First evidence for a multienzyme complex of lipid biosynthesis pathway enzymes in Cunninghamella bainieri.

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.