Production of Propane and Other Short‐Chain Alkanes by Structure‐Based Engineering of Ligand Specificity in Aldehyde‐Deformylating Oxygenase

Khara, Basile; Menon, Navya; Levy, Colin; Mansell, David; Das, Debanu; Marsh, E. Neil G.; Leys, D.; Scrutton, Nigel S.

doi:10.1002/cbic.201300307

Cited by 88 publications

(122 citation statements)

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“…However, the results demonstrated that mutation of them into the large ones caused some steric hindrance for long-chain substrates (≥C 14 ), and the activities of I27F and V28Y against n -dodecanal are about twofold higher than that of WT. Khara et al reported the similar results for V41Y (the counterpart of V28Y of cADO-1593) and WT of cADO-PMT1231 against the substrates with different chain-lengths [21]. (3) Though the side chains of both Val184 and Leu198 points towards the C 9 –C 10 position, V184F and L198F performed differently.…”

Section: Discussionmentioning

confidence: 82%

Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length

Bao

Jia

et al. 2016

Biotechnol Biofuels

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BackgroundAldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths.ResultsBased on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6–10 aldehydes and L198F for C7–10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane.ConclusionsSome amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0596-9) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 82%

Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length

Bao

Jia

et al. 2016

Biotechnol Biofuels

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“…Several studies have been conducted to improve alkane production, yet overall productivities remain low (Schirmer et al, 2010; Khara et al, 2013; Harger et al, 2013; Choi and Lee 2013; Akhtar et al, 2013; Howard et al, 2013; Andre et al, 2013). Better enzymes need to be engineered through either rational or directed evolution approaches, as well as improving the recharge rate of the diiron enzyme system.…”

Section: Discussionmentioning

confidence: 99%

“…Biological alkane production has been shown in the range from C3 to C20 alkanes (Schirmer et al, 2010; Khara et al, 2013; Harger et al, 2013; Choi and Lee, 2013; Akhtar et al, 2013; Howard et al, 2013; Andre et al, 2013), but overall titers and productivities remain lower than those of the corresponding alcohols. This biological pathway is dependent on the formation of a fatty aldehyde intermediate, which can be converted to an alkane or an alcohol by ADO or ALR, respectively (Schirmer et al, 2010; Li et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

Rodriguez

Atsumi

2014

Metabolic Engineering

126

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Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90–99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2–C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production.

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“…The biosynthesis of these alkanes by cyanobacteria is the most recently discovered biosynthetic pathway (Eq. (5)) [95] and the cyanobacterial aldehyde deformylase oxygenase (cADO) is the only decarbonylase for which a tridimensional structure has been obtained by X-ray diffraction analysis [95][96][97].…”

Section: Cyanobacterial Aldehyde Deformylase Oxygenasementioning

confidence: 99%

A growing family of O2 activating dinuclear iron enzymes with key catalytic diiron(III)-peroxo intermediates: Biological systems and chemical models

Trehoux

Mahy

Avenier

2016

Coordination Chemistry Reviews

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Production of Propane and Other Short‐Chain Alkanes by Structure‐Based Engineering of Ligand Specificity in Aldehyde‐Deformylating Oxygenase

Cited by 88 publications

References 19 publications

Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length

Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length

Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

A growing family of O2 activating dinuclear iron enzymes with key catalytic diiron(III)-peroxo intermediates: Biological systems and chemical models

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