Identification and characterization of a very long-chain fatty acid elongase gene in the methylotrophic yeast, Hansenula polymorpha

To understand the biosynthetic network of fatty acids in the methylotrophic yeast Hansenula polymorpha, which is able to produce poly-unsaturated fatty acids, we have attempted to identify genes encoding fatty acid elongase. Here we have characterized HpELO1, a fatty acid elongase gene encoding a 319-amino-acid protein containing five predicted membrane-spanning regions that is conserved throughout the yeast Elo protein family. Phylogenetic analysis of the deduced amino acid sequence suggests that HpELO1 is an ortholog of the Saccharomyces cerevisiae ELO3 gene that is involved in the elongation of very long-chain fatty acids (VLCFAs). In the fatty acid profile of the Hpelo1Delta disruptant by gas chromatography/mass spectrometry, the amount of C24:0 and C26:0 decreased to undetectable levels, whereas there was a large accumulation of C22:0, suggesting that the HpELO1 is involved in the elongation of VLCFAs and is essential for the production of C24:0. Expression of HpELO1 suppressed the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant and recovered the synthesis of VLCFAs. Similar to the S. cerevisiae elo3Delta strain, the Hpelo1Delta disruptant exhibited the extraordinary growth sensitivity to fumonisin B(1), a ceramide synthase inhibitor. Furthermore, cells of the Hpelo1Delta disruptant were more sensitive to Zymolyase and more flocculent than the wild-type cells, clumping together and falling rapidly out of suspension, suggesting that the Hpelo1Delta mutation causes changes in cell wall composition and structure.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.     

Psychrophilic <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> requires <Emphasis Type="Italic">trans</Emphasis>-monounsaturated fatty acid for growth at higher temperature

A psychrophilic bacterium, Pseudomonas syringae (Lz4W) from Antarctica, was used as a model system to establish a correlation, if any, between thermal adaptation, trans-fatty acid content and membrane fluidity. In addition, attempts were made to clone and sequence the cti gene of P. syringae (Lz4W) so as to establish its characteristics with respect to the cti of other Pseudomonas spp. and also to in vitro mutagenize the cti gene so as to generate a cti null mutant. The bacterium showed increased proportion of saturated and trans-monounsaturated fatty acids when grown at 28°C compared to cells grown at 5°C, and the membrane fluidity decreased with growth temperature. In the mutant, the trans-fatty acid was not synthesized, and the membrane fluidity also decreased with growth temperature, but the decrease was not to the extent that was observed in the wild-type cells. Thus, it would appear that synthesis of trans-fatty acid and modulation of membrane fluidity to levels comparable to the wild-type cells is essential for growth at higher temperatures since the mutant exhibits growth arrest at 28°C. In fact, the cti null mutant-complemented strain of P. syringae (Lz4W-C30b) that was capable of synthesizing the trans-fatty acid was indeed capable of growth at 28°C, thus confirming the above contention. The cti gene of P. syringae (Lz4W) that was cloned and sequenced exhibited high sequence identity with the cti of other Pseudomonas spp. and exhibited all the conserved features.     

The <Emphasis Type="Italic">Lactococcus lactis</Emphasis> FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of <Emphasis Type="Italic">Escherichia coli</Emphasis> and the FabH protein of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.     

Algicidal activity of polyunsaturated fatty acids derived from <Emphasis Type="Italic">Ulva fasciata</Emphasis> and <Emphasis Type="Italic">U. pertusa</Emphasis> (Ulvaceae,Chlorophyta) on phytoplankton

Isolation of algicidal compounds from Ulva fasciata revealed that the algicidal substances were the polyunsaturated fatty acids (PUFAs) as hexadeca-4,7,10,13-tetraenoic acid (HDTA) C16:4 n-3, octadeca-6,9,12,15-tetraenoic acid (ODTA) C18:4 n-3, α-linolenic acid (ALA) C18:3 n-3 and linoleic acid (LA) C18:2 n-6. The fatty acid composition of four species of Ulvaceae (U. fasciata, U. pertusa, U. arasakii and U. conglobota) was analyzed by capillary gas chromatography to investigate the relationship with the algicidal activity. The results indicate that highly algicidal species, U. fasciata and U. pertusa, showed higher contents of C16:4 n-3, C18:3 n-3, and C18:4 n-3. Concentrations of these PUFAs released from the seaweed in the culture medium were also analyzed. These PUFAs were found to be significantly active against Chattonella antiqua, C. marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, moderately effective against Heterocapsa circularisquama, Prorocentrum minimum, P. sigmoides, Scrippsiella trochoidea, whereas low effective against Alexandrium catenella and Cochlodinium polykrikoides. It is suggested that the PUFAs are useful mitigation agents to remove several harmful effects without causing detrimental effects on surrounding marine living organisms.     

Isolation and structural characterisation of two antibacterial free fatty acids from the marine diatom, <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>

One solution to the global crisis of antibiotic resistance is the discovery of novel antimicrobial compounds for clinical application. Marine organisms are an attractive and, as yet, relatively untapped resource of new natural products. Cell extracts from the marine diatom, Phaeodactylum tricornutum, have antibacterial activity and the fatty acid, eicosapentaenoic acid (EPA), has been identified as one compound responsible for this activity. During the isolation of EPA, it became apparent that the extracts contained further antibacterial compounds. The present study was undertaken to isolate these additional antibacterial factors using silica column chromatography and reverse-phase high-performance liquid chromatography. Two antibacterial fractions, each containing a pure compound, were isolated and their chemical structures were investigated by mass spectrometry and nuclear magnetic resonance spectroscopy. The antibacterial compounds were identified as the monounsaturated fatty acid (9Z)-hexadecenoic acid (palmitoleic acid; C16:1 n-7) and the relatively unusual polyunsaturated fatty acid (6Z, 9Z, 12Z)-hexadecatrienoic acid (HTA; C16:3 n-4). Both are active against Gram-positive bacteria with HTA further inhibitory to the growth of the Gram-negative marine pathogen, Listonella anguillarum. Palmitoleic acid is active at micro-molar concentrations, kills bacteria rapidly, and is highly active against multidrug-resistant Staphylococcus aureus. These free fatty acids warrant further investigation as a new potential therapy for drug-resistant infections.     

Analysis of the <Emphasis Type="Italic">MAT1-2-1</Emphasis> gene of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Anti-cyanobacterial fatty acids released from <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis>

This study was carried out to identify unknown allelochemicals released from Myriophyllum spicatum and to investigate their anti-cyanobacterial effects. A series of analyses of culture solutions and methanol extracts of M. spicatum using gas chromatograph equipped with a mass selective detector revealed that M. spicatum released fatty acids, specifically, nonanoic, tetradecanoic, hexadecanoic, octadecanoic, and octadecenoic acids. Nonanoic, cis-6-octadecenoic, and cis-9-octadecenoic acids significantly inhibited growth of Microcystis aeruginosa, whereas tetradecanoic, hexadecanoic, and octadecanoic acids did not show any effect. When the inhibitory effect of nonanoic acid was compared with those of 4 polyphenols and eugeniin, which are anti-cyanobacterial compounds previously reported to be released by M. spicatum, nonanoic acid was found to be the most inhibitory to M. aeruginosa. These results indicate that not only polyphenols and eugeniin but also fatty acids such as nonanoic acid must be studied to reveal how M. spicatum exerts its allelopathic effect on M. aeruginosa.     

Changes of chemical and nutrient composition of porcine blood during fermentation by <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

A solid-state fermentation (SSF) of a mixture of porcine blood and wheat bran with a ratio of 8:1 by Aspergillus oryzae was investigated. Water content, pH, crude protein, heme and total iron, free amino acids (FAA) and total fatty acids (TFA) of the fermented mixture were determined at 0, 40, 80 and 120 h, respectively, and protein hydrolysis were analyzed with SDS-PAGE accordingly. The results showed that, during the fermentation, water contents and pH decreased significantly (P < 0.05) from 68.33% to 59.94%, 7.18% to 4.48%, respectively. Heme iron content changed slightly (P > 0.05). With the degradation of large protein molecules, free amino acids in the mixture increased from 872.83 mg l⁻¹ to 11560.94 mg l⁻¹ (P < 0.05). Content of free isoleucine, methionine and cystine, deficient in fresh porcine blood increased (P < 0.05) after fermentation. Percentages of saturated fatty acids such as C14:0, C17:0 and C18:0 in the total fatty acids decreased significantly (P < 0.05), and those of polyunsaturated fatty acids C18:1, C18:2, C18:3, C20:1 and C20:2 increased significantly from 27.06% to 47.90% (P < 0.05). The results indicated that Aspergillus oryzae could ferment porcine blood and bran mixture and change its chemical and nutrient composition.     

The mutation of a novel <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> SRL4 gene rescues the Lethality of <Emphasis Type="Italic">rad53</Emphasis> and <Emphasis Type="Italic">lcd1</Emphasis> mutations by modulating dNTP levels

The SRL4 (YPL033C) gene was initially identified by the screening of Saccharomyces cerevisiae genes that play a role in DNA metabolism and/or genome stability using the SOS system of Escherichia coli. In this study, we found that the srl4Delta mutant cells were resistant to the chemicals that inhibit nucleotide metabolism and evidenced higher dNTP levels than were observed in the wild-type cells in the presence of hydroxyurea. The mutant cells also showed a significantly faster growth rate and higher dNTP levels at low temperature (16 degrees C) than were observed in the wild-type cells, whereas we detected no differences in the growth rate at 30 degrees C. Furthermore, srl4Delta was shown to suppress the lethality of mutations of the essential S phase checkpoint genes, RAD53 and LCD1. These results indicate that SRL4 may be involved in the regulation of dNTP production by its function as a negative regulator of ribonucleotide reductase.     

Environmental optimization for bioconversion of triolein into 7,10-dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3

Hydroxy fatty acids (HFAs), originally found in small amount mainly from plant systems, are well known to have special properties such as higher viscosity and reactivity compared with other normal fatty acids. Recently, various microbial strains were tested to produce HFAs from different unsaturated fatty acids. Among those microbial strains tested, Pseudomonas aeruginosa PR3 are well known to utilize various unsaturated fatty acids to produce mono-, di-, and tri-HFAs. Previously, we reported that strain PR3 could utilize triolein as a substrate for the production of 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) via the induction of lipase activity (Chang et al., Appl Microbiol Biotechnol, 74:301–306, 2007). In this study, we focused on the development of the optimal environmental conditions for DOD production from triolein by PR3. Optimal initial medium pH and incubation temperature were pH 8.0 and 25°C, respectively. Magnesium ion was essentially required for DOD production. Optimal inoculum size, time for substrate addition, and substrate concentration were 1%, 12 to 24 h, and 300 mg, respectively.     

The effect of low temperature on fatty acid composition and tocopherols of the red microalga, <Emphasis Type="Italic">Porphyridium cruentum</Emphasis>

Porphyridium cruentum was grown in 10 L batch culture at 18°C, pH 8.0 and 28‰ salinity. The cells were harvested in the stationary phase and the fatty acid composition analysed by GC and tocopherol content by HPLC. A total of 14 fatty acids were identified including saturated fatty acids (13:0, 14:0, 14:0 iso, 15:0, 16:0, 16:0iso) and monounsaturated fatty acids (MUFAs; 16:1(n-7), 18:1(n-7), 18:1(n-9). Polyunsaturated fatty acids (PUFAs) were the predominant fatty acids detected, reaching 43.7% of total fatty acids in the stationary phase of culture. Among the PUFAs, eicosapentaenoic acid (EPA, 20:5(n-3)) was dominant (25.4%), followed by 12.8% arachidonic acid (AA, 20:4(n-6)). α-Tocopherol and γ-tocopherol contents were 55.2 μg g⁻¹ dry weight and 51.3 μg g⁻¹ dry weight respectively.     

Identification of the alga known as <Emphasis Type="Italic">Nannochloropsis</Emphasis> Z-1 isolated from a prawn farm in Hainan,China as <Emphasis Type="Italic">Chlorella</Emphasis>

An alga known as “Nannochloropsis”, isolated from a prawn farm in Hainan, China, has been critically investigated and identified as Chlorella, a member of the Chlorophyceae based on fatty acid composition, ultrastructure, and 18S rDNA. Cells of this alga were spherical, measured by 1–6 μm in diameter and were enclosed in thin walls of approximately 0.04 μm thickness. They contained several small mitochondria, two to three thylakoids and had no vacuoles. There were many pyrenoids in the algal cells and their thylakoid lamellae were sparse and not translucent. Many lipid droplets were present in the cytoplasm. The total lipid content of this alga was 3% per gram dry weight and its major fatty acids were C_16:0, C_18:0, C_18:1, C_18:2, C_18:3 and C_20:0. Eicosapentaenoic acid (C_20:5, EPA) was not detected. The length of its 18S rDNA sequence was 1,712 bp. 18S rDNA sequence analyses indicated that this alga was a species of Chlorella.     

<Emphasis Type="Italic">Acinetobacter soli</Emphasis> sp. nov., isolated from forest soil

A non-motile and rod shaped bacterium, designated strain B1(T), was isolated from forest soil at Mt. Baekwoon, Republic of Korea. Cells were Gram-negative, catalase-positive, and oxidase-negative. The major fatty acids were 9-octadecenoic acid (C(18:1) omega9c; 42%) and hexadecanoic acid (C(16:0); 25.9%) and summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16:1) omega7c; 10.0%). The DNA G+C content was 44.1 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain B1(T) formed a lineage within the genus Acinetobacter and was closely related to A. baylyi DSM 14961(T) (98.6% sequence similarity), followed by A. baumannii DSM 30007(T) (97.4%), A. calcoaceticus DSM 30006(T) (97.0%) and 3 genomic species (96.8 approximately 7.6%). Phenotypic characteristics, gyrB gene sequence analysis and DNA-DNA relatedness data distinguished strain B1(T) from type strains of A. baylyi, A. baumannii, and A. calcoaceticus. On the basis of the evidence presented in this study, strain B1(T) represents a novel species of the genus Acinetobacter, for which the name Acinetobacter soli sp. nov. is proposed. The type strain is B1(T) (= KCTC 22184(T)= JCM 15062(T)).     

Lipid synthesis by <Emphasis Type="Italic">Geomyces pannorum</Emphasis> under the impact of stress factors

Lipogenic activity and fatty acid composition of two strains of Geomyces pannorum were studied in the course of fungal growth. The strains were isolated from an Arctic cryopeg lens (VKM FW-2241) and from Central Russia (VKM F-3808). The adaptive reactions in both strains towards the temperature decreasing to 2°C involved intensification of the fatty acid desaturation. The degree of lipid unsaturation increased mainly due to a higher amount of α-linolenic acid (α-C_18:3) especially in the case of strain VKM FW-2241. Elevated NaCl concentration in the medium enhanced the level of linoleic acid (C_18:2) which apparently played a specific role in osmoprotection. Strain VKM FW-2241 was more tolerant to increased salinity than strain VKM F-3808. Almost complete inhibition of the growth of strains VKM F-3808 and VKM FW-2241 occurred at salinity of 10 and 20%, respectively; however, the viability of the strains was not affected. Under the combined effect of high salinity and hypothermia, the ratio between C_18:2 and α-C_18:3 acids was intermediate, indicating that these acids were involved in two adaptation mechanisms. The inhibition of fungal growth under stress was found to result in lipid overproduction. An increased pool of energy-rich lipids in fungi possibly contributes to their strategy of cell survival.     

Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.     

Differences in growth,total lipid content and fatty acid composition among 60 clones of <Emphasis Type="Italic">Cylindrotheca fusiformis</Emphasis>

Differences between clones of the diatom Cylindrotheca fusiformi were studied with respect to growth rate, total lipid content and fatty acid composition. Sixty clones were isolated and cultivated under batch conditions. All clones were grown under identical conditions (temperature 22±1°C, light intensity 100 μmol photon m⁻² s⁻¹, salinity 28, F/2 medium) and were harvested in the late exponential growth phase for lipid and fatty acid analysis. The results show a wide variation in growth, total lipid content and fatty acid profiles among clones (p<0.05). The major fatty acids in the 60 clones were 14:0 (4.6–9.1%), 16:0 (18.2–32.0%), 16:1n-7 (21.6–33.1%), 20:4n-6 (4.1–13.5%) and 20:5n-3 (6.2–17.2%), with the highest proportion of 20:4n-6 in clone CF13 (13.5%), and the highest proportion of 20:5n-3 in clone CF5 (17.2%). The results support the view that some microalgal fatty acid variation is not restricted to interspecific variation and external factors, but also varies from clone to clone within the same species.