Commercial interest in microbial lipids is increasing due to their potential use as feedstock for biodiesel production. The supply of NADPH generated by malic enzyme (ME; NADP+-dependent; EC 1.1.1.40) has been postulated as being the rate-limiting step for fatty acid biosynthesis in oleaginous fungi, based mainly on data from the zygomycete Mucor circinelloides studies. This fungus contains five genes that code for six different ME isoforms. One of these genes, malA, codes for the isoforms III and IV, which have previously been associated with lipid accumulation. Following a strategy of targeted integration of an engineered malA gene, a stable strain overexpressing malA and showing high ME activity has been obtained, demonstrating the feasibility of this strategy to overexpress genes of biotechnological interest in M. circinelloides. This is the first report showing the integration and overexpression of a gene in Zygomycetes. Unexpectedly, the genetically modified strain showed a lipid content similar to that of a prototrophic non-overexpressing control strain, suggesting that another limiting step in the fatty acid synthesis pathway may have been revealed as a consequence of the elimination of malic enzyme-based bottleneck. Otherwise, the fact that prototrophic strains showed at least a 2.5-fold increase in lipid accumulation in comparison with leucine auxotrophic strains suggests that a wild-type leucine biosynthetic pathway is required for lipid accumulation. Moreover, increasing concentrations of leucine in culture medium increased growth of auxotrophs but failed to increase lipid content, suggesting that the leucine synthesized by the fungus is the only leucine available for lipid biosynthesis. These results support previous data postulating leucine metabolism as one of the pathways involved in the generation of the acetyl-CoA required for fatty acid biosynthesis.

Diminishing fossil fuel reserves and the increase in their consumption indicate that strategies need to be developed to produce biofuels from renewable resources. Biodiesel offers advantages over other petroleum-derived fuel substitutes, because it is comparatively environmentally friendly and an excellent fuel for existing diesel engines. Biodiesel, which consists of fatty acid methyl esters (FAMEs), is usually obtained from plant oils. However, its extensive production from oil crops is not sustainable because of the impact this would have on food supply and the environment. Microbial oils are postulated as an alternative to plant oils, but not all oleaginous microorganisms have ideal lipid profiles for biodiesel production. On the other hand, lipid profiles could be modified by genetic engineering in some oleaginous microorganisms, such as the fungus Mucor circinelloides, which has powerful genetic tools. We show here that the biomass from submerged cultures of the oleaginous fungus M. circinelloides can be used to produce biodiesel by acid-catalyzed direct transformation, without previous extraction of the lipids. Direct transformation, which should mean a cost savings for biodiesel production, increased lipid extraction and demonstrated that structural lipids, in addition to energy storage lipids, can be transformed into FAMEs. Moreover, the analyzed properties of the M. circinelloides-derived biodiesel using three different catalysts (BF 3 , H 2 SO 4 , and HCl) fulfilled the specifications established by the American standards and most of the European standard specifications.

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