Characterization of a thermotolerant aryl-alcohol oxidase from Moesziomyces antarcticus oxidizing 5-hydroxymethyl-2-furancarboxylic acid

Abstract The development of enzymatic processes for the environmentally friendly production of 2,5-furandicarboxylic acid (FDCA), a renewable precursor for bioplastics, from 5-hydroxymethylfurfural (HMF) has gained increasing attention over the last years. Aryl-alcohol oxidases (AAOs) catalyze the oxidation of HMF to 5-formyl-2-furancarboxylic acid (FFCA) through 2,5-diformylfuran (DFF) and have thus been applied in enzymatic reaction cascades for the production of FDCA. AAOs are flavoproteins that oxidize a broad range of benzylic and aliphatic allylic primary alcohols to the corresponding aldehydes, and in some cases further to acids, while reducing molecular oxygen to hydrogen peroxide. These promising biocatalysts can also be used for the synthesis of flavors, fragrances, and chemical building blocks, but their industrial applicability suffers from low production yield in natural and heterologous hosts. Here we report on heterologous expression of a new aryl-alcohol oxidase, MaAAO, from Moesziomyces antarcticus at high yields in the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). Fed-batch fermentation of recombinant P. pastoris yielded around 750 mg of active enzyme per liter of culture. Purified MaAAO was highly stable at pH 2–9 and exhibited high thermal stability with almost 95% residual activity after 48 h at 57.5 °C. MaAAO accepts a broad range of benzylic primary alcohols, aliphatic allylic alcohols, and furan derivatives like HMF as substrates and some oxidation products thereof like piperonal or perillaldehyde serve as building blocks for pharmaceuticals or show health-promoting effects. Besides this, MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FFCA, which has not been shown for any other AAO so far. Combining MaAAO with an unspecific peroxygenase oxidizing HMFCA to FFCA in one pot resulted in complete conversion of HMF to FDCA within 144 h. MaAAO is thus a promising biocatalyst for the production of precursors for bioplastics and bioactive compounds. Key points • MaAAO from M. antarcticus was expressed in P. pastoris at 750 mg/l. • MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). • Complete conversion of HMF to 2,5-furandicarboxylic acid by combining MaAAO and UPO.

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High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors

Applied Microbiology and Biotechnology ◽

10.1007/s00253-020-10878-4 ◽

2020 ◽

Vol 104 (21) ◽

pp. 9205-9218

Author(s):

Nina Jankowski ◽

Katja Koschorreck ◽

Vlada B. Urlacher

Keyword(s):

Pichia Pastoris ◽

Alcohol Oxidase ◽

Building Blocks ◽

Pleurotus Eryngii ◽

Oxidation Products ◽

Oxidative Enzymes ◽

Biotechnological Applications ◽

Positive Health ◽

Neuroprotective Effects ◽

Flavor Compound

Abstract The fungal secretome comprises various oxidative enzymes participating in the degradation of lignocellulosic biomass as a central step in carbon recycling. Among the secreted enzymes, aryl-alcohol oxidases (AAOs) are of interest for biotechnological applications including production of bio-based precursors for plastics, bioactive compounds, and flavors and fragrances. Aryl-alcohol oxidase 2 (PeAAO2) from the fungus Pleurotus eryngii was heterologously expressed and secreted at one of the highest yields reported so far of 315 mg/l using the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). The glycosylated PeAAO2 exhibited a high stability in a broad pH range between pH 3.0 and 9.0 and high thermal stability up to 55 °C. Substrate screening with 41 compounds revealed that PeAAO2 oxidized typical AAO substrates like p-anisyl alcohol, veratryl alcohol, and trans,trans-2,4-hexadienol with up to 8-fold higher activity than benzyl alcohol. Several compounds not yet reported as substrates for AAOs were oxidized by PeAAO2 as well. Among them, cumic alcohol and piperonyl alcohol were oxidized to cuminaldehyde and piperonal with high catalytic efficiencies of 84.1 and 600.2 mM−1 s−1, respectively. While the fragrance and flavor compound piperonal also serves as starting material for agrochemical and pharmaceutical building blocks, various positive health effects have been attributed to cuminaldehyde including anticancer, antidiabetic, and neuroprotective effects. PeAAO2 is thus a promising biocatalyst for biotechnological applications. Key points • Aryl-alcohol oxidase PeAAO2 from P. eryngii was produced in P. pastoris at 315 mg/l. • Purified enzyme exhibited stability over a broad pH and temperature range. • Oxidation products cuminaldehyde and piperonal are of biotechnological interest. Graphical abstract

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One-Pot Reductive Acetylation of Aldehydes using 1-Hydrosilatrane in Acetic Acid

SynOpen ◽

10.1055/s-0037-1611697 ◽

2019 ◽

Vol 03 (01) ◽

pp. 1-3 ◽

Cited By ~ 2

Author(s):

Reuben James ◽

Sharon Herlugson ◽

Sami Varjosaari ◽

Vladislav Skrypai ◽

Zainab Shakeel ◽

...

Keyword(s):

Acetic Acid ◽

Organic Synthesis ◽

Functional Group ◽

Building Blocks ◽

Flash Chromatography ◽

Primary Alcohols ◽

Facile Synthesis ◽

One Pot ◽

Aryl Aldehydes ◽

Mild Conditions

A one-pot, direct reductive acetylation of aldehydes was achieved under mild conditions using 1-hydrosilatrane as a safe and easily accessible catalyst. Described herein is a facile synthesis that produces acylated primary alcohols that can serve as valuable building blocks for organic synthesis. The method has good functional group tolerance and works for a range of aryl aldehydes, with the notable exception of electron-rich arenes. A library of esters was isolated by flash chromatography in yields as high as 92%.

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Reverse C-glycosidase reaction provides C-nucleotide building blocks of xenobiotic nucleic acids

Nature Communications ◽

10.1038/s41467-020-20035-0 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Martin Pfeiffer ◽

Bernd Nidetzky

Keyword(s):

Synthetic Biology ◽

Nucleic Acids ◽

Enzymatic Reaction ◽

Building Blocks ◽

Cascade Reactions ◽

Ring Closure ◽

One Pot ◽

Open Chain ◽

Nucleotide Synthesis ◽

Nucleotide Triphosphates

AbstractC-Analogues of the canonical N-nucleosides have considerable importance in medicinal chemistry and are promising building blocks of xenobiotic nucleic acids (XNA) in synthetic biology. Although well established for synthesis of N-nucleosides, biocatalytic methods are lacking in C-nucleoside synthetic chemistry. Here, we identify pseudouridine monophosphate C-glycosidase for selective 5-β-C-glycosylation of uracil and derivatives thereof from pentose 5-phosphate (d-ribose, 2-deoxy-d-ribose, d-arabinose, d-xylose) substrates. Substrate requirements of the enzymatic reaction are consistent with a Mannich-like addition between the pyrimidine nucleobase and the iminium intermediate of enzyme (Lys166) and open-chain pentose 5-phosphate. β-Elimination of the lysine and stereoselective ring closure give the product. We demonstrate phosphorylation-glycosylation cascade reactions for efficient, one-pot synthesis of C-nucleoside phosphates (yield: 33 – 94%) from unprotected sugar and nucleobase. We show incorporation of the enzymatically synthesized C-nucleotide triphosphates into nucleic acids by RNA polymerase. Collectively, these findings implement biocatalytic methodology for C-nucleotide synthesis which can facilitate XNA engineering for synthetic biology applications.

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Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Applied and Environmental Microbiology ◽

10.1128/aem.03740-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 1082-1090 ◽

Cited By ~ 63

Author(s):

Willem P. Dijkman ◽

Marco W. Fraaije

Keyword(s):

Building Blocks ◽

Degradation Pathway ◽

Enzyme Characterization ◽

Bacterial Degradation ◽

Primary Alcohols ◽

Content Type ◽

Wide Range ◽

Furandicarboxylic Acid ◽

Ph Range

ABSTRACTIn the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein fromMethylovorussp. strain MP688 inEscherichia coliand subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

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Synthesis of Nitrile-Bearing Quaternary Centers via an Equilibrium-Driven Transnitrilation and Anion-Relay Strategy

10.26434/chemrxiv.7834940 ◽

2019 ◽

Author(s):

Sebastien Alazet ◽

Michael West ◽

Purvish Patel ◽

Sophie Rousseaux

Keyword(s):

Building Blocks ◽

One Pot ◽

Quaternary Centers ◽

Synthetic Intermediates

The efficient preparation of nitrile-containing building blocks is of interest due to their utility as synthetic intermediates and their prevalence in pharmaceuticals. As a result, significant efforts have been made to develop methods to access these motifs which rely on safer and non-toxic sources of CN. Herein, we report that 2-methyl-2-phenylpropanenitrile is an efficient, non-toxic, electrophilic CN source for the synthesis of nitrile-bearing quaternary centers via a thermodynamic transnitrilation and anion-relay strategy. This one-pot process leads to nitrile products resulting from the gem-difunctionalization of alkyl lithium reagents.<br>

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2-Aryl-6-Polyfluoroalkyl-4-Pyrones as Promising RF-Building-Blocks: Synthesis and Application for Construction of Fluorinated Azaheterocycles

Molecules ◽

10.3390/molecules26154415 ◽

2021 ◽

Vol 26 (15) ◽

pp. 4415

Author(s):

Sergey A. Usachev ◽

Diana I. Nigamatova ◽

Daria K. Mysik ◽

Nikita A. Naumov ◽

Dmitrii L. Obydennov ◽

...

Keyword(s):

Direct Synthesis ◽

Building Blocks ◽

Oxidative Cyclization ◽

One Pot ◽

Synthetic Application ◽

General Method

A convenient and general method for the direct synthesis of 2-aryl-6-(trifluoromethyl)-4-pyrones and 2-aryl-5-bromo-6-(trifluoromethyl)-4-pyrones has been developed on the basis of one-pot oxidative cyclization of (E)-6-aryl-1,1,1-trifluorohex-5-ene-2,4-diones via a bromination/dehydrobromination approach. This strategy was also applied for the preparation of 2-phenyl-6-polyfluoroalkyl-4-pyrones and their 5-bromo derivatives. Conditions of chemoselective enediones bromination were found and the key intermediates of the cyclization of bromo-derivatives to 4-pyrones were characterized. Synthetic application of the prepared 4-pyrones has been demonstrated for the construction of biologically important CF3-bearing azaheterocycles, such as pyrazoles, pyridones, and triazoles.

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Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽

10.1093/genetics/151.4.1379 ◽

1999 ◽

Vol 151 (4) ◽

pp. 1379-1391

Author(s):

Monique A Johnson ◽

Hans R Waterham ◽

Galyna P Ksheminska ◽

Liubov R Fayura ◽

Joan Lin Cereghino ◽

...

Keyword(s):

Pichia Pastoris ◽

Allyl Alcohol ◽

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Direct Selection ◽

Toxic Exposure ◽

Peroxisome Biogenesis ◽

Yeast Pichia Pastoris ◽

Methylotrophic Yeast Pichia Pastoris ◽

Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

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1,3-Diketone Calix[4]arene Derivatives—A New Type of Versatile Ligands for Metal Complexes and Nanoparticles

Molecules ◽

10.3390/molecules26051214 ◽

2021 ◽

Vol 26 (5) ◽

pp. 1214

Author(s):

Sergey N. Podyachev ◽

Rustem R. Zairov ◽

Asiya R. Mustafina

Keyword(s):

Structural Diversity ◽

Building Blocks ◽

Structural Features ◽

Structure Property ◽

One Pot ◽

Arene Ligands ◽

Structure Property Relationships ◽

New Type ◽

Synthetic Routes ◽

Magnetic Resonance Imaging Mri

The present review is aimed at highlighting outlooks for cyclophanic 1,3-diketones as a new type of versatile ligands and building blocks of the nanomaterial for sensing and bioimaging. Thus, the main synthetic routes for achieving the structural diversity of cyclophanic 1,3-diketones are discussed. The structural diversity is demonstrated by variation of both cyclophanic backbones (calix[4]arene, calix[4]resorcinarene and thiacalix[4]arene) and embedding of different substituents onto lower or upper macrocyclic rims. The structural features of the cyclophanic 1,3-diketones are correlated with their ability to form lanthanide complexes exhibiting both lanthanide-centered luminescence and magnetic relaxivity parameters convenient for contrast effect in magnetic resonance imaging (MRI). The revealed structure–property relationships and the applicability of facile one-pot transformation of the complexes to hydrophilic nanoparticles demonstrates the advantages of 1,3-diketone calix[4]arene ligands and their complexes in developing of nanomaterials for sensing and bioimaging.

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