Pure or immobilized enzymes obtained from microorganisms could reduce the energy costs of industrial ethanol and biodiesel production. Nevertheless, the cellulases used to treat (ligno)cellulosic materials such as forestry residues, waste paper or straw are difficult to purify, like the lipases used for the transesterification of lipids yielding biodiesel. Hence, their price is still too high to make their usage economically viable (Shieh et al., 2003; Ranganathan et al., 2008). Another limiting factor for the use of enzymes is the inactivation and inhibition by reactants and substrates. These drawbacks are the object of an intensive effort to make possible the reutilization of enzymes through protein engineering (Ebrahimpour et al., 2008), in order to increase their stability and activity. Research interest is also targeted on immobilization in different supports or the usage of genetically engineered microorganisms, called whole cell catalysts, which carry the necessary enzymes, avoiding their exposure to inhibiting substrates and operating as microrefineries (Kalscheuer et al., 2006). In the case of biodiesel microbiological production that will be revealed in detail below, the authors proposing and developing this technology refer to this third-generation biofuel as ‘Microdiesel’. The microbial production of biodiesel requires the construction of genetically modified microorganisms, able to transesterificate ethanol with lipids and, if possible, able to produce it by themselves to optimize the whole process. Since their 2006 work on microdiesel production on the laboratory scale using an engineered Escherichia coli strain, Steinbuchel and collaborators have established the guidelines of microdiesel industry development. Their approach consisted of expressing heterologously in E. coli the genes from Zymomonas mobilis, encoding for piruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB), as well as the Acinetobacter baylyi non specific acyl transferase ADP1 (atfA). The obtained strain was able to carry out the aerobic ethanol fermentation from sugars, as well as the enzymatic transesterification of this alcohol with the fatty acids derived from the lipidic metabolism, yielding FAEE, referred to as ‘microdiesel’ by the authors (Kalscheuer et al., 2006). Recently, Elbahloul and Steinbuchel have used the aforementioned microdiesel producing E. coli at a pilot plant scale, using glycerol and sodium oleate as carbon and fatty acids sources respectively, with promising results (Elbahloul & Steinbuchel, 2010). Nevertheless, their conclusions for both studies indicate that there is still a long way to go to the industrial application of their findings, and that the technique needs to be modified to make the engineered strains adaptable to different lipids rich sources and to lignocellulosic raw materials. These modifications would allow the usage of forestry and agricultural wastes, making the biodiesel production process at least as versatile as chemical transesterification.