Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species

Plummer, Scott M.; Plummer, Mark A.; Merkel, Patricia; Hagen, Moira; Biddle, Jennifer F.; Waidner, Lisa A.

doi:10.1016/j.enzmictec.2016.07.011

Cited by 7 publications

(5 citation statements)

References 36 publications

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“…This approach is appealing to use for protein design because amino acid substitutions created by recombination are less disruptive than those created randomly, since the sequence blocks being swapped have already been selected by evolution for compatibility within native structures 10 . Recombination has been applied to a variety of enzymes including metal-containing oxidoreductases, such as cytochromes P450, laccases, and hydrogenases [11][12][13][14] . These studies have revealed that recombination can lead to innovation in metalloproteins, creating chimeras with distinct properties from the parent proteins being bred, including higher catalytic activity, distinct substrate specificity profiles, and altered stabilities [11][12][13][14] .…”

Section: Whilementioning

confidence: 99%

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

Campbell

Kahanda

Atkinson

et al. 2020

Preprint

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Homologous recombination can be used to create enzymes that exhibit distinct activities and stabilities from proteins in nature, allowing researchers to overcome component limitations in synthetic biology. To investigate how recombination affects the physical properties of an oxidoreductase that transfers electrons, we created ferredoxin (Fd) chimeras by recombining distantly-related cyanobacterial and cyanomyophage Fds that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also be used to build synthetic pathways that support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required to support similar levels of cellular electron transfer. These results show how recombination can be used to rapidly diversify the physical properties of protein electron carriers and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic electron transfer pathway.

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Section: Whilementioning

confidence: 99%

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

Campbell

Kahanda

Atkinson

et al. 2020

Preprint

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“…In addition to looking for the most biotechnologically suitable host for clostridial H 2 ases, direct evolution approaches can be expected in the future to enhance the yield of H 2 produced by the recombinant strains. As a proof of concept, a selected chimeric FeFe-H 2 ase which evolved from two clostridial sequences produced and purified from E. coli showed a 400%-fold increase in the H 2 ase in vitro activity in comparison with the parental sequence (Plummer et al 2016). In a recent study, a directed mutagenesis of the FeFe H 2 ase Cpl from Clostridium pasteurianum has showed that a single Cysteine substitution near the proximal delivery center significantly increased the tolerance to O 2 without lowering the amount of H 2 produced (Koo et al 2016).…”

Section: Use Of Clostridial Hydrogenases In Heterologous Systemsmentioning

confidence: 99%

Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives

Latifi

Avilán

Brugna

2018

Appl Microbiol Biotechnol

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Strictly anaerobic bacteria of the Clostridium genus have attracted great interest as potential cell factories for molecular hydrogen production purposes. In addition to being a useful approach to this process, dark fermentation has the advantage of using the degradation of cheap agricultural residues and industrial wastes for molecular hydrogen production. However, many improvements are still required before large-scale hydrogen production from clostridial metabolism is possible. Here we review the literature on the basic biological processes involved in clostridial hydrogen production, and present the main advances obtained so far in order to enhance the hydrogen productivity, as well as suggesting some possible future prospects.

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“…In another work, Denard group was able to engineer the regioselectivity of hydroxylase activity of cytochrome P450, converting cytochrome P450 from terminal-to a sub-terminal hydroxylase with 99% ee enantioselectivity ( ure 5: (a) Stereoselective conversion of racemic amines by engineered (R)-amine oxidase variant; (b) hydroxylati -octane at subterminal position to produce (S)-2-octanol with 99% ee regioselectivity, using engineered cytochrom 0 variant for highly enantioselective and regioselective activities Yasukawa et al, 2014; Dena l., 2015). Several properties of enzymes were recently improved using the directed evolution approach, some are summarized in Table 2, including enzyme thermostability (Morimoto et al, 2014 andAcevedo et al, 2017), activity (Plummer et al, 2016 andYu et al, 2016), substrate specificity (Bosshart et al, 2016 andSun et al, 2016), regio-and enantio-selectivity Denard et al, 2015). Furthermore, Arnold group recently achieved remarkable success in transforming cytochrome P450 BM3 into an efficient cyclopropanation catalyst, via carbene and nitrene transfer in vivo and in vitro, an activity for which there is no precedent in nature (Coelho et al, 2013a;b and Arnold, 2015).…”

Section: Enzyme Engineering For Enzyme Function Enhancementmentioning

confidence: 99%

Recent Trends for Discovery and Enhancement of Enzyme Function: A Review

Ibrahim

El‐Dewany

2018

Egypt. J. Microbiol.

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R ECENTLY, there is a growing recognition of enzymes utilization as ecofriendly alternative for chemical catalysts in various industrial sectors. Two main approaches are used to expand the enzymes utilization in various industrial applications including (i) Searching nature for discovery of better performing novel biocatalysts and (ii) Improvement of the enzyme function and properties. The recent advances in biocatalyst discovery and engineering have led to an increase of the efficiency and functional diversityof enzymes. Therefore, enzyme application is expanding to broader industrial processes, including those which were previously restricted to the classical chemical catalysts. Furthermore, there is a significant success in developing made-to-order enzymes that meet the industrial process conditions requirements, leading to more efficient and cost-effective processes. Here I highlight state-of-the-art strategies and tools for novel enzyme discovery and function enhancement, including enzyme engineering at the molecular level through different approaches such as directed evolution, rational design, semi rational design and computational de novo enzyme design; in addition to the recently developed field, nanobiocatalysis.

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Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species

Cited by 7 publications

References 36 publications

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives

Recent Trends for Discovery and Enhancement of Enzyme Function: A Review

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