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

Abstract

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.

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

“…Our modelling results suggest that the elimination of the C-terminal sequence has a negative effect on the substrate binding in all three candidates ( Supplementary Fig. 4), which is consistent with the notion that residues far from the active site and the substrate binding site still contribute to the enzyme activity of AD [15].…”

“…ADs are small soluble proteins, which allows them to be studied in vitro more readily, leading to their wider use in metabolic engineering [11,15,[23][24][25][26]. Although new bacterial AD is constantly being characterized and engineered for alkane production [11,22,23,27,28], our experiments showed that protein accumulation of bacterial ADs is limited in vivo.…”

“…Although new bacterial AD is constantly being characterized and engineered for alkane production [11,22,23,27,28], our experiments showed that protein accumulation of bacterial ADs is limited in vivo. It was also reported that there is no significant accumulation of most bacterial AD in E. coli during incubation [15], and even a decrease in AD abundance after 10 h of incubation [28]. The low levels of AD might be due to fast in vivo degradation, which can limit the application of AD for alkane biosynthesis.…”

A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution

“…Among these, both plant and insect ADs are membrane proteins, complicating their characterization in vitro. In contrast, bacterial ADs are small soluble proteins, which allows them to be studied in vitro more readily, leading to their wider use in metabolic engineering [11,15,[23][24][25][26]. Although new bacterial AD is constantly being characterized and engineered for alkane production [11,22,23,27,28], our experiments showed that protein accumulation of bacterial ADs is limited in vivo.…”

“…To our knowledge, the highest reported value for long chain alkane production is 2.54 g/L (C13-C17) in engineered Escherichia coli [7], but this is still insufficient for industrial scale-up. It has been reported that the low efficiency of AD is the bottleneck of alkane production [4,14,15], which warrants further investigation into how ADs behave in vivo.…”