Biotransformation of free fatty acids in mixtures to methyl ketones by Monascus purpureus

Summary Monascus purpureus converts short-chain fatty acids to methyl ketones. The regulation of the metabolic pathway is similar to that found in Penicillium roquefortii. There are differences in the actual amount of precursors metabolized. The fermentation of fatty acid mixtures led to methyl ketone mixtures. The metabolism of each fatty acid was dependent on the precursor composition. Offprint requests to: C. Kranz     

Effect of Substrate on the Fatty Acid Composition of Hydrocarbon- and Ketone-utilizing Microorganisms

The fatty acid pattern in hydrocarbon- and ketone-utilizing bacteria after growth on various substrates was examined. The fatty acid composition of one hydrocarbon-utilizing organism (Mycobacterium sp. strain OFS) was investigated in detail after growth on n-alkanes, 1-alkenes, ketones, and n-alcohols. n-Alkanes shorter than C₁₃ or longer than C₁₇ were not incorporated into cellular fatty acids without some degradation. Strain OFS incorporated C₁₄ to C₁₇ 1-alkenes into cellular fatty acids as the ω-monoenoic fatty acid. Methyl ketones were incorporated into strain OFS after removal of one- or two-carbon fragments from the carbonyl end of the molecule. An organism isolated by enrichment on methyl ketones was incapable of n-alkane utilization but could grow on, although not incorporate, ketones or long chain n-alcohols into cellular fatty acids.     

Toxicity of medium chain fatty acids to Penicillium crustosum Thom and their detoxification to methyl ketones

Conversion of medium chain length fatty acids into methyl ketones by some filamentous fungi is important in the development of flavour in the blue mould-ripened cheeses and in the promotion of ketonic rancidity in the lauric acid oils. The effect of fatty acid composition of single acid glycerides, pH, temperature and medium chain length fatty acids on conversion of fatty acids into methyl ketones has been studied with two strains of Penicillium crustosum Thom. Medium chain length fatty acids were fungitoxic. The minimum inhibitory concentration was 3776, 2884, 1723 and 3066 mg/l for the C6:0, 8:0, 10:0 and 12:0 acids, respectively, at pH 7·0 in liquid suspension culture. Evidence is provided to suggest that the cellular site for this conversion is the microbody. Growth of P. crustosum on single acid glycerides involves two distinct physiological processes; the first, a general inhibition of growth, and the second, a detoxification. The fatty acid composition and the slip point of the substrate are of paramount importance in these two processes.     

The incorporation of [1-14C]acetate into the methyl ketones that occur in steam-distillates of bovine milk fat

1. The ¹⁴C-labelling of the fatty acids and the methyl ketones in steam-distillates of milk fat from a lactating cow that had been injected intravenously with [1-¹⁴C]acetate was determined. 2. The labelling patterns of the C₆–C₁₆ fatty acids and the corresponding methyl ketones with one fewer carbon atoms were similar, particularly so for the C₅–C₁₀ compounds at 9 and 22hr. after the injection of [1-¹⁴C]acetate. The isolation of ¹⁴C-labelled methyl ketones in the range C₃–C₁₅ is evidence that the β-oxo acid precursors, which are glyceride-bound in the milk fat, are synthesized in the mammary gland from acetate. The absence of heptadecan-2-one in steam-distillates and the extremely low specific radioactivity of stearic acid are further evidence for this biosynthetic pathway. 3. The specific radioactivities of the C₅–C₁₅ methyl ketones were higher (with the exception of C₉ methyl ketone in the second milking) than the specific activities of the corresponding fatty acids with one more carbon atom. This is consistent with the methyl ketone precursors' being formed during the biosynthesis of fatty acids rather than being products of β-oxidation of fatty acids.     

Biosynthesis of flavors by Penicillium roqueforti

The development of the unique flavor of blue type cheese depends on the concerted action of numerous enzymes of Penicillium roqueforti involved in protein and lipid metabolism. Protease(s) by degrading casein modify the texture and background flavor of the ripening cheese. Lipase by hydrolyzing milk triglycerides provides flavorful fatty acids and precursors of methyl ketones. The enzyme complex involved in the partial oxidation of free fatty acids and the properties of β-ketoacyl decarboxylase which generates the major flavor components of blue cheese are discussed. Fermentation of P. roqueforti for the rapid production of methyl ketones is briefly reviewed.     

Methyl ketone formation during degradation of phenoxybutyric acid by Penicillium canescens SBUG-M 1139

Penicillium canescens SBUG-M 1139 was shown to be able to grow using phenoxybutyric acid as the sole carbon source. The rapid conversion of the phenoxyalkanoic acid resulted in the formation of phenol, which was metabolized completely. These reactions were accompanied by an accumulation of the methyl ketone phenoxypropan-2-one. Furthermore, during the metabolism of phenoxybutyric acid, 4-phenoxy-2,3-dehydrobutyric acid, 4-phenoxy-3-hydroxybutyric acid, phenoxyacetic acid, and phenoxypropan-2-ol accumulated in minor amounts. Clearly, fungi can metabolize phenoxyalkanoic acids to produce methyl ketones in a manner analogous to that used for the conversion of short- or medium-chain fatty acids by fungi. Received: 7 May 1999 / Accepted: 23 August 1999     

LIPID,FATTY ACID,AND STEROL COMPOSITION OF EIGHT SPECIES OF KARENIACEAE (DINOPHYTA): CHEMOTAXONOMY AND PUTATIVE LIPID PHYCOTOXINS1

The lipid class, fatty acid, and sterol composition of eight species of ichthyotoxic marine gymnodinioid dinoflagellate (Karenia, Karlodinium, and Takayama) species was examined. The major lipid class in all species was phospholipid (78%–95%), with low levels of triacylglycerol (TAG; 0%–16%) and free fatty acid (FFA; 1%–11%). The common dinoflagellate polyunsaturated fatty acids (PUFA), octadecapentaenoic acid (OPA 18:5ω3), and docosahexaenoic acid (DHA 22:6ω3), were present in all species in varying amounts (14%–35% and 8%–23%, respectively). The very‐long‐chain PUFA (VLC‐PUFA) 28:7ω6 and 28:8ω3 were present at low levels (<1%), and the ratio of these fatty acids may be a useful chemotaxonomic marker at the species level. The typical dinoflagellate sterol dinosterol was absent from all species tested. A predominance of the 4‐methyl and 4‐desmethyl Δ⁸⁽¹⁴⁾ sterols in all dinoflagellate species included 23‐methyl‐27‐norergosta‐8(14),22‐dien‐3β‐ol (Karenia papilionacea A. J. Haywood et Steid, 59%–66%); 27‐nor‐(24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, brevesterol, (Takayama tasmanica de Salas, Bolch et Hallegraeff 84%, Takayama helix de Salas, Bolch, Botes et Hallegraeff 71%, Karenia brevis (C. C. Davis) G. Hansen et Moestrup 45%, Karlodinium KDSB01 40%, Karenia mikimotoi (Miyake et Kominami ex Oda) G. Hansen et Moestrup 38%); and (24R)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol, gymnodinosterol, (K. mikimotoi 48%, Karenia umbella de Salas, Bolch et Hallegraeff 59%, Karlodinium veneficum (D. L. Ballant.) J. Larsen 71%–83%). In Takayama species, five steroid ketones were identified, including for the first time the 3‐keto form of brevesterol and gymnodinosterol. These results indicate a biochemical link between sterol and steroid ketone biosynthesis, suggesting that selected dinoflagellates can make a significant contribution to ketones in marine sediments. The presence of steroid ketones, specific sterols, and fatty acids, and the ratio of VLC‐PUFA may prove to be a useful chemotaxonomic tool for distinguishing between morphologically similar species. The relative levels of the PUFA, OPA, and DHA, coupled with the potential inhibitory action of Δ⁸⁽¹⁴⁾ sterols, may provide an insight into the ichthyotoxicity of these bloom‐forming dinoflagellates.     

Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H₂ and CO₂ under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO₂ as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO₂ and H₂ as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.     

Biosynthesis of acyclic methyl branched polyunsaturated hydrocarbons inPseudomonas maltophilia

Summary The hydrocarbon composition ofPseudomonas maltophilia was determined by gas chromatography-mass spectrometry. Mono-, di- and tri-unsaturated alkenes were identified with a predominance of polyunsaturated components. The carbon chains of the alkenes contained single methyl branches iniso andanteiso position and double methyl branches in theiso-iso andanteiso-anteiso configurations. The composition of the hydrocarbons from cells grown in synthetic media enriched with amino acids or volatile fatty acids demonstrated that the probable precursors incorporated into individual hydrocarbons were branched and normal fatty acid chains in the range from C₃ to C₁₆. The probable fatty acid precursors which were connected together to form the major triunsaturated hydrocarbon chains were two monounsaturated chains, whereas the major liunsaturated chains resulted from condensation of saturated and monounsaturated chains. The probable precursors for the major monounsaturated hydrocarbons were C₁₄ (C₁₅) and C₁₆ (C₁₅) fatty acids. The accumulation of hydrocarbons was not detected until the cells were in the late exponential phase of growth; the maximal levels were reached at the mid-stationary phase of growth.     

Cultivation ofCandida lipolytica 4-1 on hydrocarbons

The length of the carbon chain of the hydrocarbon substrate affects markedly the fatty acid composition in the cell lipids of the yeastCandida lipolytica 4-1. During cell growth onn-hexadecane dissolved in deparaffinated gas oil, direct incorporation of palmitic acid into lipids takes place. During growth onn-dodecane, on the other hand, an immediate synthesis and incorporation into oleic acid is observed. The cells contain only little lauric acid (maximum 11%). During fermentation of then-alkanes dissolved in deparaffinated gas oil which contains a mixture of isoalkanes, alkylated aromatic and cyclic hydrocarbons, free fatty acids accumulate in the oil phase. The fatty acid composition in the oil differs markedly according to the growth stage of cells. At the beginning of the logarithmic phase of growth, the fatty acid composition in the oil phase reflects the acid composition in the cell lipids, toward the end of cell growth, the cooxidation products of the isoalkanes accumulate. The aqueous phase contains lower fatty acids and cooxidation products of isoalkanes and of alkylated aromatic and alicyclic hydrocarbons. Part III. Oxidation and Utilization of Individual Pure Hydrocarbons—seeFolia Microbiol. 14,334 (1969).     

Conversion of coconut oil to methyl ketones by two Aspergillus species

Isolates of Aspergillus ruber and A. repens have been grown on coconut oil as the sole carbon source in shake culture. Methyl ketones (C₅-C₁₃) were isolated by solvent extraction and analysed by combined gas chromatography and mass spectrometry. 2-Undecanone was the main volatile product reflecting the high concentration of dodecanoic acid in the original coconut oil. The reactivity of the individual short chain fatty acids as substrates for production of methyl ketones would appear to decrease with increasing molecular weight of the acid after taking into account the greater volatility of the lower molecular weight homologues. 2-Hexanone and 2-octanone were produced by all isolates in low concentration (< 1%). Nonanoic acid and 2-heptanone were converted into 2-octanone. Low concentrations of secondary alcohols were formed under aerobic conditions. It is suggested that the production of methyl ketones by partial β-oxidation is too closely related to mainstream metabolism to be of use in the biochemical taxonomy of the genus.     

Metabolism of symmetrical dialkyl ethers by Acinetobacter sp. HO1-N

The growth of Acinetobacter species HO1-N on a homologous series of dialkyl ethers yielded characteristic cellular and extracellular ether fatty acids. Microbial growth on diheptyl ether resulted in the appearance of 7-n-heptoxy-1-n-heptanoic acid as a cellular fatty acid and 2-n-heptoxy-1-acetic acid as the sole extracellular fatty acid. The oxidation of dinonyl ether and didecyl ether by Acinetobacter resulted in the extracellular accumulation of 2-n-nonoxy-acetic acid and 2-n-decoxy-1-acetic acid, respectively. The 16-carbon ether fatty acid, 6-n-decoxy-1-n-hexanoic acid, was identified as a major cellular fatty acid in didecyl ether-grown cells. The extracellular ether fatty acids accumulated in an inverse relationship to the disappearance of the dialkyl ether and appeared to represent end products of metabolism. The carbon and energy required for cellular growth and metabolism resided in the terminal 5-carbons of diheptyl ether, 7-carbons of dinonyl ether and 8-carbons of didecyl ether. Glutarate, adipate, pimelate and suberate were identified from cells grown at the expense of diheptyl, dioctyl, dinonyl and didecyl ether, respectively, suggesting a role for dibasic acids as metabolic intermediates. A new and novel mechanism for the metabolism of symmetrical dialkyl ethers is suggested. Terminal methyl group oxidation of the dialkyl ether results in the formation of an alkoxy-fatty acid followed by an internal carbon-carbon scission reaction 2-carbons removed from the oxygen atom. The resulting endproducts are alkoxyacetic acid and the corresponding dibasic acid.Non-Standard Abbreviations TLC Thin Layer Chromatography - PS-DEGS · PS Diethylene glycol succinate - DHE Diheptyl ether - DOE Dioctyl ether - DNE Dionyl ether - DDE Didecyl ether     

Analysis of fatty acids in A. szechenyianum Gay. by microwave‐assisted extraction and gas chromatography‐mass spectrometry

Introduction – Aconitum szechenyianum Gay. is a traditional Chinese medicinal herb with the detumescent and styptic effects and antitumor activity. There have been only a few researches on its chemical components, but no detailed report has appeared on its fatty acids. Objective – To develop a simple and effective method for the extraction of fatty acids from A. zechenyianum Gay. and then to investigate the fatty acid components. Methodology – Microwave‐assisted extraction (MAE) was optimized with response surface methodology, and the fatty acid compositions of extract were determined by GC–MS with previous derivatisation to fatty acid methyl esters (FAMEs). The results were compared with that obtained by classical Soxhlet extraction (SE). Results – Compared with SE, MAE showed significantly higher fatty acid yields, shorter extraction time, and lower energy and solvent consumption. The major fatty acids in A. szechenyianum Gay. are linoleic acid, palmitic acid, linolenic acid, oleic acid and stearic acid, and the unsaturated fatty acids occupy 66.4% of the total fatty acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of composition and structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.     

Biodegradation and conversion of alkanes and crude oil by a marine Rhodococcus

A hydrocarbon degrader isolated from a chronically oil-polluted marine site was identified as Rhodococcus sp. on the basis of morphology, fatty acid methyl ester pattern, cell wall analysis, biochemical tests and G + C content of DNA. It degraded upto 50% of the aliphatic fraction of Assam crude oil, in seawater supplemented with 35 mM nitrogen as urea and 0.1 mM phosphorus as dipotassium hydrogen orthophosphate, after 72 h at 30 ° and 150 revolutions per minute. The relative percentage of intracellular fatty acid was higher in hydrocarbon-grown cells compared to fructose-grown cells. The fatty acids C₁₆ , C16_{16 :1} C₁₈ and C_{18 : 1} were constitutively present regardless of the growth substrate. In addition to these constitutive acids, other intracellular fatty acids varied in correlation to the hydrocarbon chain length supplied as a substrate. When grown on odd carbon number alkanes, the isolate released only monocarboxylic acids into the growth medium. On even carbon number alkanes only dicarboxylic acids were produced.     

Degradation of long-chain n-alkanes by the yeast Lodderomyces elongisporus

Summary Cells of the yeast Lodderomyces elongisporus, precultured on glycerol, were incubated with long-chain n-alkanes. The results whow that monoterminal alkane oxidation is the main pathway of alkane degradation in the investigated yeast. The amount of diterminal activity is negligible, while subterminal degradation did not occur at all.Fatty acids were the first detectable intermediates. Using different n-alkanes, in every case the fatty acids with substrate chain length predominated in the cells. The formation of radioactive fatty acids from (1-¹⁴C)-hexadecane was time-dependent and indicated that desaturation elongation and -oxidation occurred.Extracellularly, the fatty acid pattern was similar, except for the additional presence of fatty acid methyl esters and the prevalence of octadecenoic acid after growth of cells on n-hexadecane.     

Ketonic rancidity in coconut due to xerophilic fungi

A type of deterioration called ketonic rancidity occurred in coconut after inoculation with four xerophilic fungi, Eurotium amstelodami, E. chevalieri, E. herbariorum and Penicillium citrinum. The fungi were incubated at low water activity and oxygen tension. A homologous series of aliphatic methyl ketones and secondary alcohols C₅C₁₁ were isolated and identified in the rancid samples after fungal growth. Evidence is presented that odd numbered methyl ketones (C₅C₁₁) are derived from even numbered short chain fatty acids with one more carbon atoms than the ketones via a modified β-oxidation of the parent fatty acid. Heptan-2-one and heptan-2-ol are the main reaction products except in the case of E. herbariorum where even numbered hexan-2-one, hexan-2-ol, octan-2-one and octan-2-ol were produced. Other moulds grown on coconut under similar conditions—Aspergillus flavus Link and Chrysosporium farinicola (Burnside) Skou (C. fastidium Pitt)—did not cause ketonic deterioration.     

Characterization of Thiobacillus species by gas-liquid chromatography of cellular fatty acids

Summary Fatty acids of 18 strains representing 10 species of Thiobacillus were extracted from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparison of the resulting chromatograms for the presence and relative amounts of major peaks allowed rapid differentiation between such closely related species as Thiobacillus neapolitanus and T. thioparus and of eight other species. Except for a feature common to all thiobacilli tested, T. thiooxidans, T. neapolitanus and T. thioparus each possessed a characteristic fatty acid methyl ester profile that was exhibited by all the strains of that species. Hence, the thiobacilli could be divided into three distinct groups. It was possible to use the gas-liquid chromatographic patterns of the cellular fatty acids for rapid identification or grouping of these microorganisms since the fatty acid composition of the genus Thiobacillus thus appeared to be of taxonomic significance.Non-standard abbreviations GLC Gas-liquid chromatography - FAME Fatty acid methyl ester(s)