Cyanobacterial Enzymes for Bioalkane Production

Abstract

Cyanobacterial biosynthesis of alkanes is an attractive way of producing substitutes for petroleum-based fuels. Key enzymes for bioalkane production in cyanobacteria are acyl-ACP reductase (AAR) and aldehyde-deformylating oxygenase (ADO). AAR catalyzes the reduction of the fatty acyl-ACP/CoA substrates to fatty aldehydes, which are then converted into alkanes/alkenes by ADO. These enzymes have been widely used for biofuel production by metabolic engineering of cyanobacteria and other organisms. However, both proteins, particularly ADO, have low enzymatic activities, and their catalytic activities are desired to be improved for use in biofuel production. Recently, progress has been made in the basic sciences and in the application of AAR and ADO in alkane production. This chapter provides an overview of recent advances in the study of the structure and function of AAR and ADO, protein engineering of these enzymes for improving activity and modifying substrate specificities, and examples of metabolic engineering of cyanobacteria and other organisms using AAR and ADO for biofuel production.

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

“…The structure was built by homology modeling using I-TASSER [66]. The substrate binding site (yellow), iron binding sites (orange), and proton supply channel to the active center (cyan) are shown in the structure [10]. The mutation sites of 20 mutants that increased activity, 3 mutants that decreased activity by more than 20%, and 2 mutants that increased the amount of soluble ADO by more than 50% are indicated by red, blue, and green sticks, respectively.…”

“…Cyanobacteria-derived hydrocarbons have gained attention as a renewable energy source that can help limit global warming [4]. Moreover, the cyanobacterial biosynthesis of hydrocarbons is attractive because the growth rate of cyanobacteria is rapid, and their genetic manipulation is well established [5–10].…”

“…Therefore, AAR and ADO are key enzymes for the biosynthesis of hydrocarbons. Thus, AAR and ADO have been applied for the production of biohydrocarbons in several organisms [10], including cyanobacteria [15–20], E. coli [11, 21–25], yeasts [26–28], chemoautotrophic eubacteria [29], and filamentous fungi [30]. In particular, ADO has been extensively used with other enzymes because ADO can catalyze a difficult and unusual reaction in the conversion of aldehydes to hydrocarbons [10, 23, 27, 31–35].…”

“…Although ADO has been widely used for biofuel production by the metabolic engineering of cyanobacteria and other organisms, it has very low activity. Its maximum turnover number is ~ 1 min −1 [42], and the rate-limiting step in the biosynthesis of hydrocarbons in E. coli coexpressing AAR and ADO is the reaction catalyzed by ADO [10, 24]. There have been a few attempts to improve the hydrocarbon-producing activity of ADO by introducing amino acid substitutions based on the crystal structures of cyanobacterial ADOs [11, 22, 39].…”

Improving hydrocarbon production by engineering cyanobacterial acyl-(acyl carrier protein) reductase

“…Subsequently, ADO converts the fatty aldehydes (C n ) into corresponding hydrocarbons (C n −1 ) and formate (HCOOH) [10, 17]. Escherichia coli coexpressing cyanobacterial AAR and ADO can produce and secrete hydrocarbons, indicating that AAR and ADO are essential for hydrocarbon biosynthesis [10, 18].…”