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Article ; Online: CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia pastoris) Genome.

Fischer, Jasmin Elgin / Glieder, Anton

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2513, Page(s) 121–133

Abstract: Efficient targeted genome engineering of Komagataella phaffii requires balanced expression of Cas9 nuclease and a target-specific guide RNA (gRNA). In addition, correct processing of the transcribed RNA to provide the designed gRNA as a target selective ... ...

Abstract	Efficient targeted genome engineering of Komagataella phaffii requires balanced expression of Cas9 nuclease and a target-specific guide RNA (gRNA). In addition, correct processing of the transcribed RNA to provide the designed gRNA as a target selective partner of targeted Cas9 protein for binding to genomic DNA is essential for efficient genome engineering. This method describes a step-by-step procedure and recommended tools for simple and efficient design of gRNAs to introduce insertions or deletions at targeted sites by CRISPR/Cas9-directed double-strand breaks, followed by error-prone nonhomologous end-joining repair.
MeSH term(s)	CRISPR-Cas Systems/genetics ; Mutagenesis ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomycetales
Chemical Substances	RNA, Guide, CRISPR-Cas Systems
Language	English
Publishing date	2022-07-01
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2399-2_8
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Article ; Online: Bidirectional Promoter Libraries Enable the Balanced Co-expression of Two Target Genes in E. coli.

Jodlbauer, Julia / Rieder, Lukas / Glieder, Anton / Wiltschi, Birgit

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2617, Page(s) 75–86

Abstract: In this chapter, we present a bidirectional promoter library toolbox to evaluate fast and efficiently the optimal conditions for the balanced co-expression of two target genes. As a proof-of-concept, we demonstrate the co-expression of CYP505x and the ... ...

Abstract	In this chapter, we present a bidirectional promoter library toolbox to evaluate fast and efficiently the optimal conditions for the balanced co-expression of two target genes. As a proof-of-concept, we demonstrate the co-expression of CYP505x and the GroEL/ES complex, which resulted in noticeably elevated enzyme activity with one of the de-novo-designed promoters of the library. The new toolbox offers a straightforward one-pot cloning approach and is highly modular. As such, the method presented here should be of great interest to any gene co-expression study.
MeSH term(s)	Escherichia coli/genetics ; Promoter Regions, Genetic ; Gene Library
Language	English
Publishing date	2023-01-20
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2930-7_5
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Article ; Online: Multistep Biooxidation of 5-(Hydroxymethyl)furfural to 2,5-Furandicarboxylic Acid with H

Swoboda, Alexander / Zwölfer, Silvie / Duhović, Zerina / Bürgler, Moritz / Ebner, Katharina / Glieder, Anton / Kroutil, Wolfgang

ChemSusChem

2024 , Page(s) e202400156

Abstract: 5-(Hydroxymethyl)furfural (HMF) is a key platform chemical derived from renewable biomass sources, holding great potential as starting material for the synthesis of valuable compounds, thereby replacing petrochemical-derived counterparts. Among these ... ...

Abstract	5-(Hydroxymethyl)furfural (HMF) is a key platform chemical derived from renewable biomass sources, holding great potential as starting material for the synthesis of valuable compounds, thereby replacing petrochemical-derived counterparts. Among these valorised compounds, 2,5-furandicarboxylic acid (FDCA) has emerged as a versatile building block. Here we demonstrate the biocatalytic synthesis of FDCA from HMF via a one-pot three-step oxidative cascade performed via two operative steps under mild reaction conditions employing two unspecific peroxygenases (UPOs) using hydrogen peroxide as the only oxidant. The challenge of HMF oxidation by UPOs is the chemoselectivity of the first step, as one of the two possible oxidation products is only a poor substrate for further oxidation. The unspecific peroxygenase from Marasmius oreades (MorUPO) was found to oxidize 100 mM of HMF to 5-formyl-2-furoic acid (FFCA) with 95 % chemoselectivity. In the sequential one-pot cascade employing MorUPO (TON up to 13535) and the UPO from Agrocybe aegerita (AaeUPO, TON up to 7079), 100 mM of HMF were oxidized to FDCA reaching up to 99 % conversion and yielding 861 mg isolated pure crystalline FDCA, presenting the first example of a gram scale biocatalytic synthesis of FDCA involving UPOs.
Language	English
Publishing date	2024-04-03
Publishing country	Germany
Document type	Journal Article
ISSN	1864-564X
ISSN (online)	1864-564X
DOI	10.1002/cssc.202400156
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Article ; Online: Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

Weiss, Florian / Requena-Moreno, Guillermo / Pichler, Carsten / Valero, Francisco / Glieder, Anton / Garcia-Ortega, Xavier

Microbial cell factories

2024 Volume 23, Issue 1, Page(s) 116

Abstract: Background: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product ... ...

Abstract	Background: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. Results: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. Conclusions: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.
MeSH term(s)	Pichia/metabolism ; Carbon/metabolism ; Saccharomycetales/genetics ; Saccharomycetales/metabolism ; Recombinant Proteins ; Plasmids/genetics
Chemical Substances	Carbon (7440-44-0) ; Recombinant Proteins
Language	English
Publishing date	2024-04-20
Publishing country	England
Document type	Journal Article
ZDB-ID	2091377-1
ISSN	1475-2859 ; 1475-2859
ISSN (online)	1475-2859
ISSN	1475-2859
DOI	10.1186/s12934-024-02368-3
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Article ; Online: Regiospecific 7-hydroxylation of ten-carbon monoterpenes by detoxifying CYP5035S7 monooxygenase of the white-rot fungus Polyporus arcularius.

Fessner, Nico D / Weber, Hansjörg / Glieder, Anton

Biochemical and biophysical research communications

2022 Volume 595, Page(s) 35–40

Abstract: In a previous study, we identified CYP5035S7 of the white-rot fungus Polyporus arcularius with a broad activity towards monoterpenes such as p-cymene. Therefore, in this study we aimed at further exploring the substrate scope of detoxifying CYP5035S7 ... ...

Abstract	In a previous study, we identified CYP5035S7 of the white-rot fungus Polyporus arcularius with a broad activity towards monoterpenes such as p-cymene. Therefore, in this study we aimed at further exploring the substrate scope of detoxifying CYP5035S7 towards terpenes and semi-preparatively isolating some of the products via whole-cell biotransformation, in order to obtain information about the enzyme's reactivity. We noticed a clear preference for the monoterpene skeleton and elucidated a distinct regioselectivity pattern based on key structural and electronic features of its substrates. This study illustrates how minimal characterisation effort may already suffice to provide vital information on enzymatic reactivity by the comparison of structural derivatives.
MeSH term(s)	Biotransformation ; Carbon/chemistry ; Carbon/metabolism ; Cytochrome P-450 Enzyme System/metabolism ; Fungal Proteins/metabolism ; Hydroxylation ; Molecular Structure ; Monoterpenes/chemistry ; Monoterpenes/metabolism ; Polyporus/metabolism ; Stereoisomerism ; Substrate Specificity
Chemical Substances	Fungal Proteins ; Monoterpenes ; Carbon (7440-44-0) ; Cytochrome P-450 Enzyme System (9035-51-2)
Language	English
Publishing date	2022-01-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	205723-2
ISSN	1090-2104 ; 0006-291X ; 0006-291X
ISSN (online)	1090-2104 ; 0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2022.01.072
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Article: Regiospecific 7-hydroxylation of ten-carbon monoterpenes by detoxifying CYP5035S7 monooxygenase of the white-rot fungus Polyporus arcularius

Fessner, Nico D. / Weber, Hansjörg / Glieder, Anton

Biochemical and biophysical research communications. 2022 Mar. 05, v. 595

2022

Abstract	In a previous study, we identified CYP5035S7 of the white-rot fungus Polyporus arcularius with a broad activity towards monoterpenes such as p-cymene. Therefore, in this study we aimed at further exploring the substrate scope of detoxifying CYP5035S7 towards terpenes and semi-preparatively isolating some of the products via whole-cell biotransformation, in order to obtain information about the enzyme's reactivity. We noticed a clear preference for the monoterpene skeleton and elucidated a distinct regioselectivity pattern based on key structural and electronic features of its substrates. This study illustrates how minimal characterisation effort may already suffice to provide vital information on enzymatic reactivity by the comparison of structural derivatives.
Keywords	Polyporus arcularius ; biotransformation ; enzymes ; monoterpenoids ; p-cymene ; regioselectivity ; research ; white-rot fungi
Language	English
Dates of publication	2022-0305
Size	p. 35-40.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2022.01.072
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Article ; Online: Current advances in engineering tools for Pichia pastoris.

Fischer, Jasmin E / Glieder, Anton

Current opinion in biotechnology

2019 Volume 59, Page(s) 175–181

Abstract: In the past two to five years innovative DNA tools and inventive methodologies accelerated the speed of engineering of Komagataella phaffii (Pichia pastoris) for the efficient expression of intracellular and secreted proteins. Going beyond the standard ... ...

Abstract	In the past two to five years innovative DNA tools and inventive methodologies accelerated the speed of engineering of Komagataella phaffii (Pichia pastoris) for the efficient expression of intracellular and secreted proteins. Going beyond the standard approaches employing single heterologous genes or simultaneous expression of several different genes under the control of identical promoter and terminator sequences, balanced and consecutive co-expression of multiple genes (a, b) combined with simple host genome editing (c) now opens new opportunities for manufacturing of recombinant proteins or chemicals made by whole cell biocatalysis or synthetic biology.
MeSH term(s)	Gene Editing ; Pichia ; Promoter Regions, Genetic ; Recombinant Proteins ; Synthetic Biology
Chemical Substances	Recombinant Proteins
Language	English
Publishing date	2019-08-27
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2019.06.002
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Article: Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris.

Rieder, Lukas / Ebner, Katharina / Glieder, Anton / Sørlie, Morten

Biotechnology for biofuels

2021 Volume 14, Issue 1, Page(s) 122

Abstract: Background: Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry.: Results: We have established a universal ... ...

Abstract	Background: Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results: We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion: The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.
Language	English
Publishing date	2021-05-27
Publishing country	England
Document type	Journal Article
ZDB-ID	2421351-2
ISSN	1754-6834
ISSN	1754-6834
DOI	10.1186/s13068-021-01971-5
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Article ; Online: Evolution and enrichment of CYP5035 in Polyporales: functionality of an understudied P450 family.

Fessner, Nico D / Nelson, David R / Glieder, Anton

Applied microbiology and biotechnology

2021 Volume 105, Issue 18, Page(s) 6779–6792

Abstract: Bioprospecting for innovative basidiomycete cytochrome P450 enzymes (P450s) is highly desirable due to the fungi's enormous enzymatic repertoire and outstanding ability to degrade lignin and detoxify various xenobiotics. While fungal metagenomics is ... ...

Abstract	Bioprospecting for innovative basidiomycete cytochrome P450 enzymes (P450s) is highly desirable due to the fungi's enormous enzymatic repertoire and outstanding ability to degrade lignin and detoxify various xenobiotics. While fungal metagenomics is progressing rapidly, the biocatalytic potential of the majority of these annotated P450 sequences usually remains concealed, although functional profiling identified several P450 families with versatile substrate scopes towards various natural products. Functional knowledge about the CYP5035 family, for example, is largely insufficient. In this study, the families of the putative P450 sequences of the four white-rot fungi Polyporus arcularius, Polyporus brumalis, Polyporus squamosus and Lentinus tigrinus were assigned, and the CYPomes revealed an unusual enrichment of CYP5035, CYP5136 and CYP5150. By computational analysis of the phylogeny of the former two P450 families, the evolution of their enrichment could be traced back to the Ganoderma macrofungus, indicating their evolutionary benefit. In order to address the knowledge gap on CYP5035 functionality, a representative subgroup of this P450 family of P. arcularius was expressed and screened against a test set of substrates. Thereby, the multifunctional enzyme CYP5035S7 converting several plant natural product classes was discovered. Aligning CYP5035S7 to 102,000 putative P450 sequences of 36 fungal species from Joint Genome Institute-provided genomes located hundreds of further CYP5035 family members, which subfamilies were classified if possible. Exemplified by these specific enzyme analyses, this study gives valuable hints for future bioprospecting of such xenobiotic-detoxifying P450s and for the identification of their biocatalytic potential. KEY POINTS: • The P450 families CYP5035 and CYP5136 are unusually enriched in P. arcularius. • Functional screening shows CYP5035 assisting in the fungal detoxification mechanism. • Some Polyporales encompass an unusually large repertoire of detoxification P450s.
MeSH term(s)	Basidiomycota/genetics ; Cytochrome P-450 Enzyme System/genetics ; Evolution, Molecular ; Genome, Fungal ; Lentinula ; Phylogeny ; Polyporales/genetics ; Polyporus
Chemical Substances	Cytochrome P-450 Enzyme System (9035-51-2)
Language	English
Publishing date	2021-08-30
Publishing country	Germany
Document type	Journal Article
ZDB-ID	392453-1
ISSN	1432-0614 ; 0171-1741 ; 0175-7598
ISSN (online)	1432-0614
ISSN	0171-1741 ; 0175-7598
DOI	10.1007/s00253-021-11444-2
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Article ; Online: Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in

Smirnov, Kirill / Weiss, Florian / Hatzl, Anna-Maria / Rieder, Lukas / Olesen, Kjeld / Jensen, Sanne / Glieder, Anton

Journal of fungi (Basel, Switzerland)

2024 Volume 10, Issue 3

Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in ... ...

Abstract	CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast
Language	English
Publishing date	2024-03-05
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2784229-0
ISSN	2309-608X ; 2309-608X
ISSN (online)	2309-608X
ISSN	2309-608X
DOI	10.3390/jof10030197
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