A pre-genetic study of the isoforms of malic enzyme associated with lipid accumulation in Mucor circinelloides

Abstract

The oleaginous fungus Mucor circinelloides possesses at least six isoforms of malic enzyme (EC 1 .1 .1 .40), a key lipogenic enzyme in filamentous fungi. These isoforms were detected using a specific stain for activity after native PAGE of cell extracts. Only one isoform (isoform IV) was associated with lipid accumulation, appearing only after N-exhaustion from the medium (which is a pre-requisite for lipid accumulation) in glucose-growing cells. Isoforms I, II, V and VI were involved in anaerobic growth and only appeared under O 2 -limited conditions. Isoform III appeared to be constitutive and was formed under conditions of active (balanced) growth and is therefore thought to play a crucial role in basic metabolism. Growth on acetate increased the amount of cell lipid (from 25-27 % in glucose-grown cells to 37-38 % in acetate-grown cells) accumulated by M. circinelloides and this was associated with the appearance of isoform IV of malic enzyme prior to N-exhaustion in these cultures. Amino acid sequence analysis of isoforms III and IV suggests that these two malic enzymes may be encoded by a single gene and that isoform IV is formed from isoform III by post-translational modification initiated by either N-limitation (when glucose was the carbon source) or growth on acetate as the sole carbon source.

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

“…Biomass was harvested by filtration through a pre-weighed glass-fibre filter (Whatman GF/A), washed twice with distilled water and dried at 110 uC to a constant weight. Glucose in the culture medium was determined using a glucose oxidase Perid test kit (Boehringer Mannheim) and ammonium using the indophenol method (see Song et al, 2001). …”

“…Lipid was extracted from freeze-dried biomass as previously described (Song et al, 2001) and its amount determined gravimetrically. Fatty acids, as their methyl esters, were prepared and analysed by GC, as described by Wynn & Ratledge (2000).…”

“…Unlike Mc. circinelloides CBS 108.16, which produces six ME isoforms (Song et al, 2001), only two isoforms were observed in the Mc. circinelloides R7B strain, and these corresponded to isoforms III and IV of the former strain.…”

“…alpina and Mc. circinelloides, respectively, suggests that at least some of the multiple ME isoforms noted in both these fungi (Song et al, 2001;Zhang, 2005) are encoded by different genes. Thus, the sequence of malEMc in this study is completely different from that of isoform II (mce1) from the same Mc.…”

“…Although we have also shown that whilst there are several isoforms of ME present in Mc. circinelloides (Song et al, 2001), only one of these (termed isoform III) is involved in NADPH formation and is linked to the lipid-accumulation process. This isoform III is converted into isoform IV, presumably post-transciptionally, upon the commencement of lipid accumulation, which is triggered by depletion of nitrogen from the growth medium.…”