Heterologous biosynthesis and manipulation of alkanes in Escherichia coli

Biosynthesis of alkanes in microbial foundries offers a sustainable and green supplement to traditional fossil fuels. The dynamic equilibrium of fatty aldehydes, key intermediates, played a critical role in microbial alkanes production, due to the poor catalytic capability of aldehyde deformylating oxygenase (ADO). In our study, exploration of competitive pathway together with multi-modular optimization was utilized to improve fatty aldehydes balance and consequently enhance alkanes formation in Escherichia coli. Endogenous fatty alcohol formation was supposed to be competitive with alkane production, since both of the two routes consumed the same intermediate-fatty aldehyde. Nevertheless, in our case, alkanes production in E. coli was enhanced from trace amount to 58.8 mg/L by the facilitation of moderate fatty alcohol biosynthesis, which was validated by deletion of endogenous aldehyde reductase (AHR), overexpression of fatty alcohol oxidase (FAO) and consequent transcriptional assay of aar, ado and adhP genes. Moreover, alkanes production was further improved to 81.8 mg/L, 86.6 mg/L or 101.7 mg/L by manipulation of fatty acid biosynthesis, lipids degradation or electron transfer system modules, which directly referenced to fatty aldehydes dynamic pools. A titer of 1.31 g/L alkanes was achieved in 2.5 L fed-batch fermentation, which was the highest reported titer in E. coli. Our research has offered a reference for chemical overproduction in microbial cell factories facilitated by exploring competitive pathway.