dMAD7 is a promising tool for targeted gene regulation in the methylotrophic yeast Komagataella phaffii.

The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research.

Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.

Background: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

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.

Enantioselective High-Throughput Assay Showcased for the Identification of (R)- as well as (S)-Selective Unspecific Peroxygenases for C-H Oxidation.

Identifying (bio)catalysts displaying high enantio-/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, a high-throughput, stereoselective screening assay is reported that gives information on enantioselectivity, stereopreference and activity as showcased for peroxygenase-catalyzed hydroxylation. The assay is based on spectrophotometric analysis of the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec-alcohol product formed in the peroxygenase reaction.

Enabling growth-decoupled recombinant protein production based on the methanol-free P promoter.

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of () can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using has often been driven either by the methanol induced promoter (P ) and/or the constitutive promoter (P ).

Bidirectional Promoter Libraries Enable the Balanced Co-expression of Two Target Genes in E. coli.

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.

Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in : A Budding Yeast in Distress.

The industrially important non-conventional yeast suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous end joining repair, we chose a diverse set of CRISPR/Cas targets and conducted whole genome sequencing on 146 CRISPR/Cas9 engineered single colonies.

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

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.

Bioengineering a glucose oxidase nanosensor for near-infrared continuous glucose monitoring.

Single-walled carbon nanotubes (SWCNTs) emit photostable near-infrared (NIR) fluorescence that is conducive for optical glucose monitoring. Such SWCNT-based optical sensors often require the immobilization of proteins that can confer glucose selectivity and reactivity. In this work, we immobilize a glucose-reactive enzyme, glucose oxidase (GOx), onto SWCNTs using a -(1-pyrenyl)maleimide (PM) crosslinker thiol bioconjugation of engineered cysteine residues.

Preparative Production of Functionalized (- and -Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor.

Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g.

Regiospecific 7-hydroxylation of ten-carbon monoterpenes by detoxifying CYP5035S7 monooxygenase of the white-rot fungus Polyporus arcularius.

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.

Late-Stage Functionalisation of Polycyclic (-Hetero-) Aromatic Hydrocarbons by Detoxifying CYP5035S7 Monooxygenase of the White-Rot Fungus .

Functionalisation of polycyclic aromatic hydrocarbons (PAHs) and their -heteroarene analogues (NPAHs) is a tedious synthetic endeavour that requires diverse bottom-up approaches. Cytochrome P450 enzymes of white-rot fungi were shown to participate in the fungal detoxification of xenobiotics and environmental hazards via hydroxylation of PAH compounds. In this paper, the recently discovered activity of the monooxygenase CYP5035S7 towards (N)PAHs was investigated in detail, and products formed from the substrates azulene, acenaphthene, fluorene, anthracene, and phenanthrene by whole-cell biocatalysis were isolated and characterised.

Structural and biochemical studies enlighten the unspecific peroxygenase from Hypoxylon sp. EC38 as an efficient oxidative biocatalyst.

Unspecific peroxygenases (UPO) are glycosylated fungal enzymes that can selectively oxidize C-H bonds. UPOs employ hydrogen peroxide as oxygen donor and reductant. With such an easy-to-handle co-substrate and without the need of a reducing agent, UPOs are emerging as convenient oxidative biocatalysts.

Evolution and enrichment of CYP5035 in Polyporales: functionality of an understudied P450 family.

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.

Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris.

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.

Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9.

Background: Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy.

Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris.

Background: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P), and the constitutive GAP promoter (P). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P, P) was first conducted in chemostat cultures.

Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris.

Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression.

Current advances in engineering tools for Pichia pastoris.

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.

Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With .

(.) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER).

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies.

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P.

The Extreme Structural Plasticity in the CYP153 Subfamily of P450s Directs Development of Designer Hydroxylases.

CYP153s are bacterial class I P450 enzymes traditionally described as alkane hydroxylases with a high terminal regioselectivity. They have been more recently shown to also catalyze hydroxylations at nonactivated carbon atoms of small heterocycles. The aim of our work was to perform an extensive characterization of this subfamily in order to deliver a toolbox of CYP153 enzymes for further development as biocatalysts.