Tunnel Shape of Aldehyde Deformylating Oxygenase Determines Length of Alkane Products

Abstract

R&D on renewable energy has gained the centre stage again due to renewed call to tackle climate change. In this effort, drop‐in fuels like alkane will be the main biofuel candidate since they are similar to the petroleum fuel in their chemical and physical properties. Cyanobacterial AAR (acyl‐ACP reductase) ‐ ADO (aldehyde deformylating oxygenase) pathway, which converts fatty acyl‐ACP intermediate of the fatty acid synthesis cycle into alkane/alkene via an aldehyde intermediate, was the first pathway discovered for the microbial production of hydrocarbon. We have earlier reported an engineered thermostable ADO and demonstrated metabolic modelling based pathway engineering in E. coli to produce highest alkane titer reported so far. We have further performed biochemical characterization of ADOs from three different cyanobacteria (Nostoc punctiformes, Oscillatoria jasorensis, Oscillatoria sp CCC305), and found out that ADO from N. punctiforme(NpADO) yielded maximum alkane titer, with preference towards C12‐chain length aldehyde substrate. Upon structural modelling and molecular dynamics (MD) simulation, it was found that NpADO has suitable tunnel shape and size to facilitate catalysis of C12‐chain substrate. Considering the importance of the catalytic products in the aviation fuel range, we attempted to further improve the product specificity by mutating residues in the main tunnel to facilitate fitting of C12‐chain substrate in the cavity and blocking other less favorable tunnel. The engineered ADO showed 2‐fold higher catalytic activity towards C12‐chain aldehyde. MD simulations of the engineered ADOs indicated favorable tunnel structure, stabilized ADO‐substrate interactions and enhanced interaction of substrate with the di‐iron centre. These findings suggest ADO tunnels play a critical role in defining their substrate specificity and catalysis.