Lipid profiles of conidia of Aspergillus niger and a fatty acid auxotroph

Conidial lipids of the wild-type (V35) Aspergillus niger and its unsaturated fatty acid auxotroph (UFA2) were compared. The wild type contained lower levels (7.6%) of phospholipids and higher levels (28.4%) of glycolipids than the mutant (16.5 and 22.2%, respectively). Oleic (33.4%), linoleic (22.5%), palmitic (12.8%), stearic (7.4%), and linolenic (6.2%) were the main fatty acids of the wild type (V35). The mutant grew only in the presence of unsaturated fatty acid having at least one delta 9cis double bond, and its conidial fatty acid profile was influenced by the exogenous acid. Analyses of the fatty acids of UFA2 grown in the presence of different fatty acid supplements support the original view that the mutant is defective in delta 9-desaturase activity.     

Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.     

Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.     

Lipid profiles of Aspergillus niger and its unsaturated fatty acid auxotroph, UFA2

A comparative study of the mycelial lipid composition of a wild strain (V35) and one unsaturated fatty acid auxotroph (UFA2) of Aspergillus niger has been performed. The lipid composition of both strains are qualitatively the same but quantitatively different. All the strains contain the following phospholipids: cardiolipin, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine; and triglycerides, diglycerides, monoglycerides, ergosterol, and sterol esters as the neutral lipids; mono- and di-galactosyl diglyceride as the major glycolipids along with small amounts of the corresponding mannose analogs. Phosphatidylethanolamine and phosphatidylcholine constitute the bulk of the phospholipids. The mutant (UFA2) contains a higher level of glycerides and lower levels of sterol (both free and esterified form), phospholipids, and glycolipids than the wild type. Aspergillus niger contains C16 to C18 saturated and unsaturated fatty acids. Small amounts of long-chain (C20 to C24) and short-chain (C10 to C14) saturated and unsaturated acids are also present. Linoleic, oleic, and palmitic are the major acids, stearic and linolenic acids being minor ones. UFA2 grows only in the presence of unsaturated fatty acid (C16 or C18) and accumulates a higher concentration of supplemented acid which influences its fatty acid profile.     

Elo1p-dependent carboxy-terminal elongation of C14:1Delta(9) to C16:1Delta(11) fatty acids in Saccharomyces cerevisiae

Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Delta(9))-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Delta(11) fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Delta(11) was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (alpha-elongation). In wild-type cells, the C16:1Delta(11) elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Delta mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that ole1Delta mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid. Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool. Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Delta(11)) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Delta(9)) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species. These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.     

Use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae to modify the lipid composition and function of mitochondrial membranes

KD115 (ol1), an unsaturated fatty acid auxotroph of S. cerevisiae, was grown in a semi-synthetic medium supplemented with 3.3 x 10(-4) M palmitoleic (cis 16:1) or palmitelaidic (trans 16:1) acids. The parent strain S288C was studied as a control. The lipid composition (fatty acids, neutral lipids, and phospholipids), respiratory activity (O2 consumption), and ultrastructure were compared in mutant yeast grown with each unsaturated fatty acid supplement. The fatty acid supplement represented 70-80% of the yeast fatty acids. Yeast grown in trans 16:1 contained more squalene, a higher ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC), and had 10-20% of the respiratory activity compared to the same yeast grown in cis 16:1. The mitochondrial morphology of yeast in each growth supplement was notably different. The use of mixtures of cis and trans 16:1 in different proportions revealed that the PE/PC ratio, the squalene content, the respiratory defect, and the mitochondrial morphology were all similarly dependent on the fraction of trans 16:1 in the mixtures. As little as 10-20% of cis 16:1 in the mixture was sufficient to abrogate the physiological effects of trans 16:1 on each of the parameters noted above. The combined effects of high content of trans unsaturated fatty acid and the altered phospholipid composition seem to account for the decrease in lipid fluidity, the defective structure and function of the mitochondrial membrane.     

Double-Bond Requirement in a Fatty Acid Desaturase Mutant of Saccharomyces cerevisiae

A fatty acid Delta(9)-desaturase mutant of yeast was analyzed to establish the specificity of the fatty acid structural requirement. Several double-bond-containing and substituted fatty acids were tested. It was concluded that the presence of a Delta(9)cis double bond is necessary for growth. The need for a specific chain length or a specific number of double bonds, or for both, is more flexible. Tracer-containing 16:1Delta(9)cis, 18:1Delta(9)cis, 18:2Delta(9,12)cis,cis and 18:3Delta(9,12,15) all cis revealed that each of these growth-supporting components, once taken up, was not converted into other fatty acids. Concentration effects on doubling time were also considered.     

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.     

Monounsaturated but not polyunsaturated fatty acids are required for growth of the deep-sea bacterium Photobacterium profundum SS9 at high pressure and low temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.     

Linker mutagenesis of a bacterial fatty acid transport protein. Identification of domains with functional importance

The product of the fadL gene (FadL) of Escherichia coli is a multifunctional integral outer-membrane protein required for the specific binding and transport of exogenous long-chain fatty acids [C12-C18]. FadL also serves as a receptor for the bacteriophage T2. In order to define regions of functional importance within FadL, the fadL gene has been mutagenized by the insertion of single-stranded hexameric linkers into the unique SalI restriction site that lies towards the 3' end of the gene and into four HpaII restriction sites distributed throughout the coding region. The five insertion mutants were classified into three groups based on their specific growth rates (alpha) in minimal media containing the long-chain fatty acid oleate (C18:1) as a sole carbon and energy source: Oleslow, alpha = 0.035-0.045; Ole +/-, alpha = 0.020-0.035; and Ole-, alpha less than or equal to 0.005 (wild-type, alpha = 0.07-0.10). The hexameric insertion at the SalI site (fadL allele termed S1; insertion after amino acid 410) conferred an Oleslow phenotype and resulted in a reduction of long-chain fatty acid transport (36% the wild-type level). This insertion mutant, however, bound oleic acid at wild-type levels and was fully functional as a receptor for the bacteriophage T2. The modified FadL-S1 protein did not have the heat-modifiable property characteristic of wild-type FadL. Insertions in the four HpaII sites (fadL alleles termed H1, H2, H3, and H5; after amino acids 41, 81, 238, and 389, respectively) resulted in all three classes of mutants. The fadL insertion mutant H5 was defective for long-chain fatty acid transport but bound oleic acid at significant levels. Together with the S1 allele, these data suggest that the carboxyl terminus of FadL is crucial for long-chain fatty acid transport. The insertion mutants H1 and H2 were defective for both oleic acid binding and transport suggesting that the amino terminus of FadL is important for long-chain fatty acid binding and transport. The fadL linker mutant H3 was defective in oleic acid binding yet had significant levels of oleic acid transport. These studies delineated for the first time different regions of the fadL gene that encode domains of FadL implicated in the binding and transport of long-chain fatty acids.     

Genetic and biochemical characterization of a mutation (fatA) that allows trans unsaturated fatty acids to replace the essential cis unsaturated fatty acids of Escherichia coli.

Unsaturated fatty acid auxotrophs of Escherichia coli are able to use only unsaturated fatty acids of the cis configuration as the required growth supplement. A mutation in the fatA gene allows such auxotrophs to utilize unsaturated fatty acids with a trans double bond as well as fatty acids having a cis double bond. The fatA gene was mapped to min 69 near argG, and the allele studied (fatA1) was found to be dominant over the wild-type gene. fatA1 mutant strains grew at similar rates when supplemented with elaidate (trans-9-octadecenoate) or oleate (cis-9-octadecenoate). The fat+ strain, however, lysed when supplemented with the trans fatty acid. Physiological characterization of the fatA mutant strain was undertaken. The mutation appeared not to be involved with long-chain fatty acid transport. Introduction of lesions in known fatty acid transport genes abolished trans fatty acid utilization in the fatA mutant strain. Also, growth characteristics of the fat+ and the fatA1 mutant strains on elaidate as the sole carbon source were identical, which indicated comparables rate of fatty acid accumulation. The mutation appeared to be involved with recognition of the trans configuration after uptake into the cell. The levels of trans fatty acid incorporation into the phospholipids of the fat+ and the fatA strains differed considerably, with the mutant incorporating much higher levels. No significant accumulation of elaidate into nonphospholipid cellular components was observed. The fatA mutation did not appear to be involved with the cellular metabolic state, as cyclic AMP had no effect on the ability of the strains to utilize trans fatty acids.     

Significance and taxonomic value of iso and anteiso monoenoic fatty acids and branded beta-hydroxy acids in Desulfovibrio desulfuricans.

The fatty acids obtained from extractable lipids of the anaerobic sulfate bacterium Desulfovibrio desulfuricans were identified. Saturated and monoenoic iso (C15-C19) and anteiso (C15, C17) fatty acids and saturated normal (C14-C18) and monoenoic normal (C16, C18) fatty acids were shown to be shown to be present by capillary gas chromatography-mass spectrometry. Iso and anteiso beta-hydroxy fatty acids were analyzed as trimethylsilyl ethers in the same way. The position of methyl branches in the monoenoic fatty acids was determined from characteristic fragment ions in the mass spectra of their methyl esters. Disilyloxy methyl esters, prepared by derivatization of the mono unsaturated methyl esters and analyzed by capillary gas chromatography-mass spectrometry, provided the position of double bonds. The monoenoic fatty acids identified in this way were normal (delta7-C16:1, delta9-C16:1, delta9-C18:1, delta11-C18:1), iso (delta7-C15:1, delta9-C16:1, delta9-C17:1, delta11-C18:1, delta11-C19:1), and anteiso (delta7-C15:1, delta9-C17:1). Iso delta9-C17:1 fatty acid is present as the major component. The occurrence of these monoenoic fatty acids in this bacterium is of taxonomical importance.     

Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the growth of Saccharomyces cerevisiae cells deficient in Rsp5 ubiquitin ligase

The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.     

Monounsaturated but Not Polyunsaturated Fatty Acids Are Required for Growth of the Deep-Sea Bacterium Photobacterium profundum SS9 at High Pressure and Low Temperature

There is considerable evidence correlating the production of increased proportions of membrane unsaturated fatty acids (UFAs) with bacterial growth at low temperatures or high pressures. In order to assess the importance of UFAs to microbial growth under these conditions, the effects of conditions altering UFA levels in the psychrotolerant piezophilic deep-sea bacterium Photobacterium profundum SS9 were investigated. The fatty acids produced by P. profundum SS9 grown at various temperatures and pressures were characterized, and differences in fatty acid composition as a function of phase growth, and between inner and outer membranes, were noted. P. profundum SS9 was found to exhibit enhanced proportions of both monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids when grown at a decreased temperature or elevated pressure. Treatment of cells with cerulenin inhibited MUFA but not PUFA synthesis and led to a decreased growth rate and yield at low temperature and high pressure. In addition, oleic acid-auxotrophic mutants were isolated. One of these mutants, strain EA3, was deficient in the production of MUFAs and was both low-temperature sensitive and high-pressure sensitive in the absence of exogenous 18:1 fatty acid. Another mutant, strain EA2, produced little MUFA but elevated levels of the PUFA species eicosapentaenoic acid (EPA; 20:5n-3). This mutant grew slowly but was not low-temperature sensitive or high-pressure sensitive. Finally, reverse genetics was employed to construct a mutant unable to produce EPA. This mutant, strain EA10, was also not low-temperature sensitive or high-pressure sensitive. The significance of these results to the understanding of the role of UFAs in growth under low-temperature or high-pressure conditions is discussed.     

Elongation of C16:0 to C18:0 fatty acids in methylotrophic yeast Hansenula polymorpha CBS 1976 and fatty acid auxotrophic mutants

Fatty acid elongation defective mutant was isolated from the ethyl methanesulfonate treated Hansenula polymorpha based on the growth ability. Using biochemical and genetic approaches, the mutant was characterized. When compared with the fatty acid phenotype of the parental strain, the differences in profile and content of fatty acids in V1 mutant were found. In this V1 mutant, polyunsaturated fatty acids, linoleic and alpha-linolenic acids, could not be detected with a corresponding increase in the content of mono-unsaturated fatty acids. The ratio of C16/C18 fatty acids revealed that the accumulation of C16 fatty acids was increased significantly. The experiments on fatty acid supplementation indicated that the mutant required C18:0 for the proper growth. The results of genetic complementation with the elongase genes of Saccharomyces cerevisiae confirmed that the lesion was occurred at least in the extension of C16:0 to C18:0 of V1. The H. polymorpha mutant obtained in this work will be used as a useful tool for unraveling the pathway of fatty acid synthesis and the role of fatty acids on biological processes.     

Isolation and characterization of fatty acid auxotrophs from the oleaginous yeast Apiotrichum curvatum

Summary In order to improve the economic value of lipids produced by the oleaginous yeast strain Apiotrichum curvatum ATCC 20509, a search was made for mutants defective in the conversion of stearic acid to oleic acid. Mutants could be selected as unsaturated fatty acid auxotrophs, since unsaturated fatty acids are essential componenets in membrane lipids. After treatment of A. curvatum wild-type with N-methyl-N-nitro-N-nitrosoguanidine, 58 fatty-acid-requiring mutants were isolated. On the basis of (1) the growth response to saturated and unsaturated fatty acids and (2) the fatty acid composition of lipids produced by these mutants, it was concluded that only 18 of them were real unsaturated fatty acid (Ufa) mutants, while the other 40 were designated as fatty acid synthetase (Fas) mutants. It is further shown that Ufa mutants of A. curvatum are able to produce high amounts of lipids consisting of more than 90% triacylglycerols with a percentage of saturated fatty acids resembling that of cocoa butter, when grown in the presence of relatively small amounts of oleic acid in the growth medium. This may offer an economically favourable alternative in comparison with other methods that have been developed for the production of cocoa butter equivalents by microorganisms.Offprint requests to: H. Smit     

Substrate specificity of regiospecific desaturation of aliphatic compounds by a mutant Rhodococcus strain

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.