Production of Novel Tetrahydroxyfuranyl Fatty Acids from α-Linolenic Acid by Clavibacter sp. Strain ALA2

Previously, it was reported that a newly isolated microbial culture, Clavibacter sp. strain ALA2, produced trihydroxy unsaturated fatty acids, diepxoy bicyclic fatty acids, and tetrahydroxyfuranyl fatty acids (THFAs) from linoleic acid (C. T. Hou, J. Am. Oil Chem. Soc. 73:1359-1362, 1996; C. T. Hou and R. J. Forman III, J. Ind. Microbiol. Biotechnol. 24:275-276, 2000; C. T. Hou, H. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 75:1483-1487, 1998; C. T. Hou, H. W. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 78:1167-1169, 2001). In this study, we found that Clavibacter sp. strain ALA2 produced novel THFAs, including 13,16-dihydroxy-12-THFA, 15-epoxy-9(Z)-octadecenoic acid (13,16-dihydroxy-THFA), and 7,13,16-trihydroxy-12, 15-epoxy-9(Z)-octadecenoic acid (7,13,16-trihydroxy-THFA), from α-linolenic acid (9,12,15-octadecatrienoic acid). The chemical structures of these products were determined by gas chromatography-mass spectrometry and proton and ¹³C nuclear magnetic resonance analyses. The optimum incubation temperature was 30°C for production of both hydroxy-THFAs. 13,16-Dihydroxy-THFA was detected after 2 days of incubation, and the concentration reached 45 mg/50 ml after 7 days of incubation; 7,13,16-trihydroxy-THFA was not detected after 2 days of incubation, but the concentration reached 9 mg/50 ml after 7 days of incubation. The total yield of both 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA was 67% (wt/wt) after 7 days of incubation at 30°C and 200 rpm. In previous studies, it was reported that Clavibacter sp. strain ALA2 oxidized the C-7, C-12, C-13, C-16, and C-17 positions of linoleic acid (n-6) into hydroxy groups. In this case, the bond between the C-16 and C-17 carbon atoms is saturated. In α-linolenic acid (n-3), however, the bond between the C-16 and C-17 carbon atoms is unsaturated. It seems that enzymes of strain ALA2 oxidized the C-12-C-13 and C-16-C-17 double bonds into dihydroxy groups first and then converted them to hydroxy-THFAs.     

Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids

The conversion of the plant-derived omega-3 (n-3) α-linolenic acid (ALA, 18:3n-3) to the long-chain eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) can be increased by ALA sufficient diets compared to ALA deficient diets. Diets containing ALA above an optimal level result in no further increase in DHA levels in animals and humans. The present study evaluates means of maximizing plasma DHA accumulation by systematically varying both linoleic acid (LA, 18:2n-6) and ALA dietary level. Weanling rats were fed one of 54 diets for three weeks. The diets varied in the percentage of energy (en%) of LA (0.07–17.1 en%) and ALA (0.02–12.1 en%) by manipulating both the fat content and the balance of vegetable oils. The peak of plasma phospholipid DHA (>8% total fatty acids) was attained as a result of feeding a narrow dietary range of 1–3 en% ALA and 1–2 en% LA but was suppressed to basal levels (～2% total fatty acids) at dietary intakes of total polyunsaturated fatty acids (PUFA) above 3 en%. We conclude it is possible to enhance the DHA status of rats fed diets containing ALA as the only source of n-3 fatty acids but only when the level of dietary PUFA is low (<3 en%).     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

Conversion of alpha-linolenic acid to longer-chain polyunsaturated fatty acids in human adults

The principal biological role of alpha-linolenic acid (alphaLNA; 18:3n-3) appears to be as a precursor for the synthesis of longer chain n-3 polyunsaturated fatty acids (PUFA). Increasing alphaLNA intake for a period of weeks to months results in an increase in the proportion of eicosapentaenoic acid (EPA; 20:5n-3) in plasma lipids, in erythrocytes, leukocytes, platelets and in breast milk but there is no increase in docosahexaenoic acid (DHA; 22:6n-3), which may even decline in some pools at high alphaLNA intakes. Stable isotope tracer studies indicate that conversion of alphaLNA to EPA occurs but is limited in men and that further transformation to DHA is very low. The fractional conversion of alphaLNA to the longer chain n-3 PUFA is greater in women which may be due to a regulatory effect of oestrogen. A lower proportion of alphaLNA is used for beta-oxidation in women compared with men. Overall, alphaLNA appears to be a limited source of longer chain n-3 PUFA in humans. Thus, adequate intakes of preformed long chain n-3 PUFA, in particular DHA, may be important for maintaining optimal tissue function. Capacity to up-regulate alphaLNA conversion in women may be important for meeting the demands of the fetus and neonate for DHA.     

Effect of environmental factors on the production of oxygenated unsaturated fatty acids from linoleic acids by Bacillus megaterium ALA2

We identified [Hou CT (2003) New uses of vegetable oils: novel oxygenated fatty acids by biotransformation. SIM News 53:56–61] many novel oxygenated fatty acids produced from linoleic acid by Bacillus megaterium ALA2: 12,13,17-trihydroxy-9(Z)-octadecenoic acid (12,13,17-THOA); 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA); 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid. 12,13,17-THOA, the main product, has antiplant pathogenic fungal activity. To develop an industrial process for the production of these new oxygenated fatty acids by strain ALA2, the effect of environmental factors on the production and their impact on the amount of various products were studied. Dextrose at 5 g/l was the optimum amount for the carbon source. A combination of 15 g yeast extract and 10 g tryptone showed good results as nitrogen sources. Among the metal ions tested, the optimum concentrations for the reaction for the different ions were as follows (in mM): magnesium 2.0, iron 0.5, zinc 0.1, nickel 0.01, and cobalt 0.05. Copper ions did not affect the production of oxygenated products; however, manganese ions inhibited the reaction. Addition of these metal ions did not alter the distribution of products. The optimum temperature and pH for the production of THOAs were 30°C and pH 6.5. Time course studies showed 40–48 h is the optimum for the production of both THOAs. These data provide the basis for engineering scale-up production of these new products.     

Production of D-lactic acid from 1,2-propanediol byPseudomonas sp. strain TB-135

Summary A newly isolated bacterium, strain TB-135, produces solely D-lactic acid from 1,2-propanediol (1,2-PD). From taxonomical studies, the strain was concluded to belong to the genusPseudomonas. The optimal conditions for the acid production were found to be at 4% 1,2-PD and 0.2% yeast extract, when the strain produced 21mg/ml of the acid of a high optical purity (over 99% e.e.) after 4days of cultivation. Since it was considered that the acid was produced from (R)-(–)-1,2-PD, the molar yield was estimated to be 86%.     

Production of 10-ketostearic acid from oleic acid by a strain of Flavobacterium sp. 12-4A

Summary A new Flavobacterium sp., strain 12-4A, produces solely 10-ketostearic acid (KSA) from oleic acid (OA) in growing cultures. Under optimized conditions 14 mg/ml of KSA was produced from 25 mg/ml of OA after 3 days of cultivation at 30°C.     

Production of a novel copolyester of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids by Pseudomonas sp. 61-3 from sugars

 Pseudomonas sp. 61-3 (isolated from soil) produced a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA) of C₆, C₈, C₁₀ and C₁₂, when sugars of glucose, fructose and mannose were fed as the sole carbon source. The polyester produced was a blend of homopolymer and copolymer, which could be fractionated with boiling acetone. The acetone-insoluble fraction of the polyester was a homopolymer of 3-hydroxybutyrate units [poly (3HB)], while the acetone-soluble fraction was a copolymer [poly(3HB-co-3HA)] containing both short- and medium-chain-length 3-hydroxyalkanoate units ranging from C₄ to C₁₂:44 mol% 3-hydroxybutyrate, 5 mol% 3-hydroxyhexanoate, 21 mol% 3-hydroxyoctanoate, 25 mol% 3-hydroxydecanoate, 2 mol% 3-hydroxydodecanoate and 3 mol% 3-hydroxy-5-cis-dodecenoate. The copolyester was shown to be a random copolymer of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoate units by analysis of the ¹³C-NMR spectrum. The poly(3HB) homopolymer and poly (3HB-co-3HA) copolymer were produced simultaneously within cells from glucose in the absence of any nitrogen source, which suggests that Pseudomonas sp. 61-3 has two types of polyhydroxy-alkanoate syntheses with different substrate specificities. Received: 9 June 1995/Received last revision: 30 October 1995/Accepted: 6 November 1995     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

Utilization of amino acids by Chromatium sp. strain D

Summary The duration of various morphologically distinct phases in the division cycle of the marine heterotroph Cryptothecodinium cohnii was measured in cultures initiated with synchronously excysted swarmer cells. Parent cysts were selectively isolated on plastic surfaces and progeny of a narrow age distribution harvested in a specifically conditioned medium. The swarmer phase, an interval of predivisional encystment, daughter cell formation and excystment were 5.0, 3.0 and 2.0 h respectively. Two major kinds of cytokinesis (production of 2 and 4 daughter cells) were observed resulting in a mean daughter cell number of 2.7 under these conditions. Other growth parameters for this dinoflagellate are described.     

Production of ω3 fatty acids in marine cyanobacterium Synechococcus sp. strain NKBG 15041c via genetic engineering

Omega-3 fatty acids (ω3 FAs) have attracted attention because they have various health benefits for humans. Fish oils are currently major sources of ω3 FAs, but a sustainable supply of ω3 FAs based on fish oils is problematic because of the increasing demand. In this study, the production potential of a genetically engineered marine cyanobacterium, Synechococcus sp. strain NKBG 15041c, was examined as an alternative source of ω3 FAs. A change in fatty acid composition of this cyanobacterium was successfully induced by the expression of a heterologous Δ6-desaturase, and the transformants synthesized stearidonic acid, which the wild type cannot produce. As a result of optimization of culture conditions, maximal contents of stearidonic acid and total ω3 FAs reached 12.2 ± 2.4 and 118.1 ± 3.5 mg/g, respectively. The maximal ω3 FA productivity was 4.6 ± 0.7 mg/(L⋅day). These are the highest values of the contents of stearidonic acid and ω3 FAs in genetically engineered cyanobacteria reported thus far. Therefore, genetically engineered Synechococcus sp. strain NKBG 15041c may be a promising sustainable source of ω3 fatty acids.     

微小杆菌(Exiguobacterium sp.)对肉桂酸降解行为

【目的】为有效缓解自毒物质肉桂酸对西瓜等作物生长的危害,从宁夏中卫硒砂瓜连作土壤中分离筛选得到一株高效降解肉桂酸的菌株,研究其基本降解特性。【方法】分离筛选得到一株能有效利用肉桂酸生长的菌株,采用16S r RNA基因序列分析进行菌株鉴定,运用高效液相色谱法和西瓜幼苗生长毒性实验检测降解特性。【结果】从多年西瓜连作土壤中筛选得到一株高效降解肉桂酸的细菌R30,鉴定为Exiguobacterium sp.,其96 h内对肉桂酸的降解率可达99%以上,最适降解温度和p H分别为30°C、p H 7.0。除肉桂酸外,该菌也能够高效降解香豆酸、阿魏酸、苯甲酸等其他酚酸类物质,表现出一定的底物广谱性;检测96 h降解液对西瓜种子萌发直至幼苗生长阶段的影响表明,该菌株可有效缓解肉桂酸对西瓜幼苗的生长抑制作用。【结论】菌株R30在肉桂酸、香豆酸、阿魏酸、苯甲酸等酚酸类物质导致的农作物连作障碍治理领域具有潜在的开发应用价值。     

Production of volatile fatty acids from bagasse by rumen bacteria

Summary Conditions are described for converting bagasse lignocellulose to volatile fatty acids (VFA) by anaerobic fermentation. Although yields of VFA were as high as 74% by weight of digestible organic matter (or 54% of dry bagasse), limitations were imposed by both fermenter design and fibre digestibility. All fermentations were substrate-limited up to the maximum initial concentration examined of 50 g bagasse · l^-1 and no product inhibition was evident (up to 260 mM VFA produced). Maximum VFA productivities of 0.25 to 0.65 g · l^-1 · h^-1 were obtained in batch fermentations and this is greater than those previously reported using lignocellulosic substrates. Batch fermentations neared completion after 66 h.     

Production of extracellular higher fatty acids by yeasts grown on hexadecanoic acid

Production of exocellular higher fatty acids by Candida yeasts was studied during their growth in a mineral medium with hexadecane. The qualitative and quantitative composition of exocellular higher fatty acids was investigated during cultivation of Candida lipolytica VCM Y 2378(695) under the conditions of different aeration (5 and 80% saturation of the medium with oxygen). Palmitic (C16:0), palmitooleic (C16:1), oleic (C18:1) and linoleic (C18:2) acids predominated among other higher fatty acids. The overall amount of fatty acids increased and the content of unsaturated fatty acids decreased when the yeast growth was limited with oxygen.     

Lignin transformation by a versatile peroxidase from a novel Bjerkandera sp. strain

A versatile peroxidase, purified from a novel strain of Bjerkandera sp. (B33/3), was tested for its reactivity on a lignin fraction obtained from straw pulping. The effects of such processing parameters as reaction time, pH, and lignin:enzyme ratio were evaluated. Gel filtration chromatography was employed to characterise the molecular mass distribution of the lignin fragments produced by the enzyme-mediated reaction. Our results have shown that such a versatile peroxidase can directly bring about transformations of lignin, even in the absence of external mediators.     

Utilization of amino acids by Frankia sp. strain CpI1

The utilization of some amino acids, added at 1 mM and 10 mM concentrations, as the sole combined nitrogen sources by Frankia sp. strain CpI1, has been investigated. Glutamine, like NH ₄ ⁺ , provided rapid growth without N₂ fixation. Histidine at 1 mM yielded poor N₂-fixing activity but better cell growth than N₂. Aspartate, glutamate, alanine, proline, each at 1 mM concentration, supported similar levels of N₂ fixation and growth. Growth on 10 mM glutamate, proline, or histidine resulted in poor N₂-fixing activity and poor cell growth. Cells grown on 10 mM alanine had about half the N₂-fixing activity of cells grown on N₂ but growth was good. Aspartate at 10 mM concentration, however, stimulated N₂-fixing activity dramatically and promoted faster growth. Enzyme analysis suggested that asparate is catabolized by glutamate-oxaloacetate transaminase (GOT), since GOT specific activity was induced, and aspartase activity was not detected, in cells grown on aspartate as the sole combined nitrogen source. Thinlayer chromatography (TLC) of metabolites extracted from N₂-grown cells fed with [¹⁴C]-aspartate showed that label was rapidly accumulated mainly on aspartate and/or glutamate, depending on the cells' physiological state, without detectable labeling on fumarate or oxaloacetate (OAA). These findings provide evidence that aspartate is catabolized by GOT to OAA which, in turn, is rapidly converted to -ketoglutarate through the TCA cycle and then to glutamate by GOT or by glutamate synthase (GOGAT). The stimulation of N₂ fixation and growth by aspartate is probably caused by an increased intracellular glutamate pool.     

Glycerols and fatty acids isolated from Micractinium sp. KSF0031

From the collected extract from King Sejong Antarctic Station, strain Micractinium sp. KSF0031, led to the isolation of one new monoacyldigalactosyl glycerol (1) and seven known compounds (2–8). Their chemical structures were established using extensive spectroscopic techniques, including 1D, 2D-NMR, and MS, and compared with the published data. To the best of our knowledge, this is the first report to investigate the secondary metabolites from genus Micractinium. The monoacyldigalactosyl glycerol in Micractinium could serve as its chemotaxonomic markers.