Conversion of fatty aldehydes into alk (a/e)nes by in vitro reconstituted cyanobacterial aldehyde-deformylating oxygenase with the cognate electron transfer system

Abstract

Background: Biosynthesis of fatty alk(a/e)ne in cyanobacteria has been considered as a potential basis for the sunlight-driven and carbon-neutral bioprocess producing advanced solar biofuels. Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in that pathway. The heterologous or chemical reducing systems were generally used in in vitro ADO activity assay. The cognate electron transfer system from cyanobacteria to support ADO activity is still unknown.

Results: We identified the potential endogenous reducing system including ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) to support ADO activity in Synechococcus elongatus PCC7942. ADO (Synpcc7942_1593), FNR (SynPcc7942_0978), and Fd (SynPcc7942_1499) from PCC7942 were cloned, overexpressed, purified, and characterized. ADO activity was successfully supported with the endogenous electron transfer system, which worked more effectively than the heterologous and chemical ones. The results of the hybrid Fd/FNR reducing systems demonstrated that ADO was selective against Fd. And it was observed that the cognate reducing system produced less H2O2 than the heterologous one by 33% during ADO-catalyzed reactions. Importantly, kcat value of ADO 1593 using the homologous Fd/FNR electron transfer system is 3.7-fold higher than the chemical one.

Conclusions: The cognate electron transfer system from cyanobacteria to support ADO activity was identified and characterized. For the first time, ADO was functionally in vitro reconstituted with the endogenous reducing system from cyanobacteria, which supported greater activity than the surrogate and chemical ones, and produced less H2O2 than the heterologous one. The identified Fd/FNR electron transfer system will be potentially useful for improving ADO activity and further enhancing the biosynthetic efficiency of hydrocarbon biofuels in cyanobacteria.

Figure 1

ADO-catalysed reaction [[8],[12]-[16]]. Oxygen and the auxiliary reducing system (biological or chemical) providing four electrons are needed for ADO activity, one O-atom is incorporated into formate, and the aldehyde hydrogen is retained in formate.

Figure 2

Representative electron flow in conversion of fatty aldehyde into alk(a/e)ne with the reconstituted ADO/Fd/FNR system. Reducing equivalents from NADPH are transferred from FNR to Fd and then to ADO. The crystal structures of related ADO (PDB ID: 2OC5, 60% sequence identity to ADO from PCC7942) from Prochlorococcus marinus MIT9313, Fd (PDB ID: 1QT9, 82% sequence identity to Fd from PCC7942) and FNR (PDB ID: 1QUE, 67% sequence identity to FNR from PCC7942) from Anabaena sp. PCC7119 were used to demonstrate electron transfer in the reconstituted ADO/Fd/FNR system.

Figure 3

Characterization of FNR from Synechococcus elongatus PCC7942. (A) UV–vis absorption spectrum of FNR. (B) Ferricyanide reductase activity of FNR.

Figure 4

Characterization of Fd from Synechococcus elongatus PCC7942. (A) UV–vis absorption spectrum of Fd. (B) Fd-mediated cytochrome c reductase activity of FNR.

Figure 5

SDS-PAGE analysis of ADO 1593. Lane 1 and 4, low protein molecular weight marker; Lane 2, crude supernatant for over-expressed AD 1593; Lane 3, flow-through when loaded; Lane 5 and 6, eluents of buffer A + 60 mM imidazole; Lanes 7–11, eluents of buffer A + 80 mM imidazole.