Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation

Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK Correspondence Colin Ratledge c.ratledge{at}hull.ac.uk Malic enzyme (ME; NADP + -dependent; EC 1 . 1 . 1 . 40) has been postulated to be the rate-limiting step for fatty acid biosynthesis in oleaginous fungi in which the extent of lipid accumulation is below the maximum possible. The genes encoding the isoform of ME involved in fatty acid synthesis were identified in Mucor circinelloides and Mortierella alpina , two commercially useful oil-producing fungi, using degenerate primers. Both showed high similarity with ME genes from other micro-organisms. The whole-length ME gene from each source was cloned into a leucine auxotroph of Mc. circinelloides and placed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene ( gpd1 ) promoter. After confirming correct expression of the ME genes, the two recombinant strains were grown in fully controlled, submerged-culture bioreactors using a high C : N ratio medium for lipid accumulation. Activities of ME were increased by two- to threefold and the lipid contents of the cells, in both recombinants, were increased from 12 % of the biomass to 30 %. Simultaneously, the degree of fatty acid desaturation increased slightly. Thus, increased expression of the ME gene leads to both increased biosynthesis of fatty acids and formation of unsaturated fatty acids, including -linolenic acid (18 : 3 n-6). At the end of lipid accumulation (96 h), ME activity in the recombinant strains had ceased, as it had done in the parent wild-type cells, indicating that additional, but unknown, controls over its activity must be in place to account for this loss of activity: this may be due to the presence of a specific ME-cleaving enzyme. The hypothesis that the rate-limiting step of fatty acid biosynthesis is therefore the supply of NADPH, as generated specifically and solely by ME, is therefore considerably strengthened by these results. Abbreviations: ACL, ATP, citrate lyase; GLA, -linolenic acid; ME, malic enzyme Present address: National Institutes of Health, NIDCR, 9000 Rockville Pike, Bethesda, MD 20892, USA. Present address: Centre for Novel Agricultural Products (CNAP), Department of Biology, University of York, York YO10 5DD, UK. The GenBank/EMBL/DDBJ accession numbers for the ME sequences of Mc. circinelloides and Mt. alpina are DQ975377 and DQ973624, respectively. A table showing the strains and plasmids used in this study is availab