Comparing the Catalytic and Structural Characteristics of a ‘Short’ Unspecific Peroxygenase (UPO) Expressed in Pichia pastoris and Escherichia coli

Abstract

Unspecific peroxygenases (UPOs) have emerged as valuable tools for the oxygenation of non-activated carbon atoms, as they exhibit high turnovers, good stability and depend only on hydrogen peroxide as the external oxidant for activity. However, the isolation of UPOs from their natural fungal sources remains a barrier to wider application. We have cloned the gene encoding an 'artificial' peroxygenase (artUPO), close in sequence to the 'short' UPO from Marasmius rotula (MroUPO), and expressed it in both the yeast Pichia pastoris and E. coli to compare the catalytic and structural characteristics of the enzymes produced in each system. Catalytic efficiency for the UPO substrate 5-nitro-1,3-benzodioxole (NBD) was largely the same for both enzymes, and the structures also revealed few differences apart from the expected glycosylation of the yeast enzyme. However, the glycosylated enzyme displayed greater stability, as determined by nano differential scanning fluorimetry (nano-DSF) measurements. Interestingly, while artUPO hydroxylated ethylbenzene derivatives to give the (R)-alcohols, also given by a variant of the 'long' UPO from Agrocybe aegerita (AaeUPO), it gave the opposite (S)-series of sulfoxide products from a range of sulfide substrates, broadening the scope for application of the enzymes. The structures of artUPO reveal substantial differences to that of AaeUPO, and provide a platform for investigating the distinctive activity of this and related'short' UPOs.

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

“…coli systems for other UPOs’ expression (Table S5). ,, In contrast, UPOs without sfGFP fusion showed no activity for ethylbenzene hydroxylation. Overall, the E.…”

“…Consequently, protein expression ranging from 14.6 to 16.3 mg L −1 were achieved, which are higher than most of the existing E. coli systems for other UPOs' expression (Table S5). 11,21,36 In contrast, UPOs without sfGFP fusion showed no activity for ethylbenzene hydroxylation. Overall, the E. coli sf GFP-mediated secretion system presents a valuable platform for facilitating UPO secretion expression in E. coli, and it should be considered a significant tool for screening novel UPOs and evolving the existing UPOs to improve their catalytic properties.…”

“…However, the heterologous expression of UPOs (originating from fungi) in prokaryotic E. coli is challenging. Up to now, only Mro UPO, Cvi UPO, Dca UPO, and art UPO (artificial peroxygenase, shares 73% sequence identity with Mro UPO) have been expressed in soluble and active forms, 11 , 20 , 21 but with very low efficiency. Even worse, complex procedures like codon optimization, autoinduction medium, low incubation temperature, and prolonged induction time (4–5 days) are essential for facilitating the corrected folding of UPOs in E. coli .…”

Unspecific Peroxygenase (UPO) can be Tuned for Oxygenation or Halogenation Activity by Controlling the Reaction pH

“…2-Ethylanisole 22 gave no oxygenated products, which is consistent with previous observations that the ortho-substitution militates against oxygenation at the benzylic position by Compound I in the active site of the UPO. [38] Scheme 1. A: Halogenation of thymol 1 by rAaeUPO-PaDa-IÀ H at pH 3.0; % values indicate conversions measured on GC; B: Oxygenation of 1 by rAaeUPO-PaDa-IÀ H at pH 6.0; Each reaction was performed using 1 mg mL À 1 UPO in 0.1 M potassium citrate buffer with 10 % (v/v) acetone as co-solvent at RT; C: Preparative scale oxygenation of thymol by rAaeUPO-PaDa-IÀ H; reaction was performed using 5 mg mL À 1 rAaeUPO-PaDa-IÀ H. Having established the contrasting product profiles of r-AaeUPO-PaDa-IÀ H catalysis under the different conditions, factors affecting bromination conversion were studied using 2-ethylanisole 22 as a model substrate (Table S2).…”

Agar plate‐based activity assay for easy and fast screening of recombinant Pichia pastoris expressing unspecific peroxygenases

“…For instance, UPO expression in E. coli resulted in low yields and also in lower protein stability most likely due to the lack of glycosylation. [17,18] P. pastoris is an attractive host for highlevel heterologous expression and secretion of proteins and several fungal oxidative enzymes like laccases or aryl-alcohol oxidases have been expressed in P. pastoris with expression levels of up to 1 g L −1 or even higher. [19][20][21] Recently, we reported an expression yield of ∼700 mg L −1 of AbrUPO from A. brasiliensis in P. pastoris.…”