Assembly and multiple gene expression of thermophilic enzymes in <i>Escherichia coli</i> for in vitro metabolic engineering

Ninh, Pham Huynh; Honda, Kohsuke; Sakai, Takaaki; Okano, Kenji; Ohtake, Hisao

doi:10.1002/bit.25338

Cited by 53 publications

(35 citation statements)

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“…For thermostability, the enzyme was preincubated for 1 h at different temperatures (60, 70, 80, 90, and 100°C), and then the activity was measured in 50 mM CHES and 150 mM NaCl at 65°C. The kinetic parameters of the enzyme were determined using 0.2 nM enzyme in 50 mM CHES and 150 mM NaCl at 65°C with different concentrations of nicotinamide (10,20,30,40,50,80, 100, and 200 M).…”

Section: Methodsmentioning

confidence: 99%

“…In our previous studies, we recognized that the decomposition of NAD ϩ and the consequent decrease in NAD ϩ concentrations at high temperatures are major obstacles for the biocatalytic manufacturing of value-added chemicals, in particular, with thermophilic enzymes (9,10). As a solution, our group constructed an "in vitro synthetic pathway" for the salvage synthesis of NAD ϩ from its decomposition products (5).…”

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confidence: 99%

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A Key Enzyme of the NAD ⁺ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

et al. 2017

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NAD (NAD ϩ ) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD ϩ from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD ϩ de novo synthesis pathway. Herein, we report the importance of the NAD ϩ salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD ϩ salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (K m of 17 M, k cat of 50 s Ϫ1 , k cat /K m of 3.0 ϫ 10 3 s Ϫ1 · mM Ϫ1 ). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD ϩ salvage pathway in T. thermophilus. Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD ϩ and NADH.IMPORTANCE NAD ϩ and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD ϩ decomposition is, in general, more rapid. This study emphasizes that NAD ϩ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.KEYWORDS NAD ϩ , Thermus thermophilus, nicotinamidase, salvage synthesis N AD (NAD ϩ ) is an essential molecule for cellular metabolism. It serves as an electron donor/acceptor for many redox reactions in cellular metabolism. It also serves as a precursor for NADP ϩ and a substrate for both bacterial DNA ligases and ADP ribosyl transferases (1, 2). Due to its importance as a cofactor for many oxidoreductases, NAD ϩ is also an essential compound in biotechnology applications (3); however, NAD ϩ is known to be chemically unstable, especially at high temperatures, where it nonenzymatically decomposes to ADP-ribose and nicotinamide (4,5).Cells need to maintain a certain concentration of NAD ϩ for metabolism, and different organisms possess different pathways for synthesizing NAD ϩ , such as the de novo biosynthesis pathway and the salvage pathway (6). In the salvage pathway,

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

A Key Enzyme of the NAD ⁺ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

et al. 2017

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“…The expression vector for G3P dehydrogenase of T. thermophilus (G3PDH Tt , gi|55981709) was obtained from the Riken T. thermophilus HB8 expression plasmid set [22]. Gene encoding NADH oxidase of Thermococcus profundus (NOX Tp , gi|187453160) was cloned and expressed in E. coli as described elsewhere [12].…”

Section: Microorganisms and Plasmidmentioning

confidence: 99%

“…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]. Furthermore, although the detailed mechanisms remain to be clarified, many thermophilic enzymes have also been reported to display higher tolerance towards denaturants such as detergents and organic solvents than their mesophilic counterparts [13,14], and activities of some thermophilic enzymes are even improved with organic solvents [15].…”

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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“…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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Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering

Cited by 53 publications

References 31 publications

A Key Enzyme of the NAD ⁺ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

A Key Enzyme of the NAD ⁺ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

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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Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering

Cited by 53 publications

References 31 publications

A Key Enzyme of the NAD + Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

A Key Enzyme of the NAD + Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

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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A Key Enzyme of the NAD ⁺ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

A Key Enzyme of the NAD ⁺ Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures