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

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

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11557-8 ◽

2021 ◽

Author(s):

Alessa Lappe ◽

Nina Jankowski ◽

Annemie Albrecht ◽

Katja Koschorreck

Keyword(s):

Enzymatic Reaction ◽

Residual Activity ◽

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Building Blocks ◽

Oxidation Products ◽

Complete Conversion ◽

Primary Alcohols ◽

One Pot ◽

Furandicarboxylic 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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Two adjacent C-terminal mutations enable expression of aryl-alcohol oxidase from Pleurotus eryngii in Pichia pastoris

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11585-4 ◽

2021 ◽

Vol 105 (20) ◽

pp. 7743-7755

Author(s):

Nina Jankowski ◽

Vlada B. Urlacher ◽

Katja Koschorreck

Keyword(s):

Pichia Pastoris ◽

Heterologous Expression ◽

Alcohol Oxidase ◽

Functional Expression ◽

Amino Acid Identity ◽

Pleurotus Eryngii ◽

Mrna Levels ◽

Salt Bridges ◽

Primary Alcohols ◽

Gene Copies

AbstractFungal aryl-alcohol oxidases (AAOs) are attractive biocatalysts because they selectively oxidize a broad range of aromatic and aliphatic allylic primary alcohols while yielding hydrogen peroxide as the only by-product. However, their use is hampered by challenging and often unsuccessful heterologous expression. Production of PeAAO1 from Pleurotus eryngii ATCC 90787 in Pichia pastoris failed, while PeAAO2 from P. eryngii P34 with an amino acid identity of 99% was expressed at high yields. By successively introducing mutations in PeAAO1 to mimic the sequence of PeAAO2, the double mutant PeAAO1 ER with mutations K583E and Q584R was constructed, that was successfully expressed in P. pastoris. Functional expression was enhanced up to 155 U/l via further replacements D361N (variant NER) or V367A (variant AER). Fed-batch cultivation of recombinant P. pastoris yielded up to 116 mg/l of active variants. Glycosylated PeAAO1 variants demonstrated high stability and catalytic efficiencies similar to PeAAO2. Interestingly, P. pastoris expressing PeAAO1 variant ER contained roughly 13 gene copies but showed similar volumetric activity as NER and AER with one to two gene copies and four times lower mRNA levels. Additional H-bonds and salt bridges introduced by mutations K583E and Q584R might facilitate heterologous expression by enhanced protein folding.Key points• PeAAO1 not expressed in P. pastoris and PeAAO2 well-expressed in Pichia differ at 7 positions.• Expression of PeAAO1 in P. pastoris achieved through mutagenesis based on PeAAO2 sequence.• Combination of K583E and Q584R is essential for expression of PeAAO1 in P. pastoris.

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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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A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material

Journal of Fungi ◽

10.3390/jof7060426 ◽

2021 ◽

Vol 7 (6) ◽

pp. 426

Author(s):

Ander Peña ◽

Rashid Babiker ◽

Delphine Chaduli ◽

Anna Lipzen ◽

Mei Wang ◽

...

Keyword(s):

Extracellular Enzymes ◽

Fungal Growth ◽

2D Nmr ◽

Pleurotus Eryngii ◽

Glycoside Hydrolases ◽

Oxidative Enzymes ◽

Initial Growth ◽

Lignocellulosic Material ◽

Polysaccharide Lyases ◽

Polysaccharide Monooxygenases

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

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Future trends in diagnosis using laboratory-on-a-chip technologies

Parasitology ◽

10.1017/s0031182099004126 ◽

1999 ◽

Vol 117 (7) ◽

pp. 191-203 ◽

Cited By ~ 16

Author(s):

M. S. TALARY ◽

J. P. H. BURT ◽

R. PETHIG

Keyword(s):

Gene Expression ◽

Lower Cost ◽

Building Blocks ◽

Cancer Diagnostics ◽

Future Trends ◽

Biotechnological Applications ◽

Environmental Testing ◽

Current State ◽

Microelectronic Fabrication ◽

Wide Range

There has been an enormous growth in the development of biotechnological applications, where advances in the techniques of microelectronic fabrication and the technologies of miniaturization and integration in semiconductor industries are being applied to the production of Laboratory-on-a-Chip devices. The aim of this development is to create devices that will perform the same processes that are currently carried out in the laboratory in reduced timescales, at a lower cost, requiring less reagents, and with a greater resolution of detection and specificity. The expectations of this Laboratory-on-a-Chip revolution is that this technology will facilitate rapid advances in gene discovery, genetic mapping and gene expression with broader applications ranging from infectious diseases and cancer diagnostics to food quality and environmental testing. A review of the current state of development in this field reveals the scale of the ongoing revolution and serves to highlight the advances that can be perceived in the development of Laboratory-on-a-Chip technologies. Since miniaturization can be applied to such a wide range of laboratory processes, some of the sub-units that can be used as building blocks in these devices are described, with a brief description of some of the fabrication processes that can be used to create them.

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Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.1111-1121.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 1111-1121

Author(s):

S B Ellis ◽

P F Brust ◽

P J Koutz ◽

A F Waters ◽

M M Harpold ◽

...

Keyword(s):

Pichia Pastoris ◽

Carbon Source ◽

Sole Carbon Source ◽

Alcohol Oxidase ◽

Methylotrophic Yeasts ◽

Sequence Analyses ◽

Yeast Pichia Pastoris ◽

Oxidation Of Methanol ◽

Regulated Expression

The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts. The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined. The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-responsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation.

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Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications

10.21203/rs.3.rs-19704/v1 ◽

2020 ◽

Author(s):

Wissal BEN ALI ◽

Delphine Chaduli ◽

David Navarro ◽

Christian Lechat ◽

Annick Turbé-Doan ◽

...

Keyword(s):

Culture Supernatant ◽

Cost Effective ◽

Optimal Growth ◽

Dye Decolorization ◽

Growth Conditions ◽

Active Cell ◽

Oxidative Enzymes ◽

Synthetic Dyes ◽

Biotechnological Applications ◽

Fungal Strains

Abstract Background : Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost–effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization.Results : Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum , Stemphylium lucomagnoense and Aspergillus nidulans . Among these five isolates, one T. asperellum strain ( T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator.Conclusions : The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO 4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

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