The biochemistry of lipid accumulation in Mucor circinelloides and Mortierella alpina

Aidil bin Abdul Hamid

Microbiology; Biochemistry
June 1998

Thesis or dissertation

© 1998 Aidil bin Abdul Hamid. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

1. The profile and biochemistry of growth and lipid accumulation in M. circinelloides and Mt. alpina were investigated.

2. A nitrogen-limited condition was vital in triggering lipid accumulation in both fungi, which was in agreement with previous work reported in oleaginous yeasts (Botham and Ratledge, 1979; Boulton and Ratledge, 1984). Good growth and lipid production by M. circinelloides were obtained only when it was grown in fermenters. Growth in stirred bottles (whirlipots) did not result in high lipid yields. This was caused by the anaerobic nature of cultivation in the whirlipots which affected the utilization of ammonium by the cells. As a result the cultures became carbon-limited instead of nitrogen-limited. Conversely, in a fermenter culture which had an efficient aeration, the culture reached a nitrogen-limited condition at an early stage of the incubation which led to a higher lipid production of the cells.

3. The lipid production in both fungi increased in parallel with the increase in the C:N ratio of the medium but the fatty acid compositions were not affected.

4. Ten enzymes potentially linked to the regulation of lipid biosynthesis (fatty acid synthase, acetyl-CoA carboxylase, ATP:Citrate lyase, AMP:deaminase, carnitine acetyl transferase, malic enzyme, glucose-6- phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP:isocitrate dehydrogenase and NAD:isocitrate dehydrogenase) were detected in both fungi. In both fungi, the profile of all enzymes stated above was similar with the activities increased coincidentally with the depletion of ammonia in the medium.

5. The only differences in the enzymatic profiles of the two fungi was the early depletion of ME activity in M. circinelloides where it disappeared after approximately 40 h of incubation, coincident with the cessation of lipid accumulation, although other key enzymes of lipid biosynthesis (FAS, ACC and ACL) together with the activities of other NADPHgenerating enzymes were still active and the glucose was still present. Conversely, ME in Mt. alpina culture was present until the late stage of fermentation and the cell lipid continued to increase until the end of the fermentation. This suggests ME is a major provider of NADPH for lipid biosynthesis which was in agreement with previous observations in Aspergillus nidu/ans (Wynn and Ratledge, 1997) and M. circinelloides (Wynn et al., 1997).

6. The depletion of ME activity in M. circinelloides after approximately 40 h of incubation was as a result of the cessation of the protein from being synthesized, triggered by the depletion of ammonium in the culture. This - was evident as malic enzyme activity returned after its initial depletion when ammonium tartrate was added into the culture. Also, the restitution of malic enzyme activity was prevented when cycloheximide, a protein synthesis inhibitor, was added simultanously with the addition of ammonium tartrate.

7. The NAD:isocitrate dehydrogenase from both fungi showed an increased affinity for its substrate, isocitrate, in, the presence of AMP. However, the enzyme did not show an absolute requirement for AMP for its activity as it could still be activated in the absence of AMP at a saturating concentration of isocitrate.

8. ME was purified some 20-fold purification from both fungi. Both showed a similar Km values for NADP (approximately 0.04 mM) but a slightly higher Km value for malate was obtained in Mt. alpina compared to M. circinelloides (1 mM and 0.4 mM, respectively).

9. ME from both fungi showed various degrees of inhibition by tartronic acid, oxaloacetate, palmitoyl-CoA and oleoyl-CoA. At 10 mM, tartronic acid caused approximately 40 % inhibition in the activity of ME from both fungi while OAA inhibited ME from M. circinelloides more strongly (70 %) than that from Mt. alpina ( 45 % ). At a final concentration of 1 mM, palmitoyl-CoA and oleoyl-CoA caused a 100 % inhibition on ME from M. circinelloides and approximately 90 % on ME from Mt. alpina.

10. FAS purified from both fungi showed a similar Km values for malonyl-CoA and acetyl-CoA (approximately 0.013 and 0.016 mM, - respectively) while a higher Km value for NAOPH was observed in Mt. alpina compared to M. circinelloides (0.038 and 0.01 mM, respectively).

11. Despite a range of experiment using different approaches, no direct evidence of a physical association between FAS and ME was obtained when experiments were performed to observe the formation of complexes between the two enzymes in vitro.

Department of Biological Sciences, The University of Hull
Ratledge, Colin; Wynn, James Patrick
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