**4. Metabolic engineering of oleaginous yeasts**

The availability of genome data and genetic tools, such as the possibility to integrate homologous or heterologous genes, opened up the possibility to use metabolic engineering to understand the molecular mechanisms involved in lipid accumulation or to increase the yield of stored lipids in *S. cerevisiae* and *Y. lipolytica*. Whereas *S. cerevisiae* 

Getting Lipids for Biodiesel Production from Oleaginous Fungi 81

organisms (Ykema et al., 1986; Granger et al., 1993; Economou et al., 2011). *Y. lipolytica* is the oleaginous microorganism for which information about the metabolic response to different C/N ratios is most abundant (Beopoulos et al., 2009a), particularly due to the availability of molecular tools for genetic engineering of this organism. Therefore, *Y. lipolytica* is regarded as a model organism for microbial oil production and the main traits of its metabolism can be used to give a general description of the metabolic response to different C/N ratios in the majority of oleaginous yeasts. With the increase of the C/N ratio, different metabolic behaviors were observed in *Y. lipolytica*: i) growth with mobilization of storage lipids, ii) growth of fat-free biomass, iii) growth with accumulation of lipids, and iv) growth with

Fig. 6. Metabolic activity of oleaginous fungi (e.g. *Y. lipolitica*) as a function of carbon flow rate for a fixed nitrogen flow rate. Arrows indicate the consumption of nitrogen and carbon sources by the cells; squares indicate production rate. The dimension of arrows and squares

If the medium is carbon limited or when the extracellular carbon supply gets exhausted, previously stored intracellular lipid can be mobilized and utilized by oleaginous microorganisms to sustain cells generation and production of lipid-free biomass (Park et al., 1990) (Fig. 6, i). If the medium is balanced and/or furnishes just the right amount of carbon flow to satisfy the growth need, balanced growth occurs without any accumulation of storage lipids (Fig. 6, ii). In conditions of carbon excess, a part of the carbon flow, which is proportional to nitrogen availability (Granger et al., 1993), is directed toward cells generation, whereas the carbon exceeding growth needs is channeled to the production of storage lipids (Fig. 6, iii). In some oleaginous fungi, the presence of a large carbon excess leads to the production of great amounts of organic acids, such as pyruvic acid and diverse TCA-cycle intermediates, at the expenses of lipid accumulation (Fig. 6, iv). In these latter conditions, *Y. lipolytica* produces citric acid (Levinson et al., 2007) but other oleaginous

lipid accumulation and production of organic acids (Fig. 6).

is proportional to flow. (adapted from Beopoulos et al., 2009a)

yeasts have never been reported to behave this way.

has been used mostly as a model to investigate and understand the lipid metabolism, in *Y. lipolytica* several attempts have been done in order to address the carbon flux toward TAG production and accumulation. Similar approaches are precluded to other oleaginous fungi since they lack genome information and the necessary tools for gene manipulation and strain improvement.

In *Y. lipolytica*, the role of glycerol-3-phosphate (G3P) in triacylglycerol (TAG) biosynthesis and accumulation has been investigated (Beopoulos et al., 2008). In this yeast G3P is formed from glycerol by the glycerol kinase encoded by *GUT1*, or it is synthetized from dihydroxyacetone phosphate (DHAP) by the G3P dehydrogenase (*GPD1*). The antagonist reaction, which produces DHAP from G3P, is carried out in competition by a second isoform of the G3P dehydrogenase, encoded by *GUT2*. In order to force the conversion of DHAP into G3P, the gene *GPD1* was over-expressed and the gene *GUT2* was deleted.

A diverse strategy to increase lipid accumulation was based on the disruption of the βoxidative metabolism, through the deletion of the 6 *POX* genes (*POX1 to POX6*) that encode the peroxisomal acyl-coenzyme oxidases (Mlickowa et al., 2004a; Mlickowa et al., 2004b; Beopoulos et al., 2008). As a whole, the best results in terms of percentage of lipids per dry biomass, were reached coupling the increased level of G3P with the disactivation of the βoxidation pathway (65%) (Dulermo et al., 2011).

Metabolic engineering strategies have been recently exploited to expand the range of substrates used by oleaginous fungi, also through functional expression of heterologous genes. Recently, it has been found that inulin is a good material for bio-productions (Chi et al., 2009). In order to make the oleaginous yeast *Y. lipolytica* able to accumulate lipids on inulin containing materials, the *Kluyveromyces marxianus* exo-inulinase gene (*INU1*) was heterologously expressed on a high copy plasmid (Zhao et al., 2010). The inulinase was efficiently secreted by *Y. lipolytica*, and inulin was hydrolyzed, assimilated and converted into TAG.
