scholarly journals One-pot enzymatic synthesis of 2-deoxy-scyllo-inosose from d-glucose and polyphosphate

2021 ◽  
Vol 85 (1) ◽  
pp. 108-114
Author(s):  
Fumitaka Kudo ◽  
Ayaka Mori ◽  
Mai Koide ◽  
Ryo Yajima ◽  
Ryohei Takeishi ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Aromatic Compounds ◽  
Nicotinamide Adenine Dinucleotide ◽  
Enzymatic Synthesis ◽  
Enzymatic Reaction ◽  
Aminoglycoside Antibiotics ◽  
Nicotinamide Adenine ◽  
One Pot ◽  
The One ◽  
Full Conversion

Abstract 2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one) is a biosynthetic intermediate of 2-deoxystreptamine-containing aminoglycoside antibiotics, including butirosin, kanamycin, and neomycin. In producer microorganisms, 2DOI is constructed from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS) with the oxidized form of nicotinamide adenine dinucleotide (NAD+). 2DOI is also known as a sustainable biomaterial for production of aromatic compounds and a chiral cyclohexane synthon. In this study, a one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate was investigated. First, 3 polyphosphate glucokinases (PPGKs) were examined to produce G6P from d-glucose and polyphosphate. A PPGK derived from Corynebacterium glutamicum (cgPPGK) was found to be suitable for G6P production under ordinary enzymatic conditions. Next, 7 DOISs were examined for the one-pot enzymatic reaction. As a result, cgPPGK and BtrC, the latter of which is a DOIS derived from the butirosin producer Bacillus circulans, achieved nearly full conversion of d-glucose to 2DOI in the presence of polyphosphate.

Chemoenzymatic Synthesis of Ubiquitous Biological Redox Cofactors

Synlett ◽  
2017 ◽  
Vol 28 (10) ◽  
pp. 1151-1159 ◽  
Author(s):  
Amnon Kohen ◽  
Priyanka Singh ◽  
Qi Guo
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Enzymatic Synthesis ◽  
Metabolic Enzymes ◽  
Chemoenzymatic Synthesis ◽  
Nicotinamide Adenine ◽  
General Strategy ◽  
One Pot ◽  
Post Translational Modifications ◽  
One Step ◽  
Methylene Tetrahydrofolate

Redox cofactors are utilized by a myriad of proteins, ranging from metabolic enzymes to those performing post-translational modifications. Labeled redox cofactors have served as a vital tool for a broad range of studies. This account describes chemoenzymatic syntheses of the isotopically labeled, biologically important redox cofactors: nicotinamide adenine dinucleotide, methylene tetrahydrofolate, and flavin nucleotides. An overview of the general strategy is presented. These examples demonstrate the utility of enzymatic synthesis.1 Introduction2 Nicotinamide Cofactors2.1 Synthesis of Remote-Labeled 14C-NADPH2.1.1 Synthesis of [Ad-14C]NADPH2.1.2 Synthesis of [Carbonyl-14C]NADPH2.2 Synthesis of S- and R-[4-3H]NADPH2.2.1 One-Step S- and Three-Step R-[4-3H]NADPH Synthesis2.2.2 One-Pot, One-Step R-[4-3H]NADPH Synthesis2.3 Synthesis of S- and R-[Ad-14C, 4-2H]NADPH2.3.1 One-Step S-, Three-Step R-[Ad-14C, 4-2H]NADPH Synthesis2.3.2 One-Pot, One-Step R-[Ad-14C, 4-2H]NADPH Synthesis3 Methylene Tetrahydrofolate4 Flavin Nucleotides5 Conclusions and Outlook

scholarly journals Designing Modular Cell-free Systems for Tunable Biotransformation of l-phenylalanine to Aromatic Compounds

2021 ◽  
Vol 9 ◽  
Author(s):  
Chen Yang ◽  
Yushi Liu ◽  
Wan-Qiu Liu ◽  
Changzhu Wu ◽  
Jian Li
Keyword(s):  
Aromatic Compounds ◽  
Value Added ◽  
Resource Limitation ◽  
Biosynthetic Pathways ◽  
One Pot ◽  
Conversion Rates ◽  
The One ◽  
Free Modules ◽  
Enzymatic Pathways

Cell-free systems have been used to synthesize chemicals by reconstitution of in vitro expressed enzymes. However, coexpression of multiple enzymes to reconstitute long enzymatic pathways is often problematic due to resource limitation/competition (e.g., energy) in the one-pot cell-free reactions. To address this limitation, here we aim to design a modular, cell-free platform to construct long biosynthetic pathways for tunable synthesis of value-added aromatic compounds, using (S)-1-phenyl-1,2-ethanediol ((S)-PED) and 2-phenylethanol (2-PE) as models. Initially, all enzymes involved in the biosynthetic pathways were individually expressed by an E. coli-based cell-free protein synthesis (CFPS) system and their catalytic activities were confirmed. Then, three sets of enzymes were coexpressed in three cell-free modules and each with the ability to complete a partial pathway. Finally, the full biosynthetic pathways were reconstituted by mixing two related modules to synthesize (S)-PED and 2-PE, respectively. After optimization, the final conversion rates for (S)-PED and 2-PE reached 100 and 82.5%, respectively, based on the starting substrate of l-phenylalanine. We anticipate that the modular cell-free approach will make a possible efficient and high-yielding biosynthesis of value-added chemicals.

One-Pot Approach for SNAr Reaction of Fluoroaromatic Compounds with Cyclopropanol

Synlett ◽  
2019 ◽  
Vol 30 (08) ◽  
pp. 982-986
Author(s):  
Hao Jin ◽  
Zhuo Gao ◽  
Shaodong Zhou ◽  
Chao Qian
Keyword(s):  
Aromatic Compounds ◽  
Substitution Reaction ◽  
Nucleophilic Aromatic Substitution ◽  
Aromatic Substitution ◽  
One Pot ◽  
Mild Conditions ◽  
Snar Reaction ◽  
Novel Method ◽  
The One ◽  
Application Scope

A novel method for preparing aromatic compounds containing cyclopropoxy via nucleophilic aromatic substitution reaction (SNAr) of fluoroaromatic compounds with cyclopropanol under relatively mild conditions is presented. As compared to the approaches reported previously for preparing 1-(cyclopropyloxy)-2-nitrobenzene, the one proposed in this work is simplified without sacrificing the yields: When the reaction was performed at 75 °C with Cs2CO3 as the base and DMF as solvent, after 6 h the yield was up to 90%. Finally, various fluoroaromatic compounds were employed as substrates for a test that proves a wide application scope of the method.

scholarly journals A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate

Catalysts ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 60 ◽  
Author(s):  
Marina Simona Robescu ◽  
Immacolata Serra ◽  
Marco Terreni ◽  
Daniela Ubiali ◽  
Teodora Bavaro
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Enzymatic Reaction ◽  
Antiviral Drug ◽  
Product Formation ◽  
Reaction Conditions ◽  
One Pot ◽  
Medium Temperature ◽  
Phosphate Donor ◽  
The One ◽  
Nucleoside Phosphorylases

We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5′-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5′-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5′-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP.

scholarly journals Development of a Cofactor Balanced, Multi Enzymatic Cascade Reaction for the Simultaneous Production of L-Alanine and L-Serine from 2-Keto-3-deoxy-gluconate

Catalysts ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 31
Author(s):  
Benjamin Begander ◽  
Anna Huber ◽  
Josef Sperl ◽  
Volker Sieber
Keyword(s):  
Enzymatic Reaction ◽  
Value Added ◽  
Thermostable Enzymes ◽  
One Pot ◽  
Major Drawback ◽  
Significant Group ◽  
Cofactor Recycling ◽  
Sugar Degradation ◽  
The One ◽  
Reaction Cascades

Enzymatic reaction cascades represent a powerful tool to convert biogenic resources into valuable chemicals for fuel and commodity markets. Sugars and their breakdown products constitute a significant group of possible substrates for such biocatalytic conversion strategies to value-added products. However, one major drawback of sugar cascades is the need for cofactor recycling without using additional enzymes and/or creating unwanted by-products. Here, we describe a novel, multi-enzymatic reaction cascade for the one-pot simultaneous synthesis of L-alanine and L-serine, using the sugar degradation product 2-keto-3-deoxygluconate and ammonium as precursors. To pursue this aim, we used four different, thermostable enzymes, while the necessary cofactor NADH is recycled entirely self-sufficiently. Buffer and pH optimisation in combination with an enzyme titration study yielded an optimised production of 21.3 +/− 1.0 mM L-alanine and 8.9 +/− 0.4 mM L-serine in one pot after 21 h.

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