Centre for Advanced Lipid Research, 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: isoforms, continuous culture, filamentous fungi
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INTRODUCTION |
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Despite strong evidence that malic enzyme activity is a key factor in ensuring maximal lipid accumulation in filamentous fungi, the link between malic enzyme activity and lipid accumulation is uncertain. Fungi possessing a high activity of malic enzyme do not necessarily accumulate lipid (Wynn et al., 1999 ; J. P. Wynn, unpublished work). This finding, together with the presence of multiple isoforms of malic enzyme in some fungi (Savitha et al., 1997
; Zink, 1972
; J. P. Wynn, unpublished work), has led us to consider the possibility that several forms of malic enzyme may exist in Mucor circinelloides, perhaps even encoded by different genes. Specific isoforms of malic enzyme may, therefore, be associated with lipid accumulation whilst others have other cellular functions. As our ultimate aim is to clone the gene for malic enzyme, to overexpress it and so potentially boost lipid accumulation in filamentous fungi (and perhaps also in oilseed plants), it is crucial that we have a clear understanding of the number and cellular functions of the discrete malic enzyme isoforms in eukaryotic cells.
The aim of this study was to assess the impact of a range of growth conditions known to affect malic enzyme activity in micro-organisms and to determine their effect on (i) the total malic enzyme activity, (ii) the appearance of different isoforms of malic enzyme and (iii) lipid accumulation. In so doing we hoped to identify the isoform(s) of malic enzyme specifically associated with lipid accumulation in M. circinelloides.
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METHODS |
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Cultivation of fungi.
Mucor circinelloides CBS 108.16 was grown in modified Kendrick media (Kendrick & Ratledge, 1992 ). Medium for batch culture contained 50 g glucose l-1 and 2 g ammonium tartrate l-1; N-limited medium for continuous culture contained 12 g glucose l-1, 0·5 g NH4Cl l-1 and 0·1 g yeast extract l-1. Cultures were grown in either 1 l stirred bottles as previously described (Wynn et al., 1997
) or in 5 l fermenters with 4 l medium. Aeration was at 1·5 l air min-1 and pH was controlled at 5·56·5 by automatic addition of 2 M KOH and 2 M H2SO4, for glucose-grown cultures, or 50% (w/w) acetic acid for acetic-acid-grown cultures in a pH auxostat, with an initial concentration of sodium acetate of 10 g l-1 (Du Preez et al., 1997
).
Continuous cultivation was in a 5 l stirred chemostat with a working volume of 4 l, aeration at 1·7 l min-1 and stirring at 700 rev min-1. The pH was maintained between 5·5 and 6·5 and the dilution rate was adjusted by controlling the rate of medium addition. Wall growth was prevented by the absence of baffles and by reversing the direction of stirring every 60 s. Anaerobic growth was achieved by bubbling N2 or CO2 through the air inlet. The dissolved O2 tension was measured using a galvanic O2 electrode calibrated to 100% of full-scale deflection immediately prior to inoculation with the chemostat being flushed with air at 1·7 l min-1. Volume control of the fermenter was achieved by a level sensor opening and closing a solenoid valve allowing excess culture to flow directly to the waste bottle.
All cultivations were carried out at 30 °C.
Determination of cell dry weight.
Biomass was harvested by filtration through a preweighed dried filter (Whatman GF/A), washed twice with distilled water and dried at 110 °C to a constant weight.
Determination of ammonium, glucose and acetate.
The ammonium concentration in the culture medium was measured using the indophenol method of Chaney & Marbach (1962) . The glucose concentration in the culture was determined using a glucose oxidase Perid-test kit (Boehringer Mannheim) according to the manufacturers instructions.
The acetate concentration in the culture was determined by GC using an ATI Unicam 610 series gas chromatograph fitted with a flame-ionization detector and a BPX 70 capillary column packed with 10% (w/w) diethylene glycol succinate on Chromosorb W-AW. The oven temperature of 120 °C was increased at a rate of 6 °C min-1 to 165 °C. The inlet and detector temperatures were 135 °C and 250 °C, respectively. The carrier gas was helium at 5 ml min-1. Acetate peaks were identified and quantified by reference to an acetate standard.
Preparation of cell extracts.
Biomass was harvested by filtration under reduced pressure through a Whatman filter and washed three times with distilled water. Harvested cells were suspended in extraction buffer [100 mM KH2PO4/KOH, pH 7·5, containing 20% (w/v) glycerol, 1 mM benzamidine . HCl and 1 mM DTT] and disrupted by passage once through a One Shot Cell Disrupter (Constant Systems) at 64 MPa. The disrupted cell suspensions were centrifuged at 10000 g for 10 min at 4 °C and the supernatants were used for enzyme analysis. Protein concentrations were determined using the method of Bradford (1976) with BSA as a standard.
Detection of malic enzyme activity and malic enzyme isoforms.
The activity of malic enzyme was assayed according to the method of Hsu & Lardy (1969) with malate at 25 mM.
Malic enzyme isoforms were distinguished by activity staining of native-PAGE. Native-PAGE was prepared according to Hames (1985) using 10% (w/v) acrylamide with the omission of SDS. Activity staining of malic enzyme on native gels was carried out according to the method of Chang et al. (1991)
.
Analysis of cell lipid.
Biomass was harvested by filtration, rapidly frozen, then freeze-dried. Cell lipid was extracted with chloroform/methanol (2:1, v/v) (Folch et al., 1957 ) and determined gravimetrically. The fatty acid profile of the cell lipid was analysed as previously described (Wynn & Ratledge, 2000
).
Purification of malic enzyme isoforms III and IV
Step one.
A crude enzyme extract was centrifuged at 100000 g for 60 min at 4 °C and the supernatant was carried forward to step two.
Step two.
The protein precipitated at an ammonium sulphate concentration between 50 and 60% saturation was collected by centrifugation at 16000 g at 4 °C and resuspended in a minimum volume of extraction buffer.
Step three.
The resuspended protein from step two was loaded onto a Sephacryl S-200-HR gel filtration column (95 cmx2·5 cm) and eluted with 10 mM Tris/HCl buffer, pH 7·4, containing 20% (w/v) glycerol, 1 mM DTT and 1 mM benzamidine . HCl. Fractions of 3 ml were collected and the most enzymically active were retained and pooled.
Step four.
The pooled active fractions from step three were applied to a DEAE anion-exchange column (16 cmx2 cm) and proteins were eluted with the Tris/HCl buffer used in step 3 containing a magnesium diacetate gradient between 25 mM and 45 mM (300 ml). Again, 3 ml fractions were collected and the active fractions were pooled. The volume of the active fractions was reduced to approximately 2 ml by ultrafiltration through an Amicon PM 30 membrane filter.
Step five.
The concentrated sample from step four was applied to a Sephacryl S-200-HR gel filtration column (95 cmx2·5 cm) and eluted with 10 mM Tris/HCl buffer, pH 6·0, containing 20% (w/v) glycerol, 1 mM DTT and 1 mM benzamidine . HCl. Active fractions were collected and pooled.
Step six.
The active fraction was applied to a Mimetic Green column (Affinity Chromatography) (20 cmx1·5 cm) and the protein was eluted with the Tris/HCl buffer used in step 5, containing a magnesium diacetate gradient between 0 and 150 mM (350 ml). The most active fraction was retained and assessed for purity by SDS-PAGE, staining with Coomassie blue (Hames, 1985 ).
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RESULTS AND DISCUSSION |
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Effect of growth rate on malic enzyme activity and lipid accumulation in M. circinelloides
Previous work demonstrated that lipid accumulation in both M. circinelloides and Mortierella alpina (a related zygomycete fungus) occurred after N-exhaustion in batch cultures during a period when malic enzyme activity began to decrease (Wynn et al., 1999 ). However, once malic enzyme activity became undetectable, lipid accumulation in both fungi ceased. It was hypothesized that the gene encoding malic enzyme was repressed after N-exhaustion and that the period of lipogenesis after N-exhaustion, and therefore the final extent of lipid accumulation, was probably a result of the resistance (or susceptibility) of the malic enzyme protein to degradation.
To investigate this phenomenon in more detail, M. circinelloides was cultivated in continuous culture at a range of growth rates (nitrogen being the limiting culture component) to reproduce the various stages of growth in batch culture. As the specific growth rate decreased, the amount of lipid in the biomass increased from <15% (w/w) dry weight at 0·18 h-1 to >25% (w/w) dry weight at 0·04 h-1 (see Fig. 2). However, the activity of malic enzyme decreased at the lower growth rates, showing an inverse relationship to the cell lipid content. As maximum lipid accumulation was not necessarily associated with maximum malic enzyme activity, it appeared likely that malic enzyme must also be fulfilling cellular functions other than generating NADPH specifically for lipid accumulation.
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When M. circinelloides was grown under anaerobic conditions, malic enzyme activity was higher than in aerobically cultivated biomass (by three- to sixfold) and between three and five isoforms were present in the biomass (see Table 1). The only isoform that was consistently absent from biomass grown under anaerobic conditions was isoform IV. Under anaerobic conditions the cell lipid content was very low (<2%, w/w, dry wt) and the amount of unsaturated fatty acids in the cell lipid was severely restricted (see Table 2
).
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In response to O2-limited growth conditions, M. circinelloides displayed changes in the fatty acid profiles that compensate for a lack of fatty acid desaturase activity, which is an O2-dependent process. While the content of the unsaturated fatty acids in general, and the polyunsaturated fatty acids in particular, decreased, the amount of short-chain fatty acids (12:0 and 14:0) increased. A change to shorter chain fatty acids, with lower melting points, would maintain the fluidity of cell membranes under conditions where efficient desaturation could not occur. That some fatty acid desaturation occurred demonstrated that the conditions in the cultures were not completely anaerobic and that sufficient O2 was entering the system (probably dissolved in the incoming medium which was not de-oxygenated) to maintain a basal level of fatty acid desaturation.
When M. circinelloides was cultivated in an atmosphere rich in CO2, the activity of malic enzyme was particularly high and isoform VI appeared (Table 1). This correlated with the detection of a pyruvate- and
-dependent oxidation of NADPH in crude extracts which was assumed to be the result of a pyruvate carboxylating activity of isoform VI. It therefore appears that isoform VI is peculiar in that it alone can catalyse a reverse (i.e. carboxylation) malic enzyme reaction. The in vivo importance of this minor carboxylating activity is unclear (especially as it constituted only 5% of the decarboxylating activity of malic enzyme in crude extracts) but it does suggest that isoform VI is significantly different from the others found in M. circinelloides.
Effect of growth of M. circinelloides on acetate
The effect of acetate on the growth, lipid production and malic enzyme activity of M. circinelloides is of particular interest as previous work with Aspergillus nidulans (McCullough & Roberts, 1974 ) demonstrated that acetate as a sole carbon source led to an increase in the activity of malic enzyme. Furthermore, growth on acetate has been found to increase the maximum extent of lipid accumulation by M. circinelloides (Du Preez et al., 1997
).
When M. circinelloides was grown in a pH-auxostat (fed-batch) culture with acetate supplied continuously as the carbon source (viz. Du Preez et al., 1997 ), the cell lipid content was 35% greater than in glucose-grown cultures (Fig. 4a
). The malic enzyme activity in acetate-grown cultures at or before N-limitation (Fig. 4b
) was lower than that in glucose-grown cells [3035 nmol min-1 (mg protein)-1 compared to >45 nmol min-1 (mg protein)-1]. However, after N-exhaustion the activity of malic enzyme in acetate-grown cells remained higher [
15 nmol min-1 (mg protein-1)] than in glucose-grown cells [between 3 and 10 nmol min-1 (mg protein)-1]. The difference in the isoform profiles of the cells grown on the different carbon sources was probably more significant than the actual specific activity recorded for malic enzyme, which does not distinguish between the isoforms (Fig. 5
). Cells grown on glucose contained only isoform III prior to N-exhaustion. After N-exhaustion and during lipid accumulation, isoform III became less quantitatively important and isoform IV appeared, and became the predominant isoform. In contrast, cells grown on acetate possessed both isoforms III and IV even prior to N-exhaustion, although again isoform III disappeared during the lipid accumulation idiophase.
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Purification of isoforms III and IV from M. circinelloides
Both isoforms III and IV were purified to homogeneity from aerobic cultures of M. circinelloides. The same purification protocol was used for each isoform but the biomass used for the purification was harvested at different times and from cultures cultivated on different carbon sources (Table 3). Isoform III was purified from glucose-grown biomass, harvested prior to N-exhaustion, and isoform IV was purified from acetate-grown biomass, harvested after N-exhaustion. Therefore, isoform III was isolated from biomass devoid of isoform IV and isoform IV was isolated from biomass devoid of isoform III. Although the purified isoforms were clearly separated by native PAGE (Fig. 1
) after purification, they appeared identical when run on SDS-PAGE (Fig. 6
). Therefore, the two isoforms appear to be composed of subunits of the same molecular size. The difference in migration on the native gels, therefore, reflects either a difference in the number of subunits possessed by isoforms III and IV or a modification of a subunit of similar size. This could be perhaps by the possession or absence of a charged moiety such as a phosphate.
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Due to the similarity of the subunits of isoform III and IV in terms of their molecular mass and N-terminal amino acid sequence and the way in which isoform III disappears at the time that isoform IV appears, it is our hypothesis that isoform IV is produced from isoform III by a process of post-translational modification. The most likely processes to be involved are either partial proteolytic cleavage or phosphorylation or dephosphorylation. However, attempts to clarify which of these processes is the one operating in vivo have so far been inconclusive.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 December 2000;
revised 14 February 2001;
accepted 22 February 2001.
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