Acl converts cytosolic citrate CoA and ATP into acetyl

Acl converts cytosolic citrate, CoA and ATP into acetyl-CoA, ADP+Pi and oxaloacetate (Fig. 1). This cytosolic enzyme is present in few Prokaryotes and in all Eukaryotes, but not in non-oleaginous yeasts [14]. Thus, this enzyme was presumed to be essential for FA synthesis [1], [13], [15]. In most microorganisms and in plants, Acl is composed of two subunits, encoded by the ACL1 and ACL2 genes [16], while in animals and in the oleaginous Basidiomycete yeast, Rhodotorula gracilis, Acl is encoded by one gene [17], [18], [19]. In the non-oleaginous fungus Aspergillus niger, inactivation of ACL1 and/or ACL2, results in a decrease of acetyl-CoA and citric ataluren levels, vegetative growth, pigmentation, asexual conidiogenesis, and conidial germination and an increase of succinic acid level [20], [21]. Unfortunately, the effect of Acl inactivation on FA synthesis was not described in this fungus. Additionally, overexpression of ACL1 and ACL2 generated variable results depending on the organism. In Aspergillus oryzae it leads to an 1.7-fold increase of FA content [22] whereas no effect was observed when these genes were overexpressed in the wild type (WT) or in citrate overproducing strains of Saccharomyces cerevisiae, or in WT strain of Yarrowia lipolytica[9], [10]. Few studies have examined Acl in oleaginous microorganisms. Recently, Liu et al. [23] and Ochoa-Estopier and Guillouet [24] have shown that high activity of Acl is usually observed during the FA synthesis phase in Trichosporon cutaneum and in Y. lipolytica. However, no mutant inactivated for Acl has ever been described in an oleaginous organism. Mae is a cytosolic enzyme involved in conversion of L-malate and NADP+ into pyruvate, CO2 and NADPH (Fig. 1) [25]. NADPH is essential for FA synthesis and due to its capacity to produce NADPH, this enzyme was proposed to be one of the rate-limiting steps for FA synthesis [12], [26]. In oleaginous microorganisms, the role of Mae was only deduced from its activity during lipid accumulation [23], [24], [27] or from overexpression studies, never in strains inactivated for the corresponding gene. As an example, overexpression of Mae from Mucor circinelloides improved lipid accumulation by 2.5- and 2-fold in M. circinelloides and in the oleaginous Basidiomycetes yeast Rhodotorula glutinis, respectively [28], [29]. The oleaginous yeast, Y. lipolytica, does not possess cytosolic Mae, but only a mitochondrial form, which may not be involved in NADPH production [30]. The role of this mitochondrial NADH-dependent Mae remains unclear. Moreover the overexpression of cytosolic NADPH-dependent Mae from Mortierella alpina failed to improve lipid synthesis in Y. lipolytica, raising the question of NADPH supply in this yeast. The question of provision of NADPH for FA synthesis in Y. lipolytica became a “hot topic” since Wasylenko et al. [31] showed that pentose phosphate pathway is the primary source of lipogenic NADPH in Y. lipolytica and C. Ratledge [25] just proposed alternative routes to pentose phosphate pathway to provide NADPH to FA synthesis. In Ascomycetes fungi, the mannitol cycle was proposed to be a provider of reducing power [32]. This cycle consists of two pathways (Fig. 4). The first pathway involves in the reduction of fructose-6-phosphate, from glycolysis, into mannitol-1-phosphate, by the NAD(H)-dependent mannitol-1-phosphate dehydrogenase (Mpd), which is then dephosphorylated into mannitol via a mannitol-1-phosphate phosphatase. This last reaction was described as irreversible, but some data suggest that it is not the case in all fungi [33]. In the second pathway, fructose is oxidized into mannitol via a reversible NADP(H)-dependent mannitol 2-dehydrogenase (Mdh). One turn of this cycle gives the net result: NADH+NADP++ATP converted to NAD++NADPH+ADP+Pi, i.e. the same net result as the reactions needed for NADPH production from malate with the cytosolic Mae [25]. As Y. lipolytica is known to produce mannitol [34], this raises the question of a potential role of the mannitol metabolism in FA synthesis in this yeast.