Abstract
Malic enzyme (ME) plays a vital role in determining the extent of lipid accumulation in oleaginous fungi being the major provider of NADPH for the activity of fatty acid synthase (FAS). We report here the first direct evidence of the existence of a lipogenic multienzyme complex (the lipid metabolon) involving ME, FAS, ATP: citrate lyase (ACL), acetyl-CoA carboxylase (ACC), pyruvate carboxylase (PC) and malate dehydrogenase (MDH) in Cunninghamella bainieri 2A1. Cell-free extracts prepared from cells taken in both growth and lipid accumulation phases were prepared by protoplasting and subjected to Blue Native (BN)-PAGE coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS). A high molecular mass complex (approx. 3.2 MDa) consisting of the above enzymes was detected during lipid accumulation phase indicating positive evidence of multienzyme complex formation. The complex was not detected in cells during the balanced phase of growth or when lipid accumulation ceased, suggesting that it was transiently formed only during lipogenesis.
Highlights
Most microbial cells normally contain no more than 10% (w/w) lipid which occurs mainly in cell membranes as well as other membranous structures
Of NADPH, Malic enzyme (ME) has been suggested to play a key role as the sole NADPH provider for fatty acid biosynthesis catalysed by fatty acid synthase (FAS) in three oleaginous fungi; Mucor circinelloides, Mortierella alpina[9] and Cunninghamella bainieri 2A110. (The biochemistry of no other oleaginous fungus has been studied in similar detail)
Detection of lipogenic multienzyme complex using Blue Native (BN)-PAGE coupled with LC-MS/MS
Summary
Most microbial cells normally contain no more than 10% (w/w) lipid which occurs mainly in cell membranes as well as other membranous structures. A number of microorganisms, mainly fungi and some protists, produce lipids containing high contents of polyunsaturated fatty acids (PUFAs) such as γ-linolenic acid (GLA), arachidonic acid (ARA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Some of these organisms have been commercially developed as sources of PUFAs, with current sales of several thousands of tons per annum[2]. The lipid content of Mcr circinelloides decreased significantly from 24 to 2% (w/w) when sesamol (specific inhibitor of ME activity) was added to the cultures[7] These results have been interpreted as strongly implicating a direct role of ME in lipid biosynthesis in oleaginous fungi
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