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

Abstract

BackgroundBiosynthesis 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.ResultsWe 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.ConclusionsThe 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.

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…(2) Zhang et al (2013) P450s may, however, catalyze many other reactions, such as N-or S-hydroxylations, epoxidations, dealkylations, and many more (Sono et al 1996). Catalysis takes place by a complex cycle, wherein reductive activation of O 2 produces a highly reactive ferryl-oxo radical that attacks the substrate R-H bond (Rittle and Green 2010;Munro et al 2013a, b).…”

Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

“…The vectors pET28b-sefdx and pET28b-sefdR for expression of Fdx and FdR of S. elongatus PCC 7942 were gifts from Xuefeng Lu, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences. For construction of the pCDFDuet-sefdR-sefdx coexpression vector, the sefdx and sefdR genes bearing the corresponding restriction sites were PCR amplified and sequentially inserted into the NdeI-XhoI and BamHI-HindIII sites of pCDFDuet-1 (25). Plasmids pET-28b-cyp-sb21 and pCDFDuet-sefdR-sefdx were cotransformed into Escherichia coli BL21(DE3) cells to obtain whole-cell transformation strain W1.…”

Alka(e)ne synthesis in Cupriavidus necator boosted by the expression of endogenous and heterologous ferredoxin–ferredoxin reductase systems

“…Recently, (Cao et al, ) improved alkane production in E.coli from 60 to 100 mg/L by expressing the heterologous Se Fd/FNR system concomitantly with Se aar and ado genes, whereas the overexpression of the endogenous E. coli Fd/FNR had no detectable effect. The authors suggested that ADO was selective for homologous cyanobacteria other than heterologous and chemical ones as observed earlier by Zhang et al (). We demonstrated here that the Se ADO was in vivo functional with the surrogate Fd/FNR system from C. necator , even if it was not fully adequate.…”

“…The ADO activity requires oxygen, NADPH, and an auxiliary reducing system ferredoxin (Fd) and ferredoxin reductase (FNR) (N. Warui et al, 2011;Zhang, Lu, & Li, 2013). An auxiliary reducing system (biological or chemical) is needed for ADO activity indeed, as the ADO forms a peroxo intermediate as part of its reaction mechanism as demonstrated by numerous in vitro studies (Das, Eser, Han, Sciore, & Marsh, 2011;N.…”