High‐Yield Production of Dihydrogen from Xylose by Using a Synthetic Enzyme Cascade in a Cell‐Free System

Campo, Julia S. Martín del; Rollin, Joseph A.; Myung, Suwan; You, Chun; Chandrayan, Sanjeev K.; Patiño, Rodrigo; Adams, Michael W. W.; Zhang, Y.-H. Percival

doi:10.1002/anie.201300766

Cited by 112 publications

(54 citation statements)

References 31 publications

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“…In addition to HjLAD, it is likely that there are other enzymes present in the cell-free system involved in NAD + regeneration that help facilitate the reaction towards L-xylulose production. In vitro biocatalytic cell-free systems have been studied well and utilized in many applications like the production of (3R)acetoin from glycerol, 31 production of dihydrogen from xylose, 32 and transformation of nonfood biomass to starch. 33 It shows advantages in higher product yields, faster reaction rates, and elimination of cell-associated process barriers compared to in vivo biocatalytic systems.…”

Section: L-xylulose Production By Hjlad In a Cell-free Systemmentioning

confidence: 99%

Rare sugar production by coupling of NADH oxidase and l-arabinitol dehydrogenase

et al. 2016

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An efficient biocatalytic cell-free system containing L-arabinitol dehydrogenase (LAD) for L-arabinitol oxidation and NADH oxidase (Nox) for cofactor regeneration was successfully constructed and used for L-rare sugar production. The recombinant LAD (HjLAD) from Hypocrea jecorina suffered from NADH inhibition. Applying Nox to the cell-free HjLAD system drove the thermodynamically unfavorable equilibrium from L-arabinitol to L-xylulose, along with the regeneration of the oxidized cofactor NAD + . Response surface methodology was used to optimize the conditions for L-xylulose production. The optimal conditions for biocatalytic production of L-xylulose were found to be 4.9 U/mL HjLAD, 8.2 mM NAD + at pH 8.0, and 30.9 o C; a high conversion rate of 78.4% was achieved under these conditions. We demonstrate a cellfree enzyme coupling system enabling efficient cofactor recycling and providing high yields of L-xylulose. The results indicate that this coupling system provides a new biocatalytic method for L-rare sugar production.

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Section: L-xylulose Production By Hjlad In a Cell-free Systemmentioning

confidence: 99%

Rare sugar production by coupling of NADH oxidase and l-arabinitol dehydrogenase

et al. 2016

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“…Enzymatic synthesis has been used to produce numerous valuable compounds [6]–[16] and often provides significant enhancements in yield, purity, production time and cost when compared to traditional chemical synthetic methods [17], [18]. Considerable effort is being expended to develop cell-free enzymatic systems for the production of biofuels, including dihydrogen [19] and butanol [20], biomass conversion to starch [21], and high-energy-density biobatteries [22]. While enzymatic synthesis will never replace traditional synthesis, it provides a valuable adjunct to traditional approaches particularly when the objective is to build complex natural products.…”

Section: Introductionmentioning

confidence: 99%

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors

Rodriguez

Leyh

2014

PLoS ONE

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The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation.

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“…The use of enzymes from (hyper)thermophiles provides a number of advantages in constructing in vitro enzyme systems (7)(8)(9)(10)(12)(13)(14)(15)(16). The first advantage is that it is possible to remove or deactivate the activities of enzymes deriving from the host cell that might compete with the designed pathway simply by incubating the cell extracts at high temperature.…”

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confidence: 99%

An In Vitro Enzyme System for the Production of myo -Inositol from Starch

Fujisawa

Fujinaga

Atomi

2017

Appl Environ Microbiol

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We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus, and IMPase from the hyperthermophilic bacterium Thermotoga maritima. The enzymes were individually produced in Escherichia coli and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD ϩ , resulting in the production of myo-inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD ϩ was added every 2 h, we observed the production of 2.9 g myo-inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from T. maritima into the system and observed myo-inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively. IMPORTANCE myo-Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce myo-inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase, and myo-inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of myo-inositol. myo-Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo-inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo-inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of myo-inositol.

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High‐Yield Production of Dihydrogen from Xylose by Using a Synthetic Enzyme Cascade in a Cell‐Free System

Cited by 112 publications

References 31 publications

Rare sugar production by coupling of NADH oxidase and l-arabinitol dehydrogenase

Rare sugar production by coupling of NADH oxidase and l-arabinitol dehydrogenase

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors

An In Vitro Enzyme System for the Production of myo -Inositol from Starch

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