Lipid Research Group, Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK1
Author for correspondence: James P. Wynn. Tel: +44 1482 465507. Fax: +44 1482 465458. e-mail: j.p.wynn{at}biosci.hull.ac.uk
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ABSTRACT |
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Keywords: fatty acid desaturation, fatty acyl chain-elongation systems, lipid metabolism, Mortierella alpina
Abbreviations: PUFA, polyunsaturated fatty acid
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INTRODUCTION |
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There is interest in using Mort. alpina as a biocatalyst for the biotransformation of low-value oils (such as sunflower oil) rich in 18:2 into higher-value oils, such as those that are rich in 20:4(n-6). Biotransformation of exogenous oils by Mort. alpina has been reported previously (Shimizu et al., 1989a , b
; Shinmen et al., 1992
; Certik et al., 1997
). These data are somewhat contentious, however, because of: (a) the possibility of carry over of exogenous lipid substrate adhering to the cells during their harvesting; (b) the possibility of continued de novo synthesis of PUFAs by the cells (albeit at a low level, but never previously measured in this organism); or (c) selective uptake of particular PUFAs from the mixed fatty acids used as the carbon source (Certik et al., 1997
). Furthermore, work in this laboratory suggested that growth of four filamentous fungi, although not Mort. alpina itself, on exogenous lipid caused a down-regulation of all fatty acid desaturation and elongation processes (Kendrick & Ratledge, 1996
).
No studies, as far as we are aware, have been carried out using 14C- or 13C-labelled substrates, which would have provided unequivocal evidence for continuing desaturation and elongation reactions when filamentous fungi are cultivated on exogenous oils. The present study aimed to clarify some of the more equivocal findings of the previous studies by using a combination of enzymology and lipid analysis of Mort. alpina cultivated on glucose and a range of water-soluble, lipid-based carbon sources, the Tweens, that can easily be washed off cells, thereby preventing unwanted carry over of the substrate fatty acids. Tweens are polyesters of sorbitan and fatty acids; when they are hydrolysed, by lipase/esterases, only the fatty acids are available;sorbitan is not a substrate for this fungus.
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METHODS |
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Preparation of cell extracts.
Biomass was harvested by filtration (under reduced pressure) through a Whatman no. 1 paper and washed extensively with distilled H2O. The mycelia were suspended in 100 mM KH2PO4/KOH (pH 7·5), containing 20% (w/v) glycerol, 1 mM benzamidine . HCl and 1 mM dithiothreitol, and disrupted by passage, twice, through a French pressure cell at 35 MPa. The resulting extract was centrifuged at 16000 g for 10 min at 4 °C and the supernatant retained for analysis. Protein concentrations were determined using the method of Bradford with BSA as a standard.
Determination of enzyme activities.
All enzymes were assayed using previously published procedures. Isocitrate lysase (EC 4 . 1 . 3 . 1) was assayed as described by Armitt et al. (1976) , ATP:citrate lyase (EC 4 . 1 . 3 . 8), fatty acid synthase (EC 2 . 3 . 1 . 85), glucose-6-phosphate dehydrogenase (EC 1 . 1 . 1 . 49), and malic enzyme (EC 1 . 1 . 1 . 40) as described by Wynn et al. (1997)
, carnitine acetyltransferase (EC 2 . 3 . 1 . 7) as described by Kawamoto et al. (1978)
, and pyruvate kinase (EC 2 . 7 . 1 . 40) as described by Worthington Enzymes (1979)
.
Lipid analysis.
Washed biomass was freeze-dried and lipid analysis carried out using two separate protocols. In the first protocol, the cell lipid was extracted with 2:1 (v/v) chloroform/methanol (Folch et al., 1957 ) and quantified gravimetrically. If this method was employed, the fatty acyl composition of the cell lipid was determined by GC analysis of fatty acid methyl esters, prepared using trimethylsulphonium hydroxide as described by Butte (1983)
. In the second protocol, the cell lipid was extracted and methylated in a single step as described by Rodriguez-Ruiz et al. (1998)
and quantified by reference to a 17:0 internal standard. The fatty acyl composition of the cell lipid was analysed using an ATI Unicam 610 series GC fitted with a 25 m, 0·5 µm, fused silica BPX70 column (SGE). The detector and injector temperatures were 300 °C and 250 °C respectively and the oven temperature was increased from 100 °C to 200 °C in 10 min then maintained at 200 °C for a further 5 min. Fatty acid methyl esters were identified by comparison with authentic standards.
Reproducibility of data.
All experiments were carried out in triplicate at least and data are presented as mean values±standard error of the mean.
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RESULTS AND DISCUSSION |
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Carnitine acetyltransferase.
This is an enzyme involved in the translocation of acetyl-CoA inside the cell. In contrast to the energy-dependent export of acetyl-CoA from the mitochondria (culminating in the cleavage of citrate by ATP:citrate lyase to yield cytosolic acetyl-CoA), carnitine acetyltransferase is involved in a more general shuttling of acetyl-CoA between cellular compartments (Kohlaw & Tan-Wilson, 1977 ). This enzyme was detected in glucose-grown Mort. alpina, in accord with previous observations (Wynn et al., 1998
), but its activity was increased sevenfold when Tween 80 was the carbon source. The elevated activity of this enzyme was a result of the increased requirement for acetyl-CoA transportation between cellular compartments in cells grown on a fatty-acid-based carbon source.
The changes in the activities of this enzyme, and of isocitrate lyase and ATP:citrate lyase, confirm that when Mort. alpina was cultivated on Tween 80, the metabolism of the fungus underwent changes identical to those previously described when both oleaginous yeasts and filamentous fungi were grown on exogenous triacylglycerol oils (Holdsworth et al., 1988 ; Wynn, 1994
).
Fatty acid synthase.
Whilst activity of fatty acid synthase was readily detected in glucose-grown cells, indicating active de novo fatty acid biosynthesis, cells grown on Tween 80 were devoid of this enzyme activity. That the inactivity of fatty acid synthase in extracts from Tween-grown biomass was not the result of trace amounts of Tween being carried over into the extracts and inhibiting this enzyme was demonstrated by direct addition of Tween 40 and Tween 80 to the in vitro fatty acid synthase assay. Neither Tween 40 nor Tween 80 inhibited fatty acid synthase activity at 0·1% (w/v). The lack of fatty acid synthase activity in extracts from Tween-grown biomass was clear evidence of the cessation of de novo fatty acid synthesis in cells grown on a fatty-acid-based carbon source.
As the cessation of de novo fatty acid biosynthesis when Mort. alpina was grown on fatty-acid-based carbon sources is important in the evaluation of the lipid analysis data given below, the fatty acid synthase activity when the fungus was grown on Tween 20 and 40 was also determined. In all cases, when Tween compounds were the carbon source, fatty acid synthase activity was undetectable.
That no fatty acid synthesis was occurring precluded the possibility of the appearance of PUFAs as a result of de novo synthesis rather than transformation of exogenous fatty acids, as has previously been suggested (Kendrick & Ratledge, 1996 ; Certik et al., 1997
).
Malic enzyme.
Although activity of this enzyme has been detected in a number of filamentous fungi its cellular function remains contentious. In Aspergillus nidulans, where the activity of malic enzyme is low when glucose is the carbon source (McCullough & Roberts, 1974 ; Wynn & Ratledge, 1997
), this enzyme appears vital for the provision of pyruvate to allow growth on carbon sources metabolized via acetyl-CoA (including lipids) (McCullough & Roberts, 1974
). Evidence has been obtained in this laboratory to suggest that malic enzyme in filamentous fungi, and in the zygomycetes in particular, is crucial for cell lipid biosynthesis (Wynn & Ratledge, 1997
; Wynn et al., 1997
, 1999
).
When Mort. alpina was grown on glucose the activity of malic enzyme was relatively high (Table 2), tenfold greater than in Asp. nidulans (Wynn & Ratledge, 1997
). Growth of Mort. alpina on Tween 80 led to a decrease in malic enzyme activity. [The overlapping errors shown in Table 2
were a result of inter-experiment variability. During the five occasions that malic enzyme was measured in glucose- and Tween 80-grown biomass (grown at the same time, from the same inoculum) the malic enzyme activity was less in the Tween 80-grown cells on four occasions, whilst being equivalent in the fifth.] That malic enzyme activity decreased when Mort. alpina was grown on Tween 80, together with its high activity in glucose-grown cells, suggested that the major cellular role of this enzyme is not in the provision of pyruvate when the fungus is grown on carbon sources such as fatty acids that are metabolized via acetyl-CoA and therefore require the glyoxylate bypass for metabolism. In this regard Mort. alpina may be similar to Fusarium oxysporum and Neurospora crassa, where a decrease in the activity of malic enzyme was observed when these fungi were grown on acetate (Zink, 1972
; Zink & Katz, 1973
). The residual activity of malic enzyme (
70% of that in glucose-grown cells) in Mort. alpina grown on Tween 80 suggests that malic enzyme fulfils another role as well as providing NADPH for fatty acid de novo synthesis. As fatty acid metabolism (although not de novo synthesis) continued in cells grown on Tween 80 (see below) it is suggested that malic enzyme in Mort. alpina may be acting as a provider of NADPH for fatty acid desaturation, as has been suggested by Kendrick & Ratledge (1992
) with Mucor circinelloides, and even fatty acid elongation.
Pyruvate kinase.
Activity of this enzyme was detected at 1000 nmol min-1 (mg protein)-1 in glucose-grown cells, but was decreased by 90% when Mort. alpina was grown on Tween 80 (Table 2
). These observations are similar to those of McCullough & Roberts (1974)
for Asp. nidulans grown on either glucose or acetate. McCullough & Roberts (1974)
surmised that the residual pyruvate kinase activity was not involved in the provision of pyruvate for biosynthesis in Asp. nidulans. It was hypothesized that malic enzyme activity (which increased under these conditions) was responsible for the generation of pyruvate needed for biosynthetic purposes. It appears that the primary role of malic enzyme in Mort. alpina and Asp. nidulans may differ, as evidenced by the far higher activity of malic enzyme in glucose-grown cells of Mort. alpina than in Asp. nidulans (McCullough & Roberts, 1974
; Wynn & Ratledge, 1997
; Wynn et al., 1999
) and by the decrease rather than increase in malic enzyme when Mort. alpina is grown on carbon sources that induce the glyoxylate shunt. We therefore propose that in Mort. alpina, as in Mr. circinelloides (Wynn et al., 1997
, 1999
), malic enzymes primary function is the provision of NADPH for fatty acid synthesis and that biosynthetic pyruvate is probably generated by the residual pyruvate kinase activity.
Glucose-6-phosphate dehydrogenase.
This is a marker enzyme for the hexose monophosphate pathway that acts to synthesize ribose for nucleic acids and erythrose 4-phosphate for synthesis of aromatic amino acids, which must still continue in lipid-utilizing cells, as well as generating NADPH for anabolism. The lower activity of glucose-6-phosphate dehydrogenase in Mort. alpina cultivated on Tween 80 undoubtedly reflected the decreased carbon flux through the hexose monophosphate pathway when cells are grown on exogenous oils.
Growth and lipid production in Mort. alpina grown on glucose and Tween carbon sources
The problems of determining the fatty acyl composition of fungi when cultivated on lipids are principally those of removing exogenous lipid that adheres to the surface of the mycelia. These have been highlighted elsewhere (Kendrick & Ratledge, 1996 ; Certik et al., 1997
). The use of various Tweens as carbon sources during the present work has overcome these problems. Tween detergents are fatty-acid-based compounds that have the advantage of being water-soluble and can be easily washed off the cells at the end of growth. The Tweens are also easily hydrolysed to their constituent fatty acids and sorbitan, which is a non-metabolizable carbohydrate. Therefore if the fungus can hydrolyse the Tweens and is seen to grow on these carbon sources then it must be using the fatty acids as the sole source of carbon. That Mort. alpina was utilizing the fatty acid component of the Tweens as a carbon source was confirmed by the changes in the enzymology of the fungus (see above). The water solubility of the Tweens and their removal from the mycelia using an aqueous wash was important as it obviated the need for an organic solvent wash as employed in previous studies (Kendrick & Ratledge, 1996
; Certik et al., 1997
). An organic solvent wash has the potential to remove some of the intracellular lipid and, in any case, was undesirable for biomass destined for enzymological study.
Growth of Mort. alpina on Tween carbon sources resulted in less biomass being obtained than when glucose was the carbon source (see Table 3); however, the cell lipid content was greatly increased. The fatty acyl profiles of the cell lipids when Mort. alpina was grown on the Tween carbon sources were similar but, significantly, not identical, to those of the Tweens themselves (shown in Table 1
). Although the cell lipids were not as unsaturated as those in glucose-grown biomass (see Table 3
) they had higher unsaturation indexes than the starting carbon source and contained higher amounts of 18:2(n-6) as well as significant amounts of the PUFAs 18:3(n-6) and 20:4(n-6), which were entirely absent from the Tween carbon sources (see Table 1
). Thus growth on a lipid carbon source did not inhibit fatty acid desaturation and elongation even though complete repression of fatty acid synthesis occurred (Table 2
). This is in accord with the data reported by Shimizu et al. (1989a
, b
), Shinmen et al. (1992)
and Certik et al. (1997)
, but which were equivocal because of the possibility of residual substrates still being present or that mixed fatty acids in the substrate could be taken up at different rates, which would result in observed differences in the fatty acid profiles of the substrate and the cell lipid recovered from the biomass (Kendrick & Ratledge, 1996
; Certik et al., 1997
). That conversion of 16:0, and even 12:0, into C18 and C20 fatty acids occurred in Mort. alpina lacking fatty acid synthase activity indicated that the fatty acid elongation system was capable of accepting relatively short-chain fatty acids as well as long-chain fatty acids. A similarly flexible elongation system has also been demonstrated in yeasts (Dittrich et al., 1998
).
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These results (see Table 4) allow the biosynthetic capacity of the Tween-grown cells to be compared on an equal basis with that of the glucose-grown cells. The inclusion of an internal standard in the calculation of total cell lipid and therefore the biosynthetic capacities was important to avoid the potential for non-lipid material (e.g. sterols, carotenoids and other non-saponifiable lipids) extracted with cell lipid affecting the calculation. It was found that lipid analyses carried out both with and without an internal standard gave essentially identical results, indicating that there was no substantial change in the non-fatty acid components of the total lipid when glucose or Tween was the carbon source.
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It therefore appears that in the overall conversion of oleic acid to arachidonic acid it is the elongase activity that is the rate-limiting step in the biosynthesis of 20:4(n-6). Attempts to increase the productivity of 20:4(n-6) formation in commercial strains of Mort. alpina should therefore focus on increasing the elongase activity (presumably by genetic manipulation) rather than on increasing the 12 and
6 desaturases, which seemingly have an inherent capacity to desaturate twice the amount of substrate they normally handle. Even increasing the activity of the final
5 desaturase for the conversion of 20:3(n-6) to 20:4(n-6) would probably not increase the final yield of arachidonic acid significantly.
Conclusions
The data obtained when Mort. alpina was grown on Tween carbon sources shed light on two different aspects of the biochemistry of this fungus.
The analysis of the enzyme activities when Mort. alpina was cultivated on Tween compounds indicates that de novo fatty acid biosynthesis is abolished (repressed) by growth of cells on exogenous lipid.
The changes in the composition of the cell lipid indicate that Mort. alpina incorporates and modifies exogenous fatty acids into its own cell lipid. Neither fatty acid desaturation nor elongation is repressed under these conditions. The analysis of oleate incorporation implicates the elongase converting 18:3(n-6) to 20:3(n-6) as the rate-limiting step in arachidonic acid biosynthesis, rather than any fatty acid desaturase. This could have important implications when attempting to increase 20:4(n-6) production in genetically modifiable systems.
Also it appears likely that the provision of pyruvate for alanine biosynthesis during growth on fatty acids could be fulfilled by a residual activity of pyruvate kinase (i.e. from the phosphoenolpyruvate synthesized from oxaloacetate). The major function of malic enzyme, which is the sole enzyme activity associated with lipogenesis in this fungus (Wynn et al., 1999 ), would be the generation of NADPH for lipid biosynthesis. Under conditions of growth on lipids the residual activity of malic enzyme would provide NADPH for the continuing fatty acid desaturation and elongation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 February 2000;
revised 5 May 2000;
accepted 5 June 2000.