Unspecific peroxygenases (UPOs) are glycosylated fungal enzymes that can selectively oxidize C–H bonds. UPOs employ hydrogen peroxide as the oxygen donor and reductant. With such an easy-to-handle cosubstrate and without the need for a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from Hypoxylon sp. EC38 (HspUPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris. The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. HspUPO is a versatile catalyst performing various reactions, such as the oxidation of prim- and sec-alcohols, epoxidations, and hydroxylations. Semipreparative biotransformations were demonstrated for the nonenantioselective oxidation of racemic 1-phenylethanol rac-1b (TON = 13 000), giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated yield. HspUPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with substrates of variable polarities. The structural investigation of several protein–ligand complexes revealed that the active site of HspUPO is accessible to molecules of varying bulkiness with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, HspUPO is an exploitable candidate for peroxygenase-based biocatalysis.
Unravelling the core promoter sequence-function relationship is fundamental for engineering transcription initiation and thereby a feasible "tuning knob" for fine-tuning expression in synthetic biology and metabolic engineering applications. Here a systematic replacement studies of the core promoter and 5' untranslated region (5'UTR) of the exceptionally strong and tightly methanol regulated Komagataella phaffii (syn. Pichia pastoris) alcohol oxidase 1 (AOX1) promoter at unprecedented resolution is performed. Adjacent triplets of the 200 bp long core promoter are mutated at a time by changing the wild-type sequence into cytosine or adenine triplets, resulting in 130 variants that are cloned upstream of an eGFP reporter gene providing a library for expression fine-tuning. Mutations in the TATA box motif, regions downstream of the transcription start site or next to the start codon in the 5'UTR had a significant effect on the eGFP fluorescence. Surprisingly, mutations in most other regions are tolerated, indicating that yeast core promoters can show a high tolerance toward small mutations, supporting regulatory models of degenerate motifs, or redundant design. The authors exploited these neutral core promoter positions, not affecting expression, to introduce extrinsic sequence elements such as cloning sites (allowing targeted core promoter/5'UTR modifications) and bacterial promoters (applicable in multi host vectors).
Since 2004, unspecific peroxygenases, in short UPOs (EC. 1.11.2.1), have been explored. UPOs are closing a gap between P450 monooxygenases and chloroperoxidases. These enzymes are highly active biocatalysts for the selective oxyfunctionalisation of C–H, C=C and C-C bonds. UPOs are secreted fungal proteins and Komagataella phaffii (Pichia pastoris) is an ideal host for high throughput screening approaches and UPO production. Heterologous overexpression of 26 new UPOs by K. phaffii was performed in deep well plate cultivation and shake flask cultivation up to 50 mL volume. Enzymes were screened using colorimetric assays with 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (DMP), naphthalene and 5-nitro-1,3-benzodioxole (NBD) as reporter substrates. The PaDa-I (AaeUPO mutant) and HspUPO were used as benchmarks to find interesting new enzymes with complementary activity profiles as well as good producing strains. Herein we show that six UPOs from Psathyrella aberdarensis, Coprinopsis marcescibilis, Aspergillus novoparasiticus, Dendrothele bispora and Aspergillus brasiliensis are particularly active.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.