A synthetic biochemistry molecular purge valve module that maintains redox balance

Opgenorth, Paul H.; Korman, Tyler P.; Bowie, James U.

doi:10.1038/ncomms5113

Cited by 101 publications

(106 citation statements)

References 40 publications

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“…If not, it can be addressed to find out new enzymes using the same type of coenzymes from nature (Krutsakorn et al, 2013a), engineer coenzyme preferences by rational protein design (Opgenorth et al, 2014;Rollin et al, 2013) or add another enzyme transhydrogenase. Second, when in vitro pathways generate extra reduced coenzymes, such reduced coenzymes should be removed by using hydrogenase under anaerobic conditions (Wichmann and Vasic-Racki, 2005), by using NAD(H) oxidase under aerobic conditions (Opgenorth et al, 2014), or via electron mediators in enzymatic fuel cells (Zhu et al, 2012). When pathways need extra reduced coenzymes inputs, NAD(P)H can be usually generated by using a hydrogen-donor substrate and one of the following: a single enzyme, cascade enzymes, and electro-enzymes.…”

Section: General Design Principlesmentioning

confidence: 99%

“…The weight-average molecular weight and polydispersity of in vitro synthesized P3HAB are 6.64 × 10 6 and 1.36, respectively (Satoh et al, 2003). Recently, Bowie and his coworkers (Opgenorth et al, 2014) design an in vitro pathway for the production of P3HB from pyruvate without an external NAD(P)H input (Fig. 10).…”

Section: 3-propanediolmentioning

confidence: 99%

“…In this century, a few researchers propose to put more than four biocatalytic components in one vessel to implement very complicated reactions comparable to microbial cell factories. Because in vitro synthetic biosystems for future biomanufacturing are on their early stage, different names have been suggested in the literature, such as, synthetic pathway biotransformation (SyPaB) (Zhang, 2010a(Zhang, , 2011a, synthetic cascade biomanufacturing (Steffler and Sieber, 2013), cell-free bioprocessing (Swartz, 2013), synthetic biochemistry Opgenorth et al, 2014), cell-free biosystems for biomanufacturing or cell-free biomanufacturing , in vitro metabolic engineering (Honda et al, 2004), cascade enzyme factories (Zhang and Huang, 2012), and so on.…”

Section: Introductionmentioning

confidence: 99%

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Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges

Zhang

2015

Biotechnology Advances

157

101

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The largest obstacle to the cost-competitive production of low-value and high-impact biofuels and biochemicals (called biocommodities) is high production costs catalyzed by microbes due to their inherent weaknesses, such as low product yield, slow reaction rate, high separation cost, intolerance to toxic products, and so on. This predominant whole-cell platform suffers from a mismatch between the primary goal of living microbes - cell proliferation and the desired biomanufacturing goal - desired products (not cell mass most times). In vitro synthetic biosystems consist of numerous enzymes as building bricks, enzyme complexes as building modules, and/or (biomimetic) coenzymes, which are assembled into synthetic enzymatic pathways for implementing complicated bioreactions. They emerge as an alternative solution for accomplishing a desired biotransformation without concerns of cell proliferation, complicated cellular regulation, and side-product formation. In addition to the most important advantage - high product yield, in vitro synthetic biosystems feature several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this perspective review, the general design rules of in vitro synthetic pathways are presented with eight supporting examples: hydrogen, n-butanol, isobutanol, electricity, starch, lactate,1,3-propanediol, and poly-3-hydroxylbutyrate. Also, a detailed economic analysis for enzymatic hydrogen production from carbohydrates is presented to illustrate some advantages of this system and the remaining challenges. Great market potentials will motivate worldwide efforts from multiple disciplines (i.e., chemistry, biology and engineering) to address the remaining obstacles pertaining to cost and stability of enzymes and coenzymes, standardized building parts and modules, biomimetic coenzymes, biosystem optimization, and scale-up, soon.

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Section: General Design Principlesmentioning

confidence: 99%

Section: 3-propanediolmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges

Zhang

2015

Biotechnology Advances

157

101

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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].…”

Section: Introductionmentioning

confidence: 99%

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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“…In addition to its fundamental role in elucidating complicated cellular metabolism and regulation (15), this field is emerging as a promising alternative biomanufacturing platform, especially for the production of low-value and highimpact biofuels and biochemicals (16)(17)(18). In vitro metabolic engineering has a number of advantages over whole-cell biosystems: high product yield without the formation of by-products or the synthesis of cell mass (19,20); fast reaction rates, due to elimination of transport across a cell membrane (21,22); easy product separation (21,23); tolerance of compounds that would be toxic to intact cells (24); broad reaction conditions (25); and Significance Hydrogen (H 2 ) has great potential to be used to power passenger vehicles. One solution to these problems is to distribute and store renewable carbohydrate instead, converting it to hydrogen as required.…”

mentioning

confidence: 99%

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling

Rollin

Campo

Myung

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

212

132

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The use of hydrogen (H 2 ) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H 2 and CO 2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H 2 with a yield of two H 2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H 2 productivity was increased 3-fold to 32 mmol H 2 ·L. The productivity was further enhanced to 54 mmol H 2 ·L −1 ·h −1 by increasing reaction temperature, substrate, and enzyme concentrations-an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.hydrogen | biomass | in vitro metabolic engineering | metabolic network modeling | global sensitivity analysis

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A synthetic biochemistry molecular purge valve module that maintains redox balance

Cited by 101 publications

References 40 publications

Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges

Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges

In vitro conversion of glycerol to lactate with thermophilic enzymes

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling

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