Improving arachidonic acid fermentation by Mortierella alpina through multistage temperature and aeration rate control in bioreactor

Effective production of arachidonic acid (ARA) using Mortierella alpina was conducted in a 30-L airlift bioreactor. Varying the aeration rate and temperature significantly influenced cell morphology, cell growth, and ARA production, while the optimal aeration rate and temperature for cell growth and product formation were quite different. As a result, a two-stage aeration rate control strategy was constructed based on monitoring of cell morphology and ARA production under various aeration rate control levels (0.6–1.8 vvm). Using this strategy, ARA yield reached 4.7 g/L, an increase of 38.2% compared with the control (constant aeration rate control at 1.0 vvm). Dynamic temperature-control strategy was implemented based on the fermentation performance at various temperatures (13–28°C), with ARA level in total cellular lipid increased by 37.1% comparing to a constant-temperature control (25°C). On that basis, the combinatorial fermentation strategy of two-stage aeration rate control and dynamic temperature control was applied and ARA production achieved the highest level of 5.8 g/L.     

Production of arachidonic acid byMortierella fungi

The growing interest in the application of arachidonic acid (ARA) in various fields of health and dietary requirements has elicited much attention on the industrial production of ARA-containing oil by the cultivation ofMortierella fungi. For the industrial production of ARA, various studies, such as isolation of a high-potential strain and optimization of culture conditions, have been conducted. Studies including the investigation of morphology are important because ARA is accumulated in the mycelia, and thus cultivation with high biomass concentration is essential for obtaining a high ARA yield. Combining the results derived from various studies, a high ARA yield was attained in an industrial fermentor. These ARA production techniques are applicable to the production of other polyunsaturated fatty acids (PUFAs), and will contribute to the improvement of fermentation technology especially in the field of fungal cultivation.     

Transgenic production of arachidonic acid in oilseeds

We describe a transgenic microalgal Δ9-elongase pathway transformed in both Brassica napus and Arabidopsis thaliana seed resulting in the production of arachidonic acid (ARA). This pathway is noteworthy for both the production of ARA in seed tissue and the low levels of intermediate C20 fatty acids that accumulate. We also demonstrate that the arachidonic acid is naturally enriched at the sn2 position in triacylglycerol. This is the first report of ARA production by the Δ9-elongase pathway in an oilseed.     

Arachidonic-acid production by species of Mortierella

A growth-inhibiting, aspirin-containing medium was developed to select arachidonic-acid-(ARA)-producing Mortierella species and to determine the fatty-acid content of 87 Mortierella strains. ARA was detected in 66 strains from 33 species and its production may prove useful for systematic studies on Mortierella spp. The ARA content of the 66 producing strains tested ranged from 4% to 55% of total lipids. Most of the ARA-producing strains of Mortierella (59 strains) grown on potato/glucose/agar synthesized <20% ARA. Four strains produced between 20% and 25% ARA and three produced >40%. An inverse relationship was observed between ARA and oleic-acid contents.Names used in this article are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Enhancing arachidonic acid production by <Emphasis Type="Italic">Mortierella alpina</Emphasis> ME-1 using improved mycelium aging technology

Traditional mycelium aging technology was improved to enhance arachidonic acid (ARA) production by Mortierella alpina ME-1. Filtration step was skipped and additional carbon and nitrogen sources were fed during aging. The levels of the significant factors (time, temperature, ethanol, and KNO₃) affecting ARA production during improved aging process were also optimized by applying response surface methodology (RSM), and the maximum ARA yield of 19.02 g/l was achieved in a 5 l fermentor at 5.6 days, temperature 13.7 °C, ethanol 42.44 g/l, and KNO₃ 2.62 g/l. This yield was 1.55 times higher than that of traditional aging technology. The improved mycelium aging technology is considered to be a useful strategy for enhancing ARA production.     

Biochemical and Molecular Basis of Pesticide Degradation by Microorganisms

Abstract

Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.     

A novel two-step fermentation process for improved arachidonic acid production by Mortierella alpina

A novel two-step fermentation process was developed to enhance arachidonic acid (ARA) production by Mortierella alpina ME-1 in a 5 l fermentor. Agitation speed and aeration rate were adjusted from 180 to 40 rpm and from 0.6 to1 vvm, respectively, after 5 days cultivation, to decrease physical damage to the mycelia and to extend the stationary phase. Moreover, 3% (w/v) and 2% (w/v) ethanol were fed after 5 and 7 days cultivation, respectively, to enhance ARA content of total lipid. Eventually, an ARA yield of 19.8 g/l was achieved, which was 1.7 times higher than that of a one-step fed-batch cultivation.     

Quantitative trait locus analysis of symbiotic nitrogen fixation activity in the model legume <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Many legumes form nitrogen-fixing root nodules. An elevation of nitrogen fixation in such legumes would have significant implications for plant growth and biomass production in agriculture. To identify the genetic basis for the regulation of nitrogen fixation, quantitative trait locus (QTL) analysis was conducted with recombinant inbred lines derived from the cross Miyakojima MG-20 × Gifu B-129 in the model legume Lotus japonicus. This population was inoculated with Mesorhizobium loti MAFF303099 and grown for 14 days in pods containing vermiculite. Phenotypic data were collected for acetylene reduction activity (ARA) per plant (ARA/P), ARA per nodule weight (ARA/NW), ARA per nodule number (ARA/NN), NN per plant, NW per plant, stem length (SL), SL without inoculation (SLbac−), shoot dry weight without inoculation (SWbac−), root length without inoculation (RLbac−), and root dry weight (RWbac−), and finally 34 QTLs were identified. ARA/P, ARA/NN, NW, and SL showed strong correlations and QTL co-localization, suggesting that several plant characteristics important for symbiotic nitrogen fixation are controlled by the same locus. QTLs for ARA/P, ARA/NN, NW, and SL, co-localized around marker TM0832 on chromosome 4, were also co-localized with previously reported QTLs for seed mass. This is the first report of QTL analysis for symbiotic nitrogen fixation activity traits.     

Oligounsaturated fatty acid production by selected strains of micromycetes

Fifteen strains of filamentous fungi from theCulture Collection of Fungi (Charles University, Prague) were tested for their lipid production, fatty acid composition with emphasis on accumulation of oligounsaturated fatty acids. All cultures contained palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2) and γ-linolenic (18:3) acid (GLA). The mycelium ofCunninghamella elegans, Rhizopus arrhizus, Mortierella parvispora, M. elongata andM. alpina contained arachidonic acid (ARA) in the range of 2.3–33.5% of the total fatty acids. The strains used in our experiment were capable to accumulate a relatively high amount of intracellular lipid (9.6–20.1% in dry biomass). The highest content of GLA (22.3 mg/g) was found inMucor circinelloides. The strain ofM. alpina containing 47.1 mg/g of ARA could be considered as the best producer of ARA.     

Identification of a novel type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis

Arachidonic acid (ARA) is a polyunsaturated fatty acid (PUFA) and an essential component of membrane lipids. However, the PUFA synthase required for ARA biosynthesis has not been identified in any organism. To identify the PUFA synthase producing ARA, we determined the draft genome sequence of the marine bacterium Aureispira marina, which produces a high level of ARA, and found a gene cluster encoding a putative PUFA synthase for ARA production. Expression of the gene cluster in Escherichia coli induced production of ARA, demonstrating that the gene cluster encodes a PUFA synthase required for ARA biosynthesis.     

Spore germination in <Emphasis Type="Italic">Mortierella alpina</Emphasis> is associated with a transient depletion of arachidonic acid and induction of fatty acid desaturase gene expression

Mortierella alpina is an oleaginous filamentous fungus whose vegetative mycelium is known to accumulate triglyceride oil containing large amounts of arachidonic acid (ARA 20:4, n − 6). We report that the spores of Mortierella alpina also contain a large proportion of ARA, comprising 50% of total fatty acid. Fatty acid desaturase genes were not expressed in dormant spores but were induced during germination, following a significant drop in the level of ARA (down from 50% of total fatty acid to 12%) prior to germ-tube emergence. We propose that ARA serves as a reserve supply of carbon and energy that is utilised during the early stages of spore germination in Mortierella alpina.     

NADP+-Dependent D-Arabinose Dehydrogenase Shows a Limited Contribution to Erythroascorbic Acid Biosynthesis and Oxidative Stress Resistance in Saccharomyces cerevisiae

The molecular aspects and physiological significance of NADP⁺-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP⁺-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H₂O₂ and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H₂O₂ with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.     

Production of Lactones and Peroxisomal Beta-Oxidation in Yeasts

Abstract

Among aroma compounds interesting for the food industry, lactones may be produced by biotechnological means using yeasts. These microorganisms are able to synthesize lactones de novo or by biotransformation of fatty acids with higher yields. Obtained lactone concentrations are compatible with industrial production, although detailed metabolic pathways have not been completely elucidated. The biotransformation of ricinoleic acid into gamma-decalactone is taken here as an example to better understand the uptake of hydroxy fatty acids by yeasts and the different pathways of fatty acid degradation. The localization of ricinoleic acid beta-oxidation in peroxisomes is demonstrated. Then the regulation of the biotransformation is described, particularly the induction of peroxisome proliferation and peroxisomal beta-oxidation and its regulation at the genome level. The nature of the biotransformation product is then discussed (4-hydroxydecanoic acid or gamma-decalactone), because the localization and the mechanisms of the lactonization are still not properly known. Lactone production may also be limited by the degradation of this aroma compound by the yeasts which produced it. Thus, different possible ways of modification and degradation of gamma-decalactone are described.     

Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors

The blood–brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n ? 6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell^® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E₂ (PGE₂), an eicosanoid known to facilitate opening of the blood–brain barrier. Permeability following ARA or PGE₂ exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP₃ and EP₄ receptor antagonists attenuated the ARA‐mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE₂ which acts upon EP₃ and EP₄ receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.

     

Production of arachidonic acid by Mortierella fungi

Summary Various Mortierella fungi were assayed for their productivity of arachidonic acid (ARA). Only strains belonging to the subgenus Mortierella accumulated detectable amounts of ARA together with dihomo--linolenic acid. None of the strains belonging to the subgenus Micromucor tested accumulated these C-20 fatty acids, although they produced a C-18 fatty acid, -linolenic enic acid. A soil isolate, M. alpina 1S-4, was found to grow well in a liquid medium containing glucose and yeast extract as carbon and nitrogen sources, respectively. Addition of several natural oils such as olive and soybean oils to the medium increased the accumulation of ARA. Under optimal culture conditions in a 5-1 bench-scale fermentor, the fungus produced 3.6 g/l of ARA in 7 days. On cultivation for 10 days at 28°C in a 2000-1 fermentor, the same fungus produced 22.5 kg/kl mycelia (dry weight) containing 9.9 kg lipids, in which ARA comprised 31.0% of the total fatty acids. On standing the harvested mycelia for a further 6 days, major mycelial fatty acids (i.e. palmitic acid, oleic acid, linoleic acid, etc.) other than ARA rapidly decomposed and the ARA content of the total fatty acids reached nearly 70%.     

Diet‐switching experiments show rapid accumulation and preferential retention of highly unsaturated fatty acids in Daphnia

Zooplankton transfer ecologically important fatty acids (FA) from their diets to upper trophic levels. We used diet‐switching experiments with ¹³C‐labeled food sources to determine the time scale at which dietary uptake is manifested in the FA profiles of Daphnia magna. Daphnia dramatically shifted their FA composition in response to diet change within only four days, however Daphnia switched from a high quality (i.e. Cryptomonas) to a moderate quality (Scenedesmus) diet retained the most physiologically important FA from their original diet source even after 14 days. In particular, Daphnia exhibited long‐term retention of eicosapentaenoic (EPA; 20:5ω3) and arachidonic acid (ARA; 20:4ω6) when switched from Cryptomonas to Scenedesmus. Similarly, when switched from Scenedesmus to Cryptomonas, Daphnia took up a high proportion of EPA and ARA after only two days. The phospholipid fatty acid (PLFA) fraction in Daphnia was preferentially enriched with stearic (18:0), oleic (18:1ω9), and linoleic acid (LIN; 18:2ω6). In contrast with studies of marine copepods, dietary FA also strongly affected the PLFA composition (structural lipids) of Daphnia. Results of δ¹³C signatures of individual FA provided evidence of elongation and desaturation of α‐linolenic (ALA; 18:3ω3) or stearidonic acid (SDA; 18:4ω3) to EPA 10 days after a diet switch to EPA‐deficient Scenedesmus. Differences in the ARA content of Daphnia fed Cryptomonas and Scenedesmus suggest Daphnia consuming Cryptomonas synthesized ARA via retroconversion of ω6‐docosapentaenoic acid (ω6‐DPA; 22:5ω6). Daphnia preferentially accumulate and retain, as well as bioconvert, those FA that are also most physiologically important for fish production. Our results also indicate Daphnia FA composition responds to their diet on a short temporal scale and analyses of lipid biomarkers in zooplankton provide strong insights into the food sources that support their production.     

Chemical Composition and Anti‐Alzheimer's Disease‐Related Activities of a Functional Oil from the Edible Seaweed Hizikia fusiforme

Cholinergic disorder, oxidative stress, and neuroinflammation play important roles in the pathology of Alzheimer's disease. To explore the healthy potential of the edible seaweed Hizikia fusiforme on this aspect, a functional oil (HFFO) was extracted from this alga and investigated on its constituents by gas chromatography‐mass spectrometry (GC/MS) in this study. Its anti‐Alzheimer's related bioactivities including acetylcholinesterase (AChE) inhibition, antioxidation, and anti‐neuroinflammation were evaluated, traced, and simulated by in vitro and in silico methods. GC/MS analysis indicated that HFFO mainly contained arachidonic acid (ARA), 11,14,17‐eicosatrienoic acid (ETrA), palmitic acid, phytol, etc. HFFO showed moderate AChE inhibition and antioxidant activity. Bioactivity tracing using commercial standards verified that AChE inhibition of HFFO mainly originated from ARA and ETrA, whereas antioxidant activity mainly from ARA. Lineweaver?Burk plots showed that both ARA and ETrA are noncompetitive AChE inhibitors. A molecular docking study demonstrated low CDOCKER interaction energy of ?26.33 kcal/mol for ARA and ?43.70 kcal/mol for ETrA when interacting with AChE and multiple interactions in the ARA (or ETrA)?AChE complex. In the anti‐neuroinflammatory evaluation, HFFO showed no toxicity toward BV‐2 cells at 20 μg/mL and effectively inhibited the production of nitroxide and reduced the level of reactive oxygen species in lipopolysaccharide‐induced BV‐2 cells. The results indicated that HFFO could be used in functional foods for its anti‐Alzheimer's disease‐related activities.     

7 The Molecular Biology of Tryptophan Synthase: A Model for Protein–Protein Interaction

Abstract

The use of plastic produced from non-renewable resources constitutes a major environmental problem of the modern society. Polylactide polymers (PLA) have recently gained enormous attention as one possible substitution of petroleum derived polymers. A prerequisite for high quality PLA production is the provision of optically pure lactic acid, which cannot be obtained by chemical synthesis in an economical way. Microbial fermentation is therefore the commercial option to obtain lactic acid as monomer for PLA production. However, one major economic hurdle for commercial lactic acid production as basis for PLA is the costly separation procedure, which is needed to recover and purify the product from the fermentation broth. Yeasts, such as Saccharomyces cerevisiae (bakers yeast) offer themselves as production organisms because they can tolerate low pH and grow on mineral media what eases the purification of the acid. However, naturally yeasts do not produce lactic acid. By metabolic engineering, ethanol was exchanged with lactic acid as end product of fermentation. A vast amount of effort has been invested into the development of yeasts for lactic acid production since the first paper on this topic by Dequin and process insight. If pH stress is used as basis for DNA microarray analyses, in order to improve the host, what exactly is addressed? Growth? Or productivity? They might be connected, but can be negatively correlated. A better growing strain might not be a better producer. So if the question was growth, the answer might not be what was initially intended (productivity).

A major task for the future is to learn to ask the right questions – a lot of studies intended to lead to better productivity, did lead to interesting results, but NOT to better production strains.

Taking together what we learned from lactic acid production with yeasts, we see a bright future for bulk and fine chemical production with these versatile hosts.     

Influence of some metal ions on the lipid content and arachidonic acid production byMortierella sp.

The influence of Ca²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ ions on lipid accumulation, fatty acid composition and arachidonic acid (ARA) production byMortierella sp. S-17 was investigated. A beneficial effect of Mn²⁺ in the concentration range of 2–500 mg/L on lipogenesis was observed. The other elements at about 1 g/L repressed lipid accumulation and ARA yield. The highest yield of ARA (723 mg per liter or 148 mg per gram of dry mycelium) after incubation of the fungus in a glucose medium in the presence of 2 mg Mn²⁺ per liter was obtained. A strong inhibitory effect of Fe²⁺ (above 40 mg/L) on ARA formation was observed.     

Dietary linoleic acid has no effect on arachidonic acid,but increases n-6 eicosadienoic acid,and lowers dihomo-γ-linolenic and eicosapentaenoic acid in plasma of adult men

High intakes of linoleic acid (LA,18:2n-6) have raised concern due to possible increase in arachidonic acid (ARA, 20:4n-6) synthesis, and inhibition of alpha linolenic acid (ALA, 18:3n-3) desaturation to eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In healthy men, 10.5% energy compared to 3.8% energy LA with 1% energy ALA increased plasma phospholipid LA and 20:2n-6, the elongation product of LA, and decreased EPA, with no change in ARA. However, LA was inversely related to ARA at both 10.5% energy and 3.8% energy LA, (r=?0.761, r=?0.817, p<0.001, respectively). A two-fold variability in ARA among individuals was not explained by the dietary LA, ARA, ALA, or fish intake. Our results confirm LA requirements for ARA synthesis is low, <3.8% energy, and they suggest current LA intakes saturate Δ-6 desaturation and adversely affect n-3 fatty acid metabolism. Factors other than n-6 fatty acid intake are important modifiers of plasma ARA.