FAS1 Gene Research Articles

Unlocking lager's flavour palette by metabolic engineering of Saccharomyces pastorianus for enhanced ethyl ester production

Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1I306A and ScFAS2G1250S significantly improved ethyl hexanoate production and ScFAS1R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

Optimizing Baijiu fermentation with high-yield ethyl caproate-producing Candida parapsilosis strain

Ethyl caproate is an important flavor substance of Baijiu. It is effective to increase the content of ethyl caproate to improve the quality of strong-flavor Baijiu. In this study, the high-yielding ethyl caproate Candida parapsilosis strain F-5 was isolated from pit mud. Under optimized fermentation conditions, F-5 achieved an ethyl caproate yield of 53.69 mg/L. A novel strategy was employed by adding caproic acid and F-5 directly to the fermented grain. The application of F-5 in the fermentation of Xiaoqu and strong-flavor Baijiu significantly increased the ethyl caproate content by 24.86% and 17.45%, respectively. Transcriptomic analysis showed that Lip2 and esterase genes in the lipase synthesis pathway, and Fas1, Fas2, Eht1, and Faa4 genes in the acyltransferase pathway were significantly upregulated. These findings suggest that strain F-5 has the potential to contribute to the development of artificial pit mud and improve the flavor quality of strong-flavor Baijiu.

Identification and Functional Characterization of Adenosine Deaminase in Mucor circinelloides: A Novel Potential Regulator of Nitrogen Utilization and Lipid Biosynthesis.

Adenosine deaminase (ADA) is an enzyme distributed in a wide variety of organisms that cleaves adenosine into inosine. Since inosine plays an important role in nitrogen metabolism, ADA may have a critical function in the regulation of fatty acid synthesis. However, the role of ADA in oleaginous fungi has not been reported so far. Therefore, in this study, we identified one ada gene encoding ADA (with ID scaffold0027.9) in the high lipid-producing fungus, Mucor circinelloides WJ11, and investigated its role in cell growth, lipid production, and nitrogen metabolism by overexpressing and knockout of this gene. The results showed that knockout of the ada altered the efficiency of nitrogen consumption, which led to a 20% increment in the lipid content (25% of cell dry weight) of the engineered strain, while overexpression of the ada showed no significant differences compared with the control strain at the final growth stage; however, interestingly, it increased lipid accumulation at the early growth stage. Additionally, transcriptional analysis was conducted by RT-qPCR and our findings indicated that the deletion of ada activated the committed steps of lipid biosynthesis involved in acetyl-CoA carboxylase (acc1 gene), cytosolic malic acid enzyme (cme1 gene), and fatty acid synthases (fas1 gene), while it suppressed the expression of AMP-activated protein kinase (ampk α1 and ampk β genes), which plays a role in lipolysis, whereas the ada-overexpressed strain displayed reverse trends. Conclusively, this work unraveled a novel role of ADA in governing lipid biosynthesis and nitrogen metabolism in the oleaginous fungus, M. circinelloides.

Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica.

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2pI1220F ). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6mg L-1 Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7mg L-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1±13.4mg L-1 of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.

Fatty Acid Synthase Beta Dehydratase in the Lipid Biosynthesis Pathway Is Required for Conidiogenesis, Pigmentation and Appressorium Formation in Magnaporthe oryzae S6.

Lipid biosynthesis produces glycerol, which is important in fueling turgor pressure necessary for germination and penetration of plant host by fungi. As the relationship between pathogenicity and the lipid biosynthetic pathway is not fully understood, we have elucidated the role of the fatty acid synthase beta subunit dehydratase (FAS1) gene in lipid biosynthesis. The FAS1 gene was silenced through homologous double crossover in Magnaporthe oryzae strain S6 to study the effect on lipid biosynthesis. The vegetative growth of Δfas1 mutants show the highest drop on oleic acid (between 10 and 50%), while the mycelial dry weight of mutants dropped significantly on all media. Conidiation of FAS1 mutants show a ~10- and ~5-fold reduction on oatmeal and Potato Dextrose Agar (PDA), respectively. Mutants formed mycelium that were mildly pigmented, indicating that the deletion of FAS1 may have affected melanin biosynthesis. Biochemical and gene expression studies concluded that the fatty acid degradation pathway might have been interrupted by FAS1 deletion. FAS1 mutants showed no enzyme activity on glucose or olive oil, suggesting that the mutants may lack functional peroxisomes and be defective in β-oxidation of fatty acids, hence explaining the reduced lipid deposits in the spores.

Expression of a recombinant, 4'-Phosphopantetheinylated, active M. tuberculosis fatty acid synthase I in E. coli.

BackgroundFatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS).MethodsIn order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography.ResultsActivation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM.ConclusionPurified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Isolation and characterization of Aspergillus flavus strains in China.

Important staple foods (peanuts, maize and rice) are susceptible to contamination by aflatoxin (AF)-producing fungi such as Aspergillus flavus. The objective of this study was to explore non-aflatoxin-producing (atoxigenic) A. flavus strains as biocontrol agents for the control of AFs. In the current study, a total of 724 A. flavus strains were isolated from different regions of China. Polyphasic approaches were utilized for species identification. Non-aflatoxin and non-cyclopiazonic acid (CPA)-producing strains were further screened for aflatoxin B1 (AFB1) biosynthesis pathway gene clusters using a PCR assay. Strains lacking an amplicon for the regulatory gene aflR were then analyzed for the presence of the other 28 biosynthetic genes. Only 229 (32%) of the A. flavus strains were found to be atoxigenic. Smaller (S) sclerotial phenotypes were dominant (51%) compared to large (L, 34%) and non-sclerotial (NS, 15%) phenotypes. Among the atoxigenic strains, 24 strains were PCR-negative for the fas-1 and aflJ genes. Sixteen (67%) atoxigenic A. flavus strains were PCRnegative for 10 or more of the biosynthetic genes. Altogether, 18 new PCR product patterns were observed, indicating great diversity in the AFB1 biosynthesis pathway. The current study demonstrates that many atoxigenic A. flavus strains can be isolated from different regions of China. In the future laboratory as well as field based studies are recommended to test these atoxigenic strains as biocontrol agents for aflatoxin contamination.

Effects of HERV-R env Knockdown in Combination with Ionizing Radiation on Apoptosis-Related Gene Expression in A549 Lung Cancer Cells

Radiotherapy has played a key role in the management of non–small-cell lung cancer (NSCLC). However, the use of radiotherapy in treating NSCLC is limited because of the intrinsic radiation resistance of tumor cells and injury to adjacent normal tissues. Many oncogenes are reported to be involved in radioresistance. Thus, novel moleculartargeting approaches to enhance the radiosensitivity of NSCLC cells are required to improve the therapeutic efficiency of radiotherapy. In this study, we report that expression of the human endogenous retrovirus-R (HERV-R) env gene is greatly elevated in γ-irradiation resistant A549 cells compared with radiation sensitive H460 cells. In addition, the HERV-R env gene was significantly increased in A549 cells after treatment with γ-irradiation. HERV-R env knockdown by siRNA in irradiated A549 cells led to overexpression of TP53 mRNA, followed by significant elevation in the levels of CDKN1A mRNA. Moreover, the expression of the apoptosis-related FAS-1 gene was increased, whereas the expression levels of the anti-apoptotic gene BCL2 were significantly decreased in the A549 cells in which the HERV-R env was suppressed by γ-irradiation. These results suggest that knockdown of HERV-R env with γ-irradiation causes cell cycle disturbances, which in turn induces apoptosis. In conclusion, the combination of HERV-R env knockdown and γ-irradiation has the potential to improve the therapeutic efficiency of radiotherapy for NSCLC.

Quality Control of a Cytoplasmic Protein Complex: CHAPERONE MOTORS AND THE UBIQUITIN-PROTEASOME SYSTEM GOVERN THE FATE OF ORPHAN FATTY ACID SYNTHASE SUBUNIT Fas2 OF YEAST

For the assembly of protein complexes in the cell, the presence of stoichiometric amounts of the respective protein subunits is of utmost importance. A surplus of any of the subunits may trigger unspecific and harmful protein interactions and has to be avoided. A stoichiometric amount of subunits must finally be reached via transcriptional, translational, and/or post-translational regulation. Synthesis of saturated 16 and 18 carbon fatty acids is carried out by fatty acid synthase: in yeast Saccharomyces cerevisiae, a 2.6-MDa molecular mass assembly containing six protomers each of two different subunits, Fas1 (β) and Fas2 (α). The (α)6(β)6 complex carries six copies of all eight enzymatic activities required for fatty acid synthesis. The FAS1 and FAS2 genes in yeast are unlinked and map on two different chromosomes. Here we study the fate of the α-subunit of the complex, Fas2, when its partner, the β-subunit Fas1, is absent. Individual subunits of fatty acid synthase are proteolytically degraded when the respective partner is missing. Elimination of Fas2 is achieved by the proteasome. Here we show that a ubiquitin transfer machinery is required for Fas2 elimination. The major ubiquitin ligase targeting the superfluous Fas2 subunit to the proteasome is Ubr1. The ubiquitin-conjugating enzymes Ubc2 and Ubc4 assist the degradation process. The AAA-ATPase Cdc48 and the Hsp70 chaperone Ssa1 are crucially involved in the elimination of Fas2.

Trans-chalcone and quercetin down-regulate fatty acid synthase gene expression and reduce ergosterol content in the human pathogenic dermatophyte Trichophyton rubrum

BackgroundFatty acid synthase (FAS) is a promising antifungal target due to its marked structural differences between fungal and mammalian cells. The aim of this study was to evaluate the antifungal activity of flavonoids described in the scientific literature as FAS inhibitors (quercetin, trans-chalcone, ellagic acid, luteolin, galangin, and genistein) against the dermatophyte Trichophyton rubrum and their effects on fatty acid and ergosterol synthesis.MethodsThe antifungal activity of the natural products was tested by the microdilution assay for determination of the minimum inhibitory concentration (MIC). The effect of the compounds on the cell membrane was evaluated using a protoplast regeneration assay. Ergosterol content was quantified by spectrophotometry. Inhibition of FAS by flavonoids was evaluated by an enzymatic assay to determine IC50 values. Quantitative RT-PCR was used to measure transcription levels of the FAS1 and ERG6 genes involved in fatty acid and ergosterol biosynthesis, respectively, during exposure of T. rubrum to the flavonoids tested.ResultsThe flavonoids quercetin and trans-chalcone were effective against T. rubrum, with MICs of 125 and 7.5 μg/mL for the wild-type strain (MYA3108) and of 63 and 1.9 μg/mL for the ABC transporter mutant strain (ΔTruMDR2), respectively. The MICs of the fluconazole and cerulenin controls were 63 and 125 μg/mL for the wild-type strain and 30 and 15 μg/mL for the mutant strain, respectively. Quercetin and trans-chalcone also reduced ergosterol content in the two strains, indicating that interference with fatty acid and ergosterol synthesis caused cell membrane disruption. The MIC of quercetin reduced the number of regenerated protoplasts by 30.26% (wild-type strain) and by 91.66% (mutant strain). Half the MIC (0.5 MIC) of quercetin did not reduce the number of regenerated wild-type fungal colonies, but caused a 36.19% reduction in the number of mutant strain protoplasts. In contrast, the MIC and 0.5 MIC of trans-chalcone and cerulenin drastically reduced protoplast regeneration in the two strains. The FAS1 gene was repressed in the presence of MICs of quercetin, trans-chalcone, fluconazole and cerulenin. The ERG6 gene was induced in the presence of MICs of fluconazole and cerulenin and was repressed in the presence of MICs of trans-chalcone and quercetin. Trans-chalcone and quercetin inhibited the enzymatic activity of FAS, with IC50 values of 68.23 and 17.1 μg/mL, respectively.ConclusionTrans-chalcone and quercetin showed antifungal activity against T. rubrum, reducing ergosterol levels and modulating the expression of FAS1 and ERG6.

Fatty Acid Synthase Impacts the Pathobiology of Candida parapsilosis In Vitro and during Mammalian Infection

Cytosolic fungal fatty acid synthase is composed of two subunits α and β, which are encoded by Fas1 and Fas2 genes. In this study, the Fas2 genes of the human pathogen Candida parapsilosis were deleted using a modified SAT1 flipper technique. CpFas2 was essential in media lacking exogenous fatty acids and the growth of Fas2 disruptants (Fas2 KO) was regulated by the supplementation of different long chain fatty acids, such as myristic acid (14∶0), palmitic acid (16∶0), and Tween 80, in a dose-specific manner. Lipidomic analysis revealed that Fas2 KO cells were severely restricted in production of unsaturated fatty acids. The Fas2 KO strains were unable to form normal biofilms and were more efficiently killed by murine-like macrophages, J774.16, than the wild type, heterozygous and reconstituted strains. Furthermore, Fas2 KO yeast were significantly less virulent in a systemic murine infection model. The Fas2 KO cells were also hypersensitive to human serum, and inhibition of CpFas2 in WT C. parapsilosis by cerulenin significantly decreased fungal growth in human serum. This study demonstrates that CpFas2 is essential for C. parapsilosis growth in the absence of exogenous fatty acids, is involved in unsaturated fatty acid production, influences fungal virulence, and represents a promising antifungal drug target.

Transcription profiling and regulation of fat metabolism genes in diapausing adults of the mosquito Culex pipiens

Culex pipiens, the mosquito that vectors West Nile virus in North America, overwinters in an adult diapause (dormancy) that is programmed by the short day length and low temperatures of autumn. In response to these environmental signals, females cease feeding on blood and instead seek sources of nectar used to generate the huge lipid reserves required for winter survival. To identify regulatory networks that regulate fat accumulation and fat consumption during diapause, we compared expression of fat-related genes from nondiapausing females with expression of those same genes in early and late diapause and at diapause termination. Among the 31 genes we examined, 4 were expressed more highly in early diapause than in nondiapause, while 14 genes were downregulated in early diapause. In the transition from early to late diapause, 19 genes related to fat metabolism were upregulated. As reported previously, fatty acid synthase, identified as fas-1 in this study, was upregulated in early diapause. Numerous fat metabolism genes, including multiple kinetic classes and genes involved in beta-oxidation, an energy-generation step, were suppressed in early diapause but were highly expressed in late diapause and at diapause termination. RNA interference (RNAi) analysis revealed that the fas-1 gene and others (fas-3 and fabp) have important roles in fat storage during early diapause. When expression of these genes is suppressed, female mosquitoes fail to sequester the lipids needed for overwintering.

Ratios of Cells With and Without Virulence Genes in Rhodococcus fascians Populations Correlate with Degrees of Symptom Development.

Rhodococcus fascians, a gram-positive phytopathogenic bacterium, causes fasciation and leafy galls on a wide range of monocotyledonous and dicotyledonous plants for which it requires the plasmid-borne fas operon. Strains isolated from symptomatic plants over a 20-year-period exhibited a high degree of variability when their virulence was assessed on garden pea seedlings. Polymerase chain reaction amplification of the fas-1 and fasR virulence genes from randomly chosen single colonies showed that many strains consisted of two subpopulations, of which one had lost these genes. Inoculation of pea seedlings with mixtures of fas-1-positive and -negative cells that originated from the same strain demonstrated a strong correlation (Pearson's r ≥ 0.9205) between the proportion of cells in the inoculum carrying the fas-1 gene and the severity of disease symptoms. The minimal concentration of fas-1-positive cells required for the development of small lateral shoots on pea seedlings was 2.5 × 104 CFU/ml (P ≤ 0.008), while the overall suppression of main stem growth was observed at 2.5 × 105 CFU/ml (P ≤ 0.019). These observations underline that care should be taken when the virulence of clinical R. fascians strains is evaluated.

Increased Ethyl Caproate Production by Inositol Limitation in Saccharomyces cerevisiae

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2 , ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae

Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2, ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.

A downstream regulatory element located within the coding sequence mediates autoregulated expression of the yeast fatty acid synthase gene FAS2 by the FAS1 gene product.

The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are transcriptionally co-regulated by general transcription factors (such as Reb1, Rap1 and Abf1) and by the phospholipid-specific heterodimeric activator Ino2/Ino4, acting via their corresponding upstream binding sites. Here we provide evidence for a positive autoregulatory influence of FAS1 on FAS2 expression. Even with a constant FAS2 copy number, a 10-fold increase of FAS2 transcript amount was observed in the presence of FAS1 in multi-copy, compared to a fas1 null mutant. Surprisingly, the first 66 nt of the FAS2 coding region turned out as necessary and sufficient for FAS1-dependent gene expression. FAS2-lacZ fusion constructs deleted for this region showed high reporter gene expression even in the absence of FAS1, arguing for a negatively-acting downstream repression site (DRS) responsible for FAS1-dependent expression of FAS2. Our data suggest that the FAS1 gene product, in addition to its catalytic function, is also required for the coordinate biosynthetic control of the yeast FAS complex. An excess of uncomplexed Fas1 may be responsible for the deactivation of an FAS2-specific repressor, acting via the DRS.

Cloning of a fatty acid synthase component FAS1 gene from Saccharomyces kluyveri and its functional complementation of S. cerevisiae fas1 mutant.

A gene encoding a fatty acid synthase component, FAS1, has been cloned from a genomic library of the polyunsaturated fatty acid (PUFA)-producing yeast Saccharomyces kluyveri. This gene (named Sk-FAS1) was found to contain an open reading frame of 6150 bp, coding for 2049 amino acids. The deduced Sk-FAS1 protein showed significant (75-59%) homology with FAS proteins from the other yeasts, including S. cerevisiae, Candida albicans and Yarrowia lipolytica. The substrate-binding sites of the acetyl transferase and malonyl/palmitoyl transferase domains, and the FMN- and NADPH-binding sites of the enoyl reductase domain, were all highly conserved. Expression of the Sk-FAS1 gene in S. cerevisiae complemented genetic disruption of the S. cerevisiae FAS1 gene (Sc-FAS1), suggesting the formation of a heterogeneous complex of Sk-FAS1 (beta) and Sc-FAS2 (alpha), which is able to function to synthesize fatty acids. Compared with the isogenic wild-type of S. cerevisiae, as well as S. kluyveri, the S. cerevisiae fas1 mutant carrying the Sk-FAS1 gene showed an increase in the relative amount of 16-carbon fatty acids and a decrease in 18-carbon fatty acids.

Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis.

Tuberculosis treatment is shortened to six months by the indispensable addition of pyrazinamide (PZA) to the drug regimen that includes isoniazid and rifampin. PZA is a pro-drug of pyrazinoic acid (POA) (ref. 3), whose target of action has never been identified. Although PZA is active only against Mycobacterium tuberculosis, the PZA analog 5-chloro-pyrazinamide (5-Cl-PZA) displays a broader range of anti-mycobacterial activity. We have found that the eukaryotic-like fas1 gene (encoding fatty acid synthetase I, FASI) from M. avium, M. bovis BCG or M. tuberculosis confers resistance to 5-Cl-PZA when present on multi-copy vectors in M. smegmatis. 5-Cl-PZA and PZA markedly inhibited the activity of M. tuberculosis FASI, the biosynthesis of C16 to C24/C26 fatty acids from acetyl-CoA (ref. 6). Importantly, PZA inhibited FASI in M. tuberculosis in correlation with PZA susceptibility. These results indicate that FASI is a primary target of action for PZA in M. tuberculosis. Further characterization of FASI as a drug target for PZA may allow the development of new drugs to shorten the therapy against M. tuberculosis and may provide more options for treatment against M. bovis, M. avium and drug resistant M. tuberculosis.

Identification, isolation and biochemical characterization of a phosphopantetheine:protein transferase that activates the two type-I fatty acid synthases of Brevibacterium ammoniagenes.

Upon heterologous expression of the Brevibacterium ammoniagenes type-I fatty acid synthase FAS-A in Escherichia coli, only the pantetheine-free apoenzyme is synthesized. Activation of FAS-A to its holoform was achieved by transformation with a second B. ammoniagenes gene, PPT1, encoding a type-I FAS-specific phosphopantetheine transferase. PPT1 was identified as a coding sequence located immediately downstream of the second FAS gene present on the B. ammoniagenes genome, fasB. Due to this linkage, PPT1 was part of the cloned fasB DNA region and, consequently, FAS-B but not FAS-A was synthesized as holoFAS in E. coli. PPT1 encodes a protein of 153 amino acids and has a calculated molecular mass of 16,884 Da. The PPT1 gene product contains 25% identical and 42% conserved amino acids compared with the type-II acyl-carrier-protein-activating enzyme of E. coli. Although there is essentially no intergenic region between fasB and PPT1, the PPTase gene is autonomously expressed in E. coli if flanked by 200 bp of its endogenous 5' DNA. The structural independence of Ppt1p was confirmed immunologically, as specific antibodies react with the purified PPTase but not with FAS-B. Overexpression and purification of the His-tagged Ppt1p allowed the in vitro activation of apoFAS-A. This holoenzyme synthesis requires, in addition to Ppt1p, CoA and Mg2+ and leads to a specific FAS activity comparable to that of natural B. ammoniagenes FAS-A. The reactivity of the in vitro-activated FAS-A was verified by the optical FAS assay and by analysis of its in vitro products. In agreement with the known overall colinearity of B. ammoniagenes FAS-B and the Saccharomyces cerevisiae FAS1 and FAS2 gene products, a PPT1-like sequence is also observed at the C terminus of FAS2. However, in contrast to B. ammoniagenes PPT1, this sequence is an integral part of the yeast FAS2 gene. Thus, activation of type-I fatty acid synthases may be accomplished by distinct trans-acting PPTase enzymes and by intrinsic cis-acting PPTase domains.

No significant changes in the expression of the fas,fasl,bcl-2 and bax genes in apoptosis of an erythroleukemic cell line by p53

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. When these cells were cultured at 32 degrees C, the growth rate was reduced significantly and DNA fragmentation, a typical character of apoptosis, was observed. In this process, p53 migrated from cytoplasm to nucleus and protein complexes binding to the p53-responsive element were detected in nuclear extracts of the cells cultured at 32 degrees C by gel-shift assay and transactivation from the p53-responsive element was detected. The expression of the p21 (waf1/cip1/sdi1), cyclin G and gadd45 genes was increased (about 3- to 4-fold that at 37 degrees C), when the cells were cultured at 32 degrees C. However, no significant change was detected in expression of the mdm2, bax and fas genes. No change in the amount of Fas antigen was observed by flow cytometric analysis. Transcripts of the bcl-2 and fas1 gene were not detected in the cells either at 37 degrees C or 32 degrees C. These results suggest that other gene(s) than bax, fas, fas1 and bcl-2 may be important for induction of apoptosis of this erythroleukemic cell line by p53.