Development of a synthetic pathway to convert glucose to hydrogen using cell free extracts

Lu, Franklin J.; Smith, PR; Mehta, Kunal K.; Swartz, James R.

doi:10.1016/j.ijhydene.2015.05.121

Cited by 29 publications

(38 citation statements)

References 35 publications

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“…These higher H 2 generation rates are mainly due to (1) the higher specific activities of hyper‐thermostable enzymes (i.e., G6PDH, NROR, and SH1) on their preferred substrates (Table and Table ), and (2) the lower water solubility of H 2 at high temperature, decreasing possible end‐product inhibition. The volumetric H 2 productivity from NADPH via the biomimetic ETC was nearly six times higher than the other in vitro synthetic pathway based on FNR, ferredoxin, and [FeFe]‐hydrogenase …”

Section: Figurementioning

confidence: 95%

“…Along with ferredoxin, ferredoxin: NADP + oxidoreductase (FNR) plays a role in transferring electrons between ferredoxin and NADPH . Mimicked by the natural electron‐transport chain (ETC) that features high‐speed bio‐H 2 generation rate, an in vitro synthetic pathway composed of FNR, ferredoxin and [FeFe]‐hydrogenase was developed . However, this ETC consisting of ferredoxin and its specific FNR did not exhibit very high H 2 production rates because (1) both reduced ferredoxin and FNR are not stable to O 2 , (2) in many cases the physiological role of FNR is the oxidation of ferredoxin rather than its reduction, and (3) ferredoxins often show selectivity in their interaction with the target enzymes.…”

Section: Figurementioning

confidence: 99%

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Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

Kim

Adams

et al. 2016

Chemistry A European J

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Hydrogen production by water splitting energized by biomass sugars is one of the most promising technologies for distributed green H production. Direct H generation from NADPH, catalysed by an NADPH-dependent, soluble [NiFe]-hydrogenase (SH1) is thermodynamically unfavourable, resulting in slow volumetric productivity. We designed the biomimetic electron transport chain from NADPH to H by the introduction of an oxygen-insensitive electron mediator benzyl viologen (BV) and an enzyme (NADPH rubredoxin oxidoreductase, NROR), catalysing electron transport between NADPH and BV. The H generation rates using this biomimetic chain increased by approximately five-fold compared to those catalysed only by SH1. The peak volumetric H productivity via the in vitro enzymatic pathway comprised of hyperthermophilic glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase, NROR, and SH1 was 310 mmol H /L h , the highest rate yet reported. The concept of biomimetic electron transport chains could be applied to both in vitro and in vivo H production biosystems and artificial photosynthesis.

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Section: Figurementioning

confidence: 95%

Section: Figurementioning

confidence: 99%

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

Kim

Adams

et al. 2016

Chemistry A European J

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“…This is a promising approach that invites future research. Success will primarily depend on the effectiveness in blocking O 2 without restricting electron delivery to the hydrogenase enzyme, which is often mediated by ferredoxin molecules (Eilenberg et al, ; Lu et al, ). Too tight blockage of O 2 diffusion to the active site of the hydrogenase may hinder H 2 diffusion as well as adequate buffering.…”

Section: Engineering Variants With Enhanced O2 Tolerancementioning

confidence: 99%

“…Turnover number (TON) of the enzyme and the yield are two metrics that can be used to assess feasibility of proposed systems. To match the productivity of the bioethanol production processes (20–40 kJ·L −1 ·hr −1 ; Lu et al, ), H 2 needs to be produced at 70–140 mmol·L −1 ·hr −1 . For 1 μM hydrogenase, this translates to TON of roughly 20–40 s −1 .…”

Section: Examples Of and Perspectives On Industrial Applicationsmentioning

confidence: 99%

O₂ sensitivity and H₂ production activity of hydrogenases—A review

Koo

2019

Biotech & Bioengineering

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Hydrogenases are metalloproteins capable of catalyzing the interconversion between molecular hydrogen and protons and electrons. The iron–sulfur clusters within the enzyme enable rapid relay of electrons which are either consumed or generated at the active site. Their unparalleled catalytic efficiency has attracted attention, especially for potential use in H2 production and/or fuel cell technologies. However, there are limitations to using hydrogenases, especially due to their high O2 sensitivity. The subclass, called [FeFe] hydrogenases, are particularly more vulnerable to O2 but proficient in H2 production. In this review, we provide an overview of mechanistic and protein engineering studies focused on understanding and enhancing O2 tolerance of the enzyme. The emphasis is on ongoing studies that attempt to overcome O2 sensitivity of the enzyme while it catalyzes H2 production in an aerobic environment. We also discuss pioneering attempts to utilize the enzyme in biological H2 production and other industrial processes, as well as our own perspective on future applications.

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“…erredoxins are iron-sulfur proteins found in many anaerobic bacteria and archaea and mediate electron transfer in various metabolic processes, including photosynthesis (1, 2), alcohol production (3,4), nitrogen fixation (5,6), and hydrogen production (7). Lack of knowledge about these ferredoxin-dependent pathways currently limits our ability to incorporate them into metabolic engineering strategies.…”

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confidence: 99%

Ferredoxin:NAD ⁺ Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

Tian

Shao

et al. 2016

Appl Environ Microbiol

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Ferredoxin:NAD؉ oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD ؉ . This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum. In wild-type T. saccharolyticum, deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum. When both NADH-and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol. IMPORTANCERedox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD ؉ , a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms. Ferredoxins are iron-sulfur proteins found in many anaerobic bacteria and archaea and mediate electron transfer in various metabolic processes, including photosynthesis (1, 2), alcohol production (3, 4), nitrogen fixation (5, 6), and hydrogen production (7). Lack of knowledge about these ferredoxin-dependent pathways currently limits our ability to incorporate them into metabolic engineering strategies. The ferredoxin:NAD ϩ /NADP ϩ oxidoreductase (FNOR) enzyme (EC 1.18.1.2/EC 1.18.1.3) forms a key bridge in metabolism between the nicotinamide cofactor (i.e., NAD ϩ , NADH, NADP ϩ , and NADPH)-dependent pathways and ferredoxin-dependent pathways (Fig. 1, equation a) (8-11). Recently, the thioredoxin reductase-like (TrxR-type) FNORs were found, and they are widely distributed among the bacteria and archaea (12)(13)(14). Even these types of FNORs are more homologous to bacterial NADPH-TrxRs, but they have catalytic properties similar to those of FNOR (14). FNOR enzymes are widely believed to be of central importance for the bioenergetics of anaerobic bacteria due to their ability to couple electron transport with ion or Na ϩ gradient generation (15) or transhydrogenation. Furthermore, they are the key enzymes for many biochemical and biofuel pathways, including isopropanol, ethanol, and n-butanol (3,4,16). Since ferredoxin has a lower standard reduction potential than...

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Development of a synthetic pathway to convert glucose to hydrogen using cell free extracts

Cited by 29 publications

References 35 publications

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

O₂ sensitivity and H₂ production activity of hydrogenases—A review

Ferredoxin:NAD ⁺ Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

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Development of a synthetic pathway to convert glucose to hydrogen using cell free extracts

Cited by 29 publications

References 35 publications

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways

O2 sensitivity and H2 production activity of hydrogenases—A review

Ferredoxin:NAD + Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

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O₂ sensitivity and H₂ production activity of hydrogenases—A review

Ferredoxin:NAD ⁺ Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation