scholarly journals Identification and Characterization of the CYP52 Family of Candida tropicalis ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids

2003 ◽  
Vol 69 (10) ◽  
pp. 5983-5991 ◽  
Author(s):  
David L. Craft ◽  
Krishna M. Madduri ◽  
Mark Eshoo ◽  
C. Ron Wilson
Keyword(s):  
Fatty Acids ◽  
Cytochrome P450 ◽  
Fatty Acid ◽  
Candida Tropicalis ◽  
Strain Improvement ◽  
Dicarboxylic Acids ◽  
Fold Increase ◽  
Isolation And Characterization ◽  
Unique Genes

ABSTRACT Candida tropicalis ATCC 20336 excretes α,ω-dicarboxylic acids as a by-product when cultured on n-alkanes or fatty acids as the carbon source. Previously, a β-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the ω-hydroxylase enzymes catalyzing the first step in the ω-oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of ω-oxidation of n-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (CYP52) of C. tropicalis ATCC20336 as well as the accompanying NCP were cloned and sequenced. The 10 CYP genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes, CYP52A13 and CYP52A14 showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair CYP52A17 and CYP52A18 was also induced under all three conditions but to a lesser extent. Moderate induction of CYP52A12 was observed. These results identify the CYP52 and NCP genes as being involved in α,ω-dicarboxylic acid production by C. tropicalis and provide the foundation for biocatalyst improvement.

scholarly journals Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

2003 ◽  
Vol 69 (10) ◽  
pp. 5992-5999 ◽  
Author(s):  
William H. Eschenfeldt ◽  
Yeyan Zhang ◽  
Hend Samaha ◽  
Lucy Stols ◽  
L. Dudley Eirich ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Cytochrome P450 ◽  
Oleic Acid ◽  
Candida Tropicalis ◽  
Saturated Fatty Acids ◽  
Dicarboxylic Acids ◽  
Initial Step ◽  
Cytochrome P450s ◽  
Commercial Development ◽  
P450 Monooxygenase

ABSTRACT Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.

scholarly journals Isolation and Characterization of Unsaturated Fatty Acid Auxotrophs of Streptococcus pneumoniae and Streptococcus mutans

2007 ◽  
Vol 189 (22) ◽  
pp. 8139-8144 ◽  
Author(s):  
Silvia Altabe ◽  
Paloma Lopez ◽  
Diego de Mendoza
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Streptococcus Pneumoniae ◽  
Unsaturated Fatty Acid ◽  
Saturated Fatty Acids ◽  
Monounsaturated Fatty Acids ◽  
Chemotherapeutic Agents ◽  
Isolation And Characterization ◽  
Membrane Structure And Function

ABSTRACT Unsaturated fatty acid (UFA) biosynthesis is essential for the maintenance of membrane structure and function in many groups of anaerobic bacteria. Like Escherichia coli, the human pathogen Streptococcus pneumoniae produces straight-chain saturated fatty acids (SFA) and monounsaturated fatty acids. In E. coli UFA synthesis requires the action of two gene products, the essential isomerase/dehydratase encoded by fabA and an elongation condensing enzyme encoded by fabB. S. pneumoniae lacks both genes and instead employs a single enzyme with only an isomerase function encoded by the fabM gene. In this paper we report the construction and characterization of an S. pneumoniae 708 fabM mutant. This mutant failed to grow in complex medium, and the defect was overcome by addition of UFAs to the growth medium. S. pneumoniae fabM mutants did not produce detectable levels of monounsaturated fatty acids as determined by gas chromatography-mass spectrometry and thin-layer chromatography analysis of the radiolabeled phospholipids. We also demonstrate that a fabM null mutant of the cariogenic organism Streptococcus mutants is a UFA auxotroph, indicating that FabM is the only enzyme involved in the control of membrane fluidity in streptococci. Finally we report that the fabN gene of Enterococcus faecalis, coding for a dehydratase/isomerase, complements the growth of S. pneumoniae fabM mutants. Taken together, these results suggest that FabM is a potential target for chemotherapeutic agents against streptococci and that S. pneumoniae UFA auxotrophs could help identify novel genes encoding enzymes involved in UFA biosynthesis.

scholarly journals A novel omega3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid

2004 ◽  
Vol 378 (2) ◽  
pp. 665-671 ◽  
Author(s):  
Suzette L. PEREIRA ◽  
Yung-Sheng HUANG ◽  
Emil G. BOBIK ◽  
Anthony J. KINNEY ◽  
Kevin L. STECCA ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Eicosapentaenoic Acid ◽  
Fatty Acid Desaturase ◽  
Pcr Amplification ◽  
Oilseed Crops ◽  
Isolation And Characterization ◽  
Carbon Substrates ◽  
Nutritional Benefits

Long-chain n−3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5n−3) have important therapeutic and nutritional benefits in humans. In plants, cyanobacteria and nematodes, ω3-desaturases catalyse the formation of these n−3 fatty acids from n−6 fatty acid precursors. Here we describe the isolation and characterization of a gene (sdd17) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel ω3-desaturase. This gene was isolated by PCR amplification of an S. diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known ω3-desaturases. Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n−6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4n−6), converting it into EPA. This activity differs from that of the known ω3-desaturases from any organism. Plant and cyanobacterial ω3-desaturases exclusively desaturate 18-carbon n−6 PUFAs, and a Caenorhabditis elegans ω3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast. The sdd17-encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops.

Characterization of Aeromonas genomic species by using quinone, polyamine, and fatty acid patterns

1994 ◽  
Vol 40 (10) ◽  
pp. 844-850 ◽  
Author(s):  
Peter Kämpfer ◽  
Klaus Blasczyk ◽  
Georg Auling
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Octadecenoic Acid ◽  
Hexadecenoic Acid ◽  
Genomic Species ◽  
Great Homogeneity ◽  
Hydroxylated Fatty Acids ◽  
Fatty Acid Patterns ◽  
Chemotaxonomic Study

A chemotaxonomic study was carried out on representative strains of 13 Aeromonas genomic species. Quinone, polyamine, and fatty acid patterns were found to be very useful for an improved characterization of the genus and an improved differentiation from members of the families Enterobacteriaceae and Vibrionaceae. The Q-8-benzoquinone was the predominant ubiquinone, and putrescine and diaminopropane were the major poly amines of the genus. The fatty acid patterns of 181 strains, all characterized by DNA–DNA hybridization, showed a great homogeneity within the genus, with major amounts of hexadecanoic acid (16:0), hexadecenoic acid (16:1), and octadecenoic acid (18:1), and minor amounts of the hydroxylated fatty acids (3-OH 13:0, 2-OH 14:0, 3-OH 14:0) in addition to some iso and anteiso branched fatty acids (i-13:0, i-17:1, i-17:0, and a-17:0). Although some differences in fatty acid profiles between the genomic species could be observed, a clearcut differentiation of all species was not possible.Key words: Aeromonas, polyamines, quinones, fatty acids, differentiation.

scholarly journals Up-regulation of the expression of the gene for liver fatty acid-binding protein by long-chain fatty acids

1996 ◽  
Vol 319 (2) ◽  
pp. 483-487 ◽  
Author(s):  
Claire MEUNIER-DURMORT ◽  
Hélène POIRIER ◽  
Isabelle NIOT ◽  
Claude FOREST ◽  
Philippe BESNARD
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Binding Protein ◽  
Fold Increase ◽  
Long Chain Fatty Acids ◽  
Liver Fatty Acid ◽  
Fatty Acid Binding ◽  
Acid Binding Protein ◽  
Fabp Gene ◽  
Long Chain

The role of fatty acids in the expression of the gene for liver fatty acid-binding protein (L-FABP) was investigated in the well-differentiated FAO rat hepatoma cell line. Cells were maintained in serum-free medium containing 40 µM BSA/320 µM oleate. Western blot analysis showed that oleate triggered an approx. 4-fold increase in the cytosolic L-FABP level in 16 h. Oleate specifically stimulated L-FABP mRNA in time-dependent and dose-dependent manners with a maximum 7-fold increase at 16 h in FAO cells. Preincubation of FAO cells with cycloheximide prevented the oleate-mediated induction of L-FABP mRNA, showing that protein synthesis was required for the action of fatty acids. Run-on transcription assays demonstrated that the control of L-FABP gene expression by oleate was, at least in part, transcriptional. Palmitic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid were similarly potent whereas octanoic acid was inefficient. This regulation was also found in normal hepatocytes. Therefore long-chain fatty acids are strong inducers of L-FABP gene expression. FAO cells constitute a useful tool for studying the underlying mechanism of fatty acid action.

scholarly journals Characterization of planktonic and biofilm fatty acid profiles and evaluation of their trophic transfer potential to Hyalella Azteca

2021 ◽  
Author(s):  
Jerry Chien-Yao Chao
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Bacterial Species ◽  
Saturated Fatty Acids ◽  
Hyalella Azteca ◽  
Fatty Acid Profiles ◽  
Fa Composition ◽  
Branched Chain ◽  
Transfer Potential

Fatty acid (FA) composition between biofilms and batch planktonic cultures were compared for two bacterial species Pseudomonas aeruginosa and Staphylococcus aureaus. Biofilm cultures exhibited decrease in saturated fatty acids (SAFA) that potentially conform to a more fluidic biophysical membrane property. The amount of FA in the biofilms' extracellular polymeric substance was not sufficient to consider it having a major contribution to the observed differences between biofilms and batch planktonic cultures. While biofilm grazing by the amphipod Hyalella azteca was evident, only certain bacteria-specific FA appeared to have the potential to be retained (odd-number SAFA and branched-chain FA). H. azteca with diet strictly consisted of bacteria biofilms did not demonstrate significant changes in their nutritional condition in terms of ω-3 and ω-6 polyunsaturated fatty acids (PUFA): combined with the results from fasting trials, H. azteca appears to have the capacity to retain ω-3 and ω-6 PUFAs up to 10 days.

scholarly journals Atlantic salmon require long-chain n-3 fatty acids for optimal growth throughout the seawater period

2016 ◽  
Vol 5 ◽  
Author(s):  
Grethe Rosenlund ◽  
Bente E. Torstensen ◽  
Ingunn Stubhaug ◽  
Nafiha Usman ◽  
Nini H. Sissener
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Atlantic Salmon ◽  
Fold Increase ◽  
Optimal Growth ◽  
Dietary Fatty Acids ◽  
Nutritional Requirement ◽  
Retention Efficiency ◽  
Long Chain

AbstractThe nutritional requirement for n-3 long-chain PUFA in fast-growing Atlantic salmon (Salmo salar) during grow out in the sea is not well documented. Diets were formulated with levels of EPA (20 : 5n-3) and DHA (22 : 6n-3) ranging from 1·3 to 7·4 % of fatty acids (4–24 g/kg feed). Two long-term trials were conducted through the seawater phase, the first at 6 and 12°C, and the second at 12°C. In the first trial, growth at both temperatures was significantly lower in fish fed 1·4 % EPA+DHA of total fatty acids compared with the 5·2 % EPA+DHA group. In the second trial, growth was significantly lower in fish fed 1·3 and 2·7 % compared with 4·4 and 7·4 % EPA + DHA. Fatty acid composition in the fish reflected diet composition, but only after a 7-fold increase in body weight did the fatty acid profile of the fish stabilise according to dietary fatty acids (shown for EPA and DHA). The retention efficiency of DHA increased with decreasing dietary levels, and was 120–190 and 120–200 % in trials 1 and 2, respectively. The retention efficiency of EPA was lower (60–200 %), and values >100 % were only achieved at the lowest dietary levels in both trials. Temperature did not affect fatty acid retention efficiency. These results suggest that Atlantic salmon have a specific requirement for EPA + DHA >2·7 % of fatty acids for optimal long-term growth in seawater, and that short-term growth trials with less weight increase would not show these effects.

scholarly journals Characterization of the oils present in acid and fermented silages produced from Tilapia filleting residue

2011 ◽  
Vol 40 (2) ◽  
pp. 240-244 ◽  
Author(s):  
Rose Meire Vidotti ◽  
Maria Teresa Bertoldo Pacheco ◽  
Giovani Sampaio Gonçalves
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Nutritional Value ◽  
Lipid Fraction ◽  
Monounsaturated Fatty Acids ◽  
Degree Of Saturation ◽  
Degree Of Hydrolysis ◽  
Animal Feeding ◽  
Hydrolysis Of

The objective of this study was to determine the quality and composition of fatty acid in the lipid fraction of silages obtained from the residue of tilapia processing. Stratification of the lipid layer of the silages occurred at different times among the two types of silage (acid and fermented) and the greatest volume of oil was observed in acid silage (8.67% p/p). Although acid silage was more oxidized, it showed lower contents of free fatty acids probably because the degree of hydrolysis of its components is lower than that of fermented silage. Fatty acid composition did not differ among processes inasmuch as level of ϖ-3 was slightly higher in fermented silage. According to the degree of saturation, monounsaturated fatty acids stood out as the predominant category in acid and fermented silages with values of 39.69% and 33.39%, respectively. The use of antioxidants in the silage is needed because the process of production is carried out at temperatures higher than room temperature. The oil in the silages has excellent nutritional value and contains fatty acids essential for animal feeding.

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