Cell‐Free Metabolic Engineering: Production of Chemicals by Minimized Reaction Cascades

Guterl, Jan‐Karl; Garbe, Daniel; Carsten, Jörg; Steffler, Fabian; Sommer, Bettina; Reiße, Steven; Philipp, Anja; Haack, Martina; Rühmann, Broder; Koltermann, Andre; Kettling, Ulrich; Brück, Thomas; Sieber, Volker

doi:10.1002/cssc.201200365

Cited by 223 publications

(292 citation statements)

References 38 publications

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“…In another effort, hyper-thermophilic glycolysis enzymes were heterologously expressed, heat purified and assembled to convert glucose to n-butanol in 82% yield 18 . In another study, an elegantly simplified non-phosphorylative Entner-Doudoroff pathway from hyper-thermophilic archaea was constructed to produce ethanol and isobutanol in B55% yields 19 . These pioneering studies illustrate the flexibility of synthetic biochemistry and the potential for high yields.…”

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confidence: 99%

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A synthetic biochemistry molecular purge valve module that maintains redox balance

2014

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“…For example, in the Sieber pathway to generate ethanol and isopropanol from glucose 19 , the pathway was beautifully designed to reduce only 2 mol of NAD þ to NADH during the catabolic phase and to reoxidize 2 mol of NADH in the anabolic phase. Thus, the utilization of NAD þ was perfectly balanced allowing multiple passes through the pathway.…”

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A synthetic biochemistry molecular purge valve module that maintains redox balance

2014

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“…A possible solution to this problem is to avoid the use of living microorganisms and to construct an in vitro artificial metabolic pathway in which only a limited number of enzymes are involved. Until now, a variety of in vitro synthetic pathways have been designed and constructed for the production of alcohols [3,4], organic acids [5,6], carbohydrates [7], hydrogen [8,9], bioplastic [10], and even electricity [11]. Particularly, employment of enzymes derived from thermophiles and hyperthermophiles enables the simple preparation of catalytic modules with excellent selectivity and thermal stability [5,12].…”

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In vitro conversion of glycerol to lactate with thermophilic enzymes

Jaturapaktrarak

Napathorn

Cheng

et al. 2014

Bioresour. Bioprocess.

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Background: In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction. Particularly, employment of thermophilic and hyperthermophilic enzymes enables us a simple preparation of highly stable and selective biocatalytic modules and the construction of in vitro metabolic pathways with an excellent operational stability. In this study, we designed and constructed an artificial in vitro metabolic pathway consisting of nine (hyper)thermophilic enzymes and applied it to the conversion of glycerol to lactate. We also assessed the compatibility of the in vitro bioconversion system with methanol, which is a major impurity in crude glycerol released from biodiesel production processes. Results: The in vitro artificial pathway was designed to balance the intrapathway consumption and regeneration of energy and redox cofactors. All enzymes involved in the in vitro pathway exhibited an acceptable level of stability at high temperature (60°C), and their stability was not markedly affected by the co-existing of up to 100 mM methanol. The one-pot conversion of glycerol to lactate through the in vitro pathway could be achieved in an almost stoichiometric manner, and 14.7 mM lactate could be produced in 7 h. Furthermore, the in vitro bioconversion system exerted almost identical performance in the presence of methanol. Conclusions: Many thermophilic enzymes exhibit higher stability not only at high temperatures but also in the presence of denaturants such as detergents and organic solvents than their mesophilic counterparts. In this study, compatibilities of thermophilic enzymes with methanol were demonstrated, indicating the potential applicability of in vitro bioconversion systems with thermophilic enzymes in the conversion of crude glycerol to value-added chemicals.

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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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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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Cell‐Free Metabolic Engineering: Production of Chemicals by Minimized Reaction Cascades

Cited by 223 publications

References 38 publications

A synthetic biochemistry molecular purge valve module that maintains redox balance

A synthetic biochemistry molecular purge valve module that maintains redox balance

In vitro conversion of glycerol to lactate with thermophilic enzymes

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

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