Identification of non-conserved residues essential for improving the hydrocarbon-producing activity of cyanobacterial aldehyde-deformylating oxygenase

BACKGROUND: Cyanobacteria produce hydrocarbons corresponding to diesel fuels by means of aldehyde-deformylating oxygenase (ADO). ADO catalyzes a difficult and unusual reaction in the conversion of aldehydes to hydrocarbons and has been widely used for biofuel production in metabolic engineering; however, its activity is low. A comparison of the amino acid sequences of highly active and less active ADOs will elucidate non-conserved residues that are essential for improving the hydrocarbon-producing activity of ADOs. RESULTS: Here, we measured the activities of ADOs from 10 representative cyanobacterial strains by expressing each of them in Escherichia coli and quantifying the hydrocarbon yield and amount of soluble ADO. We demonstrated that the activity was highest for the ADO from Synechococcus elongatus PCC 7942 (7942ADO). In contrast, the ADO from Gloeobacter violaceus PCC 7421 (7421ADO) had low activity but yielded high amounts of soluble protein, resulting in a high production level of hydrocarbons. By introducing 37 single amino acid substitutions at the non-conserved residues of the less active ADO (7421ADO) to make its sequence more similar to that of the highly active ADO (7942ADO), we found 20 mutations that improved the activity of 7421ADO. In addition, 13 other mutations increased the amount of soluble ADO while maintaining more than 80% of wild-type activity. Correlation analysis showed a solubility-activity trade-off in ADO, in which activity was negatively correlated with solubility. CONCLUSIONS: We succeeded in identifying non-conserved residues that are essential for improving ADO activity. Our results may be useful for generating combinatorial mutants of ADO that have both higher activity and higher amounts of the soluble protein in vivo, thereby producing higher yields of biohydrocarbons. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13068-019-1409-8) contains supplementary material, which is available to authorized users.