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Title:	Regulation of lipogenesis in Cunninghamella bainieri 2A1 : a possible involvement of malic enzyme
Authors:	Ekhlass Mohialdeen Taha (P42083)
Supervisor:	Aidil Abd Hamid, Assoc. Prof. Dr.
Keywords:	Regulation of lipogenesis Cunninghamella bainieri 2A1 Involvement of malic enzyme Malic enzyme Fatty acids--Synthesis
Issue Date:	2-Dec-2012
Description:	Regulation of lipid biosynthesis in Cunninghamella bainieri 2A1 was studied. The importance of Nitrogen-limitation to initiate lipid accumulation, the role of malic enzyme isoforms as well as the physical association between malic enzyme and fatty acid synthase were investigated. The effect of N-limitation on lipid synthesis was studied by cultivation the fungus in two different conditions. One of the cultures was intermittently fed with ammonium tartrate to maintain the ammonium concentration above 0.5 g/l throughout the experiment thereby providing conditions inappropriate for lipid accumulation. The second culture was performed without any feeding to allow N limitation to occur at the early phase of the cultivation, hence promoting lipid accumulation. Cultivation was carried out for 120h at 250 rpm and 30°C and lipid content as well as six lipogenic enzymes (FAS, ACL, ME,NAD:ICDH,6-PGDH and G6-PDH) were assayed. The N-limited culture achieved a maximum lipid content of up to 35% (g/g biomass) at 48h with no further increase observed until 120h, whereas the culture which was fed with ammonium tartrate produced a lower lipid content 15% (g/g biomass). All six enzymes showed high activity during growth in both media with significant decrease in ME activity which coincided with the cessation of lipid accumulation at 48h. This showed that N-limitation is important for enhanced lipid accumulation but the fungus was still capable of producing more than 10% lipid even nitrogen limitation did not occur. When grown in N-limited media, the fungus showed to have at least five isoforms of malic enzyme (A,B,C,D and E), where only one isoform (isoform E) was associated with lipogenesis, showing a parallel increase in activity with the increase of lipid production. All five isoforms were also present in the nitrogen-controlled cultures which accumulated 15% lipid; but with a more pronounced activity of isoform D with trace activity isoform E. This showed that isoform E is associated with lipid biosynthesis. These are further supported when the presence of isoform E was detected in oleaginous (P.chrysogenum and T.virens) and non oleaginous (A. niger, and R. oligospora) fungi. All the oleaginous fungi studied showed high activity of isoform E, whereas the non oleaginous showed only trace activity of E forms. Experiments to prolong the lipid accumulation phase after 48h of cultivation were also carried out by, feeding the culture with complete media and media devoid of trace elements and yeast extract in the stationary phase at 72 h. The cultures which are fed with complete media showed increased lipid content (42%) while no increase in lipid content was observed in the culture fed with media devoid of trace element and yeast extract, in spite of the activation of all lipogenic enzymes as well as to ME isoforms. This showed that the cessation of lipid accumulation after 48h of cultivation was caused by the depletion of trace element and yeast extract. Further investigation on the effects of two of the trace elements, Mg2+, Mn2+ showed that ME isoforms totally dependent on Mg2+ and Mn2+ for its activity. Finally the possible associated between ME, ACL and FAS has been investigated using extracts prepared by gentle disruption of cells. Gel filtration of the cell free extract using Superdex 200 showed that the three enzymes associated into a cluster as ME and ACL were eluted in the same fractions as those containing FAS. Similar results were obtained when a broad molecular weight column Sepharose CL-2B was used. ME activity of the cluster was represented by the activity of isoforms D and E only. We conclude that, the key to lipid accumulation in C. bainieri 2A1 is the role of E form of ME that physically associated with FAS as well as ACL to form multienzyme complex.,PhD
Pages:	148
Call Number:	QK898.F3 .T334 2012
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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