Structure-guided engineering of α-ketoisocaproate dioxygenase increases isobutene production inSynechocystissp. PCC 6803

Isobutene is a promising precursor for jet fuel due to its high energy density and favorable combustion properties. Light-driven bioproduction of isobutene has recently been investigated as an alternative strategy to crude oil refinement or fermentation-based manufacturing processes by harnessing the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the α-ketoisocaproate dioxygenase ( Rn KICD) from Rattus norvegicus . However, the obtained production level was not sufficient, partially due the promiscuous activity of Rn KICD. The enzyme catalyzes both the reaction with ρ-hydroxyphenylpyruvate (HPP) for homogentisate formation, as well as the reaction with α-ketoisocaproate (KIC), the precursor for isobutene synthesis. Here, to overcome this bottleneck step in the isobutene biosynthesis, protein engineering was employed to improve Rn KICD activity and in vivo isobutene production. Purified Rn KICD variants were characterized by measuring in vitro KIC and HPP consumption rates, as well as isobutene formation rate. The active site mutations F336V, N363A altered the KIC and HPP consumption rates, while the KIC-to-isobutene conversion ratio was only marginally affected. Besides, the Rn KICD variants F336V, N363A and F336V/N363A exhibited a substantially enhanced substrate selectivity for KIC over HPP. Among the examined engineered Synechocystis strains, Syn-F336V showed a 4-fold improvement in isobutene production, compared to the base strain (Syn- Rn KICD). Our findings reveal that residues F336 and N363 play a crucial role in substrate interactions, as targeted mutations at these sites shifted the substrate selectivity towards KIC and elevated the in vivo isobutene production levels significantly. We conclude that engineering the active site of Rn KICD is a potent tool for improving isobutene bioproduction in Synechocystis . Highlights Promiscuous α-ketoisocaproate dioxygenase (KICD) from Rattus norvegicus was overproduced in Synechocystis sp. PCC 6803. Protein engineering was used to improve substrate selectivity. The generated F336V variant led to a 4-fold increase in isobutene production in vivo .