Yasushi Kamisaka

Improvement of Docosahexaenoic Acid Production in a Culture of Thraustochytrium aureum by Medium Optimization

Growth optimization of a docosahexaenoic acid (DHA) producing marine fungus, Thraustochytrium aureum (ATCC 34304), resulted in cell productivity of 5.7 g/l and 460 mg/l of total lipid in 69 h flask culture, almost twice the level of cell productivity obtained for the strain reported previously. The lipid extracted from the cells was primarily composed of triacylglycerol and contained about 40% DHA. The fermentor culture showed a lower growth rate than the flask culture indicating growth inhibition due to mechanical stirring. Cells grown in the fermentor showed a coagulant tendency. ATCC 34304, in culture, seems to reproduce asexually and the life cycle of the cells consists of a zoospore stage, trophic cell stage and sporangium stage. Zoospore release was observed once during the early growth phase throughout growth period both in flask and fermentor culture.

DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the Δsnf2 disruptant of Saccharomyces cerevisiae

We previously found that SNF2, a gene encoding a transcription factor forming part of the SWI/SNF (switching/sucrose non-fermenting) chromatin-remodelling complex, is involved in lipid accumulation, because the Deltasnf2 disruptant of Saccharomyces cerevisiae has a higher lipid content. The present study was conducted to identify other factors that might further increase lipid accumulation in the Deltasnf2 disruptant. First, expression of LEU2 (a gene encoding beta-isopropylmalate dehydrogenase), which was used to select transformed strains by complementation of the leucine axotroph, unexpectedly increased both growth and lipid accumulation, especially in the Deltasnf2 disruptant. The effect of LEU2 expression on growth and lipid accumulation could be reproduced by adding large amounts of leucine to the culture medium, indicating that the effect was not due to Leu2p (beta-isopropylmalate dehydrogenase) itself, but rather to leucine biosynthesis. To increase lipid accumulation further, genes encoding the triacylglycerol biosynthetic enzymes diacylglycerol acyltransferase (DGA1) and phospholipid:diacylglycerol acyltransferase (LRO1) were overexpressed in the Deltasnf2 disruptant. Overexpression of DGA1 significantly increased lipid accumulation, especially in the Deltasnf2 disruptant, whereas LRO1 overexpression decreased lipid accumulation in the Deltasnf2 disruptant. Furthermore, the effect of overexpression of acyl-CoA synthase genes (FAA1, FAA2, FAA3 and FAA4), which each supply a substrate for Dga1p (diacylglycerol acyltransferase), was investigated. Overexpression of FAA3, together with that of DGA1, did not further increase lipid accumulation in the Deltasnf2 disruptant, but did enhance lipid accumulation in the presence of exogenous fatty acids. Lastly, the total lipid content in the Deltasnf2 disruptant transformed with DGA1 and FAA3 overexpression vectors reached approx. 30%, of which triacylglycerol was the most abundant lipid. Diacylglycerol acyltransferase activity was significantly increased in the Deltasnf2 disruptant strain overexpressing DGA1 as compared with the wild-type strain overexpressing DGA1; this higher activity may account for the prominent increase in lipid accumulation in the Deltasnf2 disruptant with DGA1 overexpression. The strains obtained have a lipid content that is high enough to act as a model of oleaginous yeast and they may be useful for the metabolic engineering of lipid production in yeast.

Lipid bodies and lipid body formation in an oleaginous fungus, Mortierella ramanniana var. angulispora

Mortierella ramanniana var. angulispora accumulates triacylglycerol (TG) in lipid bodies. Studies on lipid transport into lipid bodies are essential for elucidating mechanisms of lipid body formation. We used fluorescent dyes and fluorescent lipid analogs to visualize lipid body formation with a confocal laser scanning microscope. Different sizes of lipid bodies were stained by Nile red, a lipid body marker - one with a diameter of about 1 micrometer and the other with a diameter of about 2-3 micrometers. Lipid bodies matured into larger ones with culture. To metabolically monitor lipid bodies, we used 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-phosphatidic acid (C5-DMB-PA), and C5-DMB-phosphatidylcholine (C5-DMB-PC). These were taken up into fungal cells and incorporated into intracellular organelles at 30 degrees C. C5-DMB-PA was quickly incorporated into lipid bodies while C5-DMB-PC was initially incorporated into internal membranes, presumably endoplasmic reticulum membranes, and fluorescence was then gradually transported into lipid bodies. The transport of fluorescent lipids accompanied their metabolism into diacylglycerol (DG) and TG, which, taken together with the fluorescence distribution, suggested that conversion to TG was not necessary for transport into lipid bodies. It is likely that the synthesized DG was mainly located in lipid bodies and the conversion to TG took place in lipid bodies. C5-DMB-PA and C5-DMB-PC were converted to DG and TG in the membrane and lipid body fractions of this fungus, which agreed with in vivo metabolism of these fluorescent lipids and in vitro enzyme activity related to PA and PC metabolism. These results indicate that transport and metabolism of C5-DMB-PA and C5-DMB-PC represent two different routes for lipid body formation in this fungus.

Isolation of Δ12 and ω3‐fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and α‐linolenic acids in Saccharomyces cerevisiae

Two clones with homology to known fatty acid desaturase genes were isolated from the yeast Kluyveromyces lactis. The first gene, which we designate KlFAD2, consists of 411 amino acids with an overall identity of 73.0% to FAD2 from Saccharomyces kluyveri. It exhibited Δ12 fatty acid desaturase activity when expressed in S. cerevisiae under the control of ADH1 promoter and produced endogenous linoleic acid. The second clone, which we designate KlFAD3, consists of 415 amino acids with an overall identity of 79.3% to FAD3 from S. kluyveri. It exhibited ω3 fatty acid desaturase activity in S. cerevisiae when expressed under the control of ADH1 promoter in the presence of the exogenous substrate linoleic acid and produced α‐linolenic acid. Co‐expression of KlFAD2 and KlFAD3 resulted in the endogenous production of both linoleic and α‐linolenic acids. The yield of α‐linolenic acid reached 0.8% of total fatty acids and its production was not increased by adding exogenous oleic acid; α‐linolenic acid reached 8.7% when exogenous linoleic acid was available. Copyright

Optimization and scale-up of γ-linolenic acid production by Mortierella ramanniana MM 15-1, a high γ-linolenic acid producing mutant

Abstract Optimum conditions for cultivation of Mortierella ramanniana mutant MM 15-1 were investigated with an aim of producing a lipid which contains high γ-linolenic acid (GLA). The optimum initial glucose concentration and pH control level were 300 g/ l and 4.0, respectively. Although both pellet and filamentous forms of this mutant were observed during the cultivation, the pellets accumulated lipid with higher GLA content than the filamentous forms. The effects of culture conditions on pellet formation were therefore investigated in a 30- l jar fermentor. The results showed that with an inoculum spore concentration of 5.0 × 10 3 /ml and an agitation speed of 800 rpm, 0.15–0.5 mm pellets were formed and consequently, the GLA content of the lipid increased to a very high value of 18.3%. Furthermore, when this process was scaled up to a 10-kl fermentor, using the impeller tip velocity as the scale-up parameter, both the pellet formation and the lipid production as well as the GLA content of the lipid were consistent with those obtained in the 30- l jar fermentor.

Identification of genes affecting lipid content using transposon mutagenesis in Saccharomyces cerevisiae.

Genes involved in lipid accumulation were identified in Saccharomyces cerevisiae using transposon insertion mutagenesis. Five ORFs, such as SNF2, IRA2, PRE9, PHO90, and SPT21 were found from the analysis of the insertion sites in transposon insertion mutants with higher lipid content. Since these ORFs are not directly involved in storage lipid biosynthesis, we speculate that they are involved in carbon fluxes into storage lipids in response to nutrient conditions. Lipid analysis of disruptants of these ORFs indicated that the Δsnf2, and Δira2 disruptants had significantly higher lipid content. Cultivation in a nitrogen-limited medium increased the lipid content in all disruptants, among which the Δpre9 disruptant was the most sensitive to nitrogen limitation. We then focused on the Δsnf2 disruptant due to its higher lipid content and its function as a regulator of phospholipid synthesis. Lipid class analysis indicated that triacylglycerol and free fatty acids contributed to the increase in total lipids of the Δsnf2 disruptant. The addition of exogenous fatty acids was not so effective at increasing the lipid content in the Δsnf2 disruptant as it was in the wild type. It should be noticed that exogenous free linoleic acid was much higher in the Δsnf2 disruptant than in the wild type, as in the case of endogenous free fatty acids. In addition, the incorporation of exogenous fatty acids into cells increased in the disruptant, suggesting that fatty acid transporters were regulated by SNF2. The results suggest that metabolic fluxes into storage lipids, which are activated in the Δsnf2 disruptant, is repressed by the incorporation of exogenous fatty acids. They provide new insight into the biosynthesis of storage lipids in yeast.

Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH tolerance.

Saccharomyces cerevisiae produces saturated and monounsaturated fatty acids of 16‐ and 18‐carbon atoms and no polyunsaturated fatty acids (PUFAs) with more than two double bonds. To study the biological significance of PUFAs in yeast, we introduced Kluyveromyces lactis Δ12 fatty acid desaturase (KlFAD2) and ω3 fatty acid desaturase (KlFAD3) genes into S. cerevisiae to produce linoleic and α‐linolenic acids in S. cerevisiae. The strain producing linoleic and α‐linolenic acids showed an alkaline pH‐tolerant phenotype. DNA microarray analyses showed that the transcription of a set of genes whose expressions are under the repression of Rim101p were downregulated in this strain, suggesting that Rim101p, a transcriptional repressor which governs the ion tolerance, was activated. In line with this activation, the strain also showed elevated resistance to Li+ and Na+ ions and to zymolyase, a yeast lytic enzyme preparation containing mainly β‐1,3‐glucanase, indicating that the cell wall integrity was also strengthened in this strain. Our findings demonstrate a novel influence of PUFA production on transcriptional control that is likely to play an important role in the early stage of alkaline stress response. The Accession No. for microarray data in the Center for Information Biology Gene Expression database is CBX68. Copyright

Incorporation of linoleic acid and its conversion to λ-linolenic acid in fungi

The incorporation of [1-14C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6-desaturation of [1-14C]LA. [1-14C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-14C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1-14C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1-14C]LA into λ-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-14C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.

Characterization of triacylglycerol biosynthesis in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora

Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [14C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [14C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-14C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPAT

Characterization of the diacylglycerol acyltransferase activity in the membrane fraction from a fungus

In an attempt to clarify the mechanism of lipid accumulation inMortierella ramanniana var.angulispora, diacylglycerol acyltransferase (DGAT) in the membrane fraction from this fungus was characterized. The enzyme had an optimum pH of 7.0–7.5, and enzyme activity was blocked by SH-reagents. Metal ions were not essential for maintaining DGAT activity.n-Octyl-β-d-glucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and Tween 80 were found to preserve activity, while Triton X-100 and sucrose monolaurate inhibited it. As the inhibition of DGAT activity by Triton X-100 was overcome by the addition of diacylglycerol (DG), the dependency of DGAT activity on exogenous DG was determined in the presence of 0.1% Triton X-100. DGAT activity in the membrane fraction was traced in fungi cultured for different time periods or in media at different carbon to nitrogen (C/N) ratios. Although the increase in total lipid content with culture time was accompanied by an increase in DGAT activity, total lipid changes related to changes in C/N ratio did not correlate with DGAT activity. Factors other than DGAT activity in the membrane fraction would appear to be involved in the regulation of total lipid content in this fungus.