Background

Research on alkane production using engineered microbes has gained significant popularity as it provides an attractive alternative to reduce dependence on fossil fuels while mitigating the climate change effects (Lee et al., 2008; Knothe, 2010; Lu, 2010; Schirmer et al., 2010; Tan et al., 2011). Various pathways have been uncovered or artificially assembled for the production of alka(e)nes in microbes (Schirmer et al., 2010; Mendez-Perez et al., 2011; Rude et al., 2011; Akhtar et al., 2013; Howard et al., 2013; Rui et al., 2014). However the highest reported titres for alkane production so far have been in E. coli using the AAR-ADO pathway (Figure 1) (Fatma et al., 2018). AAR catalyzes the reduction of fatty acyl-ACP or fatty acyl-CoA into fatty aldehydes using NADPH which is subsequently converted into alkanes by ADO (Marsh et al., 2013). E. coli is the most widely used host for heterologous production of biofuel candidates due to a broader knowledge of its cellular metabolic network as compared to other hosts. Heterologous co-expression of cyanobacterial AAR and ADO in E. coli results in production and secretion of alkanes (Schirmer et al., 2010). However, quantification of alkanes by the ADO enzyme and determination of the in vivo efficacy of the ADO enzyme cannot be determined due to the variation in the substrate availability by the first enzyme AAR of the pathway. Reports on differences in the solubility of AAR and hence its activity (Kudo et al., 2016) indicate that using AAR as a source of substrate for measuring in vivo activity of ADO is not a suitable approach. Hence, we developed an in vivo enzyme assay, which involves addition of the substrate aldehyde exogenously to the medium that is taken up by the growing cells and gets converted to alkane by the heterologously expressed intracellular ADO enzyme, thus giving a more reliable and accurate measurement of its in vivo efficacy.