 N-terminal amino acid truncating and M593I/Y700F mutation) produced 6.92 mg/L of LP, which were 23-fold higher than the strain containing LPS. Next, t79LPSMM was expressed in a new metabolically engineered chassis, and the final LP production increased 164-folds to 49.21 mg/L. Three cytochrome P450 reductases (CPRs) were co-expressed with CYP720B1 (the enzyme responsible for LA production from LP) in yeast to evaluate their LA producing abilities, and the CPR from Taxus cuspidata (TcCPR) was found to be the best (achieved 23.13 mg/L of LA production). CYP720B1 and TcCPR genes overexpression in the multi-copy site of the S.cerevisiae genome led to a 1.9-fold increase in LA production to 45.24 mg/L in a shake-flask culture. Finally, LA production was improved to 400.31 mg/L via fed-batch fermentation in a 5-L bioreactor.ConclusionsThis is the first report to produce LA in a yeast cell factory and the highest titer of LA is achieved.
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Project description:Carnosic acid (CA), a phenolic tricyclic diterpene, has many biological effects, including anti-inflammatory, anticancer, antiobesity, and antidiabetic activities. In this study, an efficient biosynthetic pathway was constructed to produce CA in Saccharomyces cerevisiae. First, the CA precursor miltiradiene was synthesized, after which the CA production strain was constructed by integrating the genes encoding cytochrome P450 enzymes (P450s) and cytochrome P450 reductase (CPR) SmCPR. The CA titer was further increased by the coexpression of CYP76AH1 and SmCPR ∼t28SpCytb5 fusion proteins and the overexpression of different catalases to detoxify the hydrogen peroxide (H2O2). Finally, engineering of the endoplasmic reticulum and cofactor supply increased the CA titer to 24.65 mg/L in shake flasks and 75.18 mg/L in 5 L fed-batch fermentation. This study demonstrates that the ability of engineered yeast cells to synthesize CA can be improved through metabolic engineering and synthetic biology strategies, providing a theoretical basis for microbial synthesis of other diterpenoids.
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Project description:Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H(+) -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.
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Project description:BackgroundProtein interaction networks aim to summarize the complex interplay of proteins in an organism. Early studies suggested that the position of a protein in the network determines its evolutionary rate but there has been considerable disagreement as to what extent other factors, such as protein abundance, modify this reported dependence.ResultsWe compare the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans with those of closely related species to elucidate the recent evolutionary history of their respective protein interaction networks. Interaction and expression data are studied in the light of a detailed phylogenetic analysis. The underlying network structure is incorporated explicitly into the statistical analysis. The increased phylogenetic resolution, paired with high-quality interaction data, allows us to resolve the way in which protein interaction network structure and abundance of proteins affect the evolutionary rate. We find that expression levels are better predictors of the evolutionary rate than a protein's connectivity. Detailed analysis of the two organisms also shows that the evolutionary rates of interacting proteins are not sufficiently similar to be mutually predictive.ConclusionIt appears that meaningful inferences about the evolution of protein interaction networks require comparative analysis of reasonably closely related species. The signature of protein evolution is shaped by a protein's abundance in the organism and its function and the biological process it is involved in. Its position in the interaction networks and its connectivity may modulate this but they appear to have only minor influence on a protein's evolutionary rate.
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Project description:Phosphoketolases catalyze an energy- and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could largely prevent this breakdown. An evaluation of in vivo function of a subset of phosphoketolases was conducted by monitoring acetate levels during growth, confirming that candidates showing high activity in vitro indeed showed increased acetate accumulation, but expression also decreased cellular fitness. The study shows that expression of several bacterial phosphoketolase candidates in S. cerevisiae can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds.
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