Applied and Industrial Microbiology
Research Article
10 September 2021

Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations


Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings.
IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.


Fungal unspecific peroxygenases (UPOs) (EC are extracellular heme-thiolate enzymes that are of particular interest in the field of industrial and environmental biocatalysis (1). UPOs act as promiscuous mono(per)oxygenases, inserting oxygen into nonactivated C-H bonds (peroxygenase activity) and thereby unlocking a palette of two-electron oxidation reactions that have a wide range of applications in organic synthesis. Accordingly, UPOs can drive the hydroxylation of aromatic and aliphatic alkanes, the epoxidation of alkenes, O- and N-dealkylations, N- and S-oxygenations, and halogenations (2). In addition to their peroxygenase activity, UPOs carry out one-electron oxidation of phenolics (peroxidase activity), and thus, they are considered hybrid/chimeric enzymes that combine the mechanism of action of generic peroxidases with the oxygenation mechanism described for P450 monooxygenases through the peroxide shunt pathway (3). Indeed, UPO oxyfunctionalization chemistry is supported simply by H2O2, which acts as both the final electron acceptor and the oxygen donor, representing an attractive and less complex alternative to the more intensely studied P450s. Unlike the latter, UPOs are soluble extracellular enzymes that need neither expensive redox cofactors nor auxiliary flavoproteins, and they do not undergo O2 uncoupling. For these reasons, UPOs are generating enormous interest in applied biocatalysis (4).
However, to establish UPOs as practical biocatalysts in the pharma, chemical, and environmental sectors, it is paramount to develop robust heterologous-expression platforms that guarantee their recombinant production and engineering by directed evolution (5). With more than 4,000 putative UPO sequences deposited into genomic databases, peroxygenases have been phylogenetically sorted into two families: family I, short-type UPOs, which typically form homodimers of ∼26-kDa monomers and carry a His residue as a charge stabilizer, and family II, long-type UPOs, which are ∼44-kDa monomeric enzymes with an Arg residue as a charge stabilizer (3). Their different geometries in the heme channel along with the above-mentioned characteristics are responsible for the different substrate profiles and product selectivities among both UPO families.
Despite their ubiquitous abundance in the fungal kingdom, UPOs are difficult to express in heterologous organisms, with only limited success in yeasts, filamentous fungi, and bacteria to date (6). In yeast, the best-secreted peroxygenase is the long-type UPO from Agrocybe aegerita (syn. Cyclocybe aegerita) (AaeUPO), with production levels of over 200 mg/liter in a bioreactor. This UPO was engineered for expression in Saccharomyces cerevisiae and Pichia pastoris by directed evolution (7, 8), and the evolved AaeUPO mutant obtained (referred to as the PaDa-I variant) currently serves as a model expression system for many peroxygenase-based biocatalysis studies, along with tailored mutants for different applications (6, 816). Along similar lines, the long-type UPO from Coprinopsis cinerea (rCciUPO) has been produced recombinantly in Aspergillus oryzae by Novozymes A/S (Bargsvaerd, Denmark), and it has also been widely studied, although the details of this expression system are not freely available (17). More recently, 9 short-type UPOs were added to this pool by applying a modular secretion system based on Golden Gate cloning in yeast together with 5 long-type UPO chimeras based on the PaDa-I mutant as the main secretion scaffold (1820). In addition, three more short-type UPO proteins were expressed in Escherichia coli, although the weak protein production seems to preclude their engineering by directed evolution (2123).
Thus, at present, only two long-type UPOs have been heterologously produced at reasonable titers in robust expression systems (the PaDa-I variant in yeast and rCciUPO in A. oryzae), which is why scientists are mining for more UPOs with new biochemical attributes that can be recombinantly expressed to open up new industrial possibilities and research opportunities. In this regard, all the UPOs hitherto reported display a pH profile for oxygen transfer reactions close to neutral (pH 5.0 to 7.0), along with poor stability under acidic conditions (pH 2.0 to 3.0) (2, 3). This narrow pH window may limit their applications should they have to be combined with other enzymes in cascade reactions or used under more drastic conditions. Two years ago, two new long-type UPO genes from Candolleomyces (Psathyrella) aberdarensis (PabUPO-I and PabUPO-II), East African ink cap (24, 25), were identified and characterized. These peroxygenases have singular biochemical properties relative to previously described UPOs in terms of their pH activity and stability profiles, showing an unusually broad pH range for peroxygenase activity (from pH 2.0 to 9.0) and strong stability at alkaline or acidic pHs (depending on the isoform). These physicochemical properties of the PabUPOs pinpoint specific adaptations of the native fungi to grow in the voluble fungal microenvironment that could be harnessed for different oxyfunctionalization reactions at acidic/basic pH (3, 26).
In the current study, we describe the heterologous functional expression in two yeast species of these two new long-type PabUPO genes. By replacing their original signal peptides and constructing and screening site-directed recombination (SDR) mutant libraries, the most beneficial secretion mutations from the PaDa-I variant were incorporated into PabUPOs for their successful expression in S. cerevisiae. The best secretion clones were further transferred to P. pastoris for overproduction in a bioreactor and characterized biochemically. While the exclusive pH activity profiles of native PabUPOs were conserved in the recombinant variants, production was boosted to approximately 300 mg/liter, paving the way for the future industrial use of these unusual acidic peroxygenases.


Adopting mutations from the evolved PaDa-I variant.

AaeUPO was engineered by directed evolution for heterologous functional expression, and to the best of our knowledge, it has the strongest recombinant UPO expression reported to date in yeast (7, 8). The wild-type AaeUPO gene encodes a 328-amino-acid mature protein preceded by a 43-amino-acid signal peptide. We previously applied directed evolution to the signal peptide by focused mutagenesis and to the mature AaeUPO protein by DNA shuffling and random mutation, through which we generated a final secretion mutant, PaDa-I, with improved expression by S. cerevisiae (8 mg/liter) and by P. pastoris in a bioreactor (217 mg/liter) (7, 8). The PaDa-I variant carries mutations F12Y-A14V-R15G-A21D-V57A-L67F-V75I-I248V-F311L (mutations in the signal peptide are underlined) that induced a total activity improvement (TAI) (the joint increase of specific activity and secretion) 3,250-fold that of wild-type AaeUPO. Since PabUPOs share ∼64 to 66% protein sequence identity with the PaDa-I mutant (see Table S1 in the supplemental material), we reasoned that some of the key elements that boosted heterologous AaeUPO expression could enhance PabUPO secretion by yeast.
The PabUPO-I and PabUPO-II genes encode mature proteins of 333 and 348 amino acids, plus 45- and 38-amino-acid signal peptides, respectively (26) (Fig. S1). As such, we first prepared four expression constructs of PabUPOs that were benchmarked for heterologous production by S. cerevisiae, Nt-PI and Nt-PII, which correspond to PabUPO-I and PabUPO-II with their native signal peptides (Nt), and their counterparts Ev-PI and Ev-PII, in which the corresponding Nt was replaced by the evolved signal peptide (Ev) from the PaDa-I mutant (previously seen to improve native AaeUPO functional expression 27-fold) (7). Despite the weak sequence identity between the Nt PabUPO signal peptides and the Ev peptide from PaDa-I (12.5 and 41% for PabUPO-I and PabUPO-II, respectively) (Fig. S2), replacing the native signal peptides with the evolved leader sequence from PaDa-I had no deleterious effects, but rather, it significantly enhanced secretion. Flask production of the different constructs indicated that the Ev peptide enhanced expression from 10 U/liter for Nt-PI to 33 U/liter for Ev-PI and from 300 U/liter for Nt-PII to 542 U/liter for Ev-PII when 2,6-dimethoxyphenol (DMP) was used as a substrate. Thus, replacing the Nt PabUPO signal peptides with the Ev peptides from PaDa-I improved secretion by 3.3- and 1.8-fold, with production levels in flasks of 2.1 and 5.4 mg/liter for Ev-PI and Ev-PII, respectively.
It is worth noting that the native, nonevolved AaeUPO is hardly secreted in S. cerevisiae (0.007 mg/liter), and thus, the expression of PabUPOs with the Nt signal peptides suggested that some of the mutations that improved the secretion and activity of PaDa-I might already be present in PabUPOs. Hence, we mapped these substitutions by aligning the PabUPO-I, PabUPO-II, and PaDa-I protein sequences (Fig. 1), identifying several matches in the PabUPO sequences to the five mutations acquired by directed evolution in the mature PaDa-I variant (V57A-L67F-V75I-I248V-F311L). In particular, there were three coincidences (Ala57, Phe67, and Ile75) in PabUPO-II, whereas only Phe67 was present in PabUPO-I. Moreover, Val248 was represented by a similar Leu residue in PabUPO-II but by an Ala in PabUPO-I. In PaDa-I, Phe311 was mutated to Leu, and at this position, there was a Phe or a Trp in PabUPO-I and PabUPO-II, respectively (Fig. 2). Taken together, we reasoned that the weaker expression of PabUPO-I in S. cerevisiae than that of PabUPO-II could be related to the fewer secretion mutations, coinciding with those in PaDa-I. Consequently, we introduced the Ala57, Ile75, and Val/Leu248 substitutions into PabUPO-I (replacing the original Ser61, Leu79, and Ala252 residues in the PabUPO-I sequence) by in vivo site-directed recombination (SDR). The F311L mutation from PaDa-I was not included in these experiments because this position is critical for regulating the trafficking of substrates to the heme, as recently observed in the crystal structure of the PaDa-I mutant by soaking experiments (27). SDR is a mutant library creation method based on the recombination machinery of S. cerevisiae that allows potential beneficial mutations, together with their reversions, to be rapidly recombined in a direct manner, allowing the identification of the optimal combination of substitutions upon screening (28). After two rounds of SDR combined with site-directed mutagenesis (SDM) (Fig. 3), we identified a PabUPO-I triple mutant, S61A-L79I-A252L (named Grogu here), with expression levels boosted from 2.1 to 14 mg/liter.
FIG 1 Sequence alignment of mature PabUPOs and the PaDa-I enzyme. The mutations acquired during the directed-evolution campaign of PaDa-I are highlighted in gray, and their corresponding positions in the PabUPOs are in square boxes (created by Clustal Omega []).
FIG 2 Homology models of PabUPO-I (cyan) and PabUPO-II (magenta) compared to the PaDa-I crystal structure (yellow). The models were generated using SWISS-MODEL (, and they are based on the crystal structure of the PaDa-I mutant (PDB accession no. 6EKZ) visualized with PyMOL ( The five secretion mutations in PaDa-I are depicted as bars and sticks.
FIG 3 Engineering of the Grogu mutant. Stronger expression (2.1 mg/liter) was achieved by replacing Nt with Ev. The Ev-PI SDR mutant library (SDR 1) included the S61A-L79I-A252V mutations and their corresponding reversions. The best selected mutant (2.6 mg/liter) incorporated only the A252V mutation. Given that PabUPO-II has a Leu residue at this position, we introduced the A252L substitution by site-directed mutagenesis (SDM), which increased secretion to 8 mg/liter. Finally, we created a new SDR mutant library (SDR 2) for the mutations S61A-L79I-A252L and their reversions, yielding the triple mutant (Grogu variant) expressed at 14 mg/liter. The stars indicate mutations, and the squares (F71) represent the exact match between wild-type PabUPO-I and PaDa-I. TAI, total activity improvement over Nt-EPI measured with DMP as a substrate; n.d., not determined.

Bioreactor production of recombinant PabUPOs in P. pastoris and their biochemical characterization.

The expression of Grogu and PabUPO-II in S. cerevisiae can aid future directed-evolution campaigns, while the industrial production of these variants could be carried out in the methylotrophic yeast P. pastoris (Komagataella phaffii) given the high cell titers that can be achieved in this yeast together with the regulation by strong promoters. Accordingly, the production of the Grogu mutant and PabUPO-II (both preceded by the Ev signal peptide) by P. pastoris was evaluated in a 5-liter fed-batch bioreactor (1.7-liter culture volume). The most strongly secreted and most stable multicopy variants were selected and fermented according to our previously described protocols (8), producing 240 mg/liter Grogu and 290 mg/liter PabUPO-II. While these expression levels are both remarkable, the production of recombinant PabUPO-II is particularly outstanding, since, to the best of our knowledge, it constitutes the highest volumetric activity of a peroxygenase (60,000 U/liter measured with veratryl alcohol) described to date. The differences in the expression of Grogu and PabUPO-II between S. cerevisiae and P. pastoris (i.e., heterologous expression in S. cerevisiae was in the order Grogu > PabUPO-II, whereas that in P. pastoris was in the order PabUPO-II > Grogu) may be related to the selection of the variants from the strain screening, with different copy numbers integrated into the P. pastoris genome.
Both the Grogu mutant and PabUPO-II from P. pastoris were purified to homogeneity (Reinheitszahl value [Rz] [A418/A280ratio] of ∼2) and characterized biochemically (Table 1; Fig. S3). The molecular masses of Grogu and PabUPO-II estimated by the average molecular mass measured by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry were 42,491 and 41,247 Da, respectively, with an ∼9 to 14% contribution of glycosylation deduced from their deglycosylated size (Table 1; Fig. S3 to S5). Kinetic thermostability was determined by measuring the T50 (the temperature at which the enzyme retained 50% of its activity after a 10-min incubation), obtaining T50 values of 56°C for Grogu and 61°C for recombinant PabUPO-II (Fig. 4A), both in the range of that reported for the PaDa-I mutant (55°C) (7, 8). In terms of the pH profiles for peroxygenase activity, both recombinant UPOs produced curves similar to those of the wild-type PabUPOs from the original fungus. The Grogu mutant retained roughly 60% relative activity in the pH range from 3.0 to 9.0, measured with veratryl alcohol, and >80% activity at pH 4.0 to 8.0, with an optimum value at pH 6.0. In contrast, optimal recombinant PabUPO-II activity was evident at pH 5.0, retaining over 40% of its relative activity in the pH range from 4.0 to 7.0 (Fig. 4B). In terms of their pH stability, recombinant PabUPO-II was stable at alkaline pH, but like wild-type PabUPO-II, it rapidly lost activity at acidic pHs (3, 26). In contrast, the Grogu mutant was fairly stable at both alkaline and acidic pHs (Fig. 4C and D). With a sequence identity of 65% between the Grogu mutant and PabUPO-II, the differences in pH activity/stability profiles and kinetic thermostability are likely to be produced in the regions of the protein with lower similarity, such as the N termini, which differ significantly in the two variants. Such regions could be of interest as targets for future protein engineering studies (Fig. 1).
FIG 4 Biochemical characterization. (A and B) Kinetic thermostability (T50) (A) and pH activity profiles measured with 5 mM veratryl alcohol and 2 mM H2O2 in 100 mM Britton-Robinson buffer at different pHs from 2.0 to 9.0 (B). Black circles, Grogu mutant; white circles, PabUPO-II. (C and D) pH stability profiles for Grogu (C) and PabUPO-II (D). Appropriate enzyme dilutions were incubated at different times over a range of pH values in 20 mM Britton-Robinson buffer. Aliquots were removed at different times to measure activity with 5 mM veratryl alcohol and 2 mM H2O2 in 100 mM phosphate buffer (pH 7.0) at room temperature. Each point represents the mean and standard deviation from 3 independent experiments.
TABLE 1 Biochemical features of recombinant PabUPOs expressed in P. pastorisa
Biochemical or spectroscopy featureValue
MW (Da)b47,00045,000
MW (Da)c42,49141,247
MW (Da)d36,50037,700
Glycosylation degree (%)149
Thermal stability (T50) (°C)5661
Sp act (U/mg)e82.9205.9
Production level (mg/liter) in bioreactor240290
Optimum pH for peroxygenase activityf6.05.0
pH range for peroxygenase activitye3–94–7
Rz (A410/A280)2.631.71
Soret region (nm)417420
CT1 (nm)567570
CT2 (nm)538537
MW, molecular weight; pI, isoelectric point; CT1 and CT2, charge transference bands 1 and 2, respectively.
Estimated by SDS-PAGE.
Estimated by MALDI-TOF mass spectrometry.
Estimated from the amino acid composition (ProtParam).
Measured with veratryl alcohol.
pH range in which the UPO variant keeps over 40% of the relative activity measured with veratryl alcohol (see Fig. S3 to S5 in the supplemental material).
Kinetic constants were measured with prototypical peroxygenase substrates (veratryl alcohol, benzyl alcohol, and 5-nitro-1,3-benzodioxole [NBD]) as well as classic peroxidase substrates [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and DMP] (Table 2). The values with ABTS were close to each other and to those of the wild-type enzymes, whereas the catalytic efficiency of recombinant PabUPO-II with DMP was increased 3-fold relative to the Grogu mutant, following the same tendency as that of wild-type PabUPOs (3, 26). The catalytic efficiencies with veratryl alcohol were also quite similar, with Grogu showing numbers similar to those of wild-type PabUPO-I, while the Km for recombinant PabUPO-II increased 3-fold. With the O-dealkylation substrate NBD, both Grogu and recombinant PabUPO-II shared virtually the same values, whereas PabUPO-II had a higher Km with benzyl alcohol. H2O2 catalytic parameters were measured with veratryl alcohol as a reducing substrate, with a higher Km for recombinant PabUPO-II but similar kcat/Km values for both enzymes, specifically due to a 3.4-fold increase in the kcat of recombinant PabUPO-II with respect to Grogu.
TABLE 2 Steady-state kinetic parameters of wild-type and recombinant PabUPOs
Variant and kinetic constantMean value ± SD
ABTSDMPNBDVeratryl alcoholBenzyl alcoholH2O2
    Km (μM)201 ± 10357 ± 44434 ± 531,277 ± 2882,259 ± 441107 ± 12
    kcat (s−1)117 ± 227 ± 129 ± 128 ± 3254 ± 3233 ± 1
    kcat/Km (mM−1 s−1)582766722112308
    Km (μM)243 ± 5397 ± 89354 ± 484,006 ± 2166,675 ± 348411 ± 60
    kcat (s−1)97 ± 187 ± 722 ± 1106 ± 4214 ± 7114 ± 7
    kcat/Km (mM−1 s−1)679219622632277
Wild-type PabUPO-Ia      
    Km (μM)105656 856  
    kcat (s−1)2023 20  
    kcat/Km (mM−1 s−1)19035 23  
Wild-type PabUPO-IIa      
    Km (μM)128143 1,291  
    kcat (s−1)8550 104  
    kcat/Km (mM−1 s−1)664350 80  
Values from reference 26.


Unspecific peroxygenases (UPOs), and in particular long-type UPOs, are difficult to express in heterologous hosts. Indeed, after more than 15 years of research, only two successful examples of recombinant long-type UPOs with high production levels have been reported so far, the evolved PaDa-I mutant from AaeUPO and rCciUPO, making these attractive blueprints for engineering and industrial applications. In contrast, several short-type UPOs have been produced heterologously (from Marasmius rotula, Humicola insolens, Chaetomium globosum, Thielavia terrestris, Myceliophthora thermophila, Myceliophthora fergusii, Myceliophthora hinnulea, Collariela virescens, and Daldinia caldariorum) (19, 29). In view of their higher structural complexity, it is somewhat paradoxical to see more recombinant short UPOs than long UPOs, particularly given their general dimeric nature and the intermonomer disulfide bridge that may demand even more complex posttranslational modifications than their long UPO counterparts (with the exceptions of M. fergusii, M. hinnulea, and T. terrestris UPOs, which lack the C-terminal Cys residue needed for the disulfide bridge and, hence, the potential dimeric form). While it remains unclear why the expression of long-type UPOs is so elusive, it may be related to the characteristic geometry of the heme cavity or the need for an internal disulfide bridge at the C terminus for correct folding. Here, we overcame the shortcomings in expressing long-type UPOs by adopting the mutational backbone of the evolved PaDa-I secretion mutant. Thereby, we achieved the highest volumetric activity level (for PabUPO-II, with 60,000 U/liter using veratryl alcohol as the substrate to be oxyfunctionalized) reported so far for both recombinant and wild-type UPOs. A comparable volumetric activity (up to 41,000 U/liter) has been demonstrated only for the short-type wild-type UPO secreted by the basidiomycetous fungus Marasmius rotula (30).
Wild-type PabUPO-I and PabUPO-II are expressed by yeast, which is of note for long-type UPOs, and this expression was further increased by switching their signal peptide for the evolved signal sequence of PaDa-I. Hence, some of the secretion-related mutations introduced into the mature PaDa-I protein may be incorporated into PabUPOs to enhance their expression. Of the five mutations in PaDa-I, PabUPO-II carried three exact matches and one synonymous change, whereas PabUPO-I carried only one exact match. By constructing and screening SDR mutant libraries, we found an optimal combination of secretion mutations that improved PabUPO-I, yielding the Grogu variant, surpassing the expression of PaDa-I in S. cerevisiae. Of the five PaDa-I secretion mutations, V57A and L67F are overrepresented in long-type UPOs. Unlike Ile248, the original Val75 and in particular the Phe311 change are also predominant (Fig. 5). Given that recombinant PabUPO-II, Grogu, and PaDa-I variants share similar secretion backbones, we hypothesize that UPO expression in the original fungus was a function of its physiological needs, and it may have been regulated in the course of evolution by removing (in the case of AaeUPO) or partially conserving (PabUPOs) key residues for expression. The use of paleoenzymology by ancestral sequence reconstruction and resurrection is providing a new twist in enzyme engineering, while it is also an invaluable strategy to mine the timeline of evolution (31, 32). By applying such methods, we have recently inferred several ancestral nodes from long-type UPOs that may help us to understand the natural role played by the secretion mutations of PaDa-I and PabUPO counterparts as well as the evolutionary trajectory within this enzyme family (our unpublished material).
FIG 5 Sequence logo of the regions harboring functional expression mutations calculated with 50 UPO sequences from GenBank ( (created with
At a structural level, we found that the heme environment of the PabUPOs has a characteristic PCP motif (Pro39/51-Cys40/52-Pro41/53 in Grogu/PabUPO-II) that places the thiolate (Cys-SH) of the proximal ligand (5th) Cys toward the heme iron (Fig. 6). This conserved feature, together with the acid-base pair typical of long-type UPOs that is formed by the glutamic acid residue and an arginine (Glu199-Arg193/Glu212-Arg205 in Grogu/PabUPO-II), is mainly responsible for the peroxide shunt pathway mechanism. The heme access channel is lined by aromatic amino acids, as occurs in PaDa-I yet with some interesting differences. At the entrance of the heme channel, the pair of protruding Phe residues that permits the substrate to access the active site in PaDa-I is present in Grogu, whereas these residues are replaced by two aliphatic amino acids in PabUPO-II (Val92-Ile207). Moreover, the aromatic triad formed by Phe69, Phe121, and Phe199 in PaDa-I, involved in orienting the substrate for catalysis (27, 33), is conserved in PabUPO-II (Phe85, Phe137, and Phe215). However, the first of these residues is replaced by methionine in Grogu (Met73, Phe125, and Phe203), as also occurs in rCciUPO (17).
FIG 6 Details of the active site of the Grogu mutant (A) and PabUPO-II (B). Acid-base pairs are depicted in yellow, the proximal ligand (5th) Cys is in blue, and the amino acids involved in orienting the substrate for catalysis are in pink. The amino acids at the entrance of both channels are shown in green. Both models were generated by SWISS-MODEL ( using the crystal structure of the PaDa-I mutant as a template (PDB accession no. 6EKZ), visualized with PyMOL (
In summary, our models seem to indicate that both PabUPOs are similar to PaDa-I in terms of dimensions and the amino acid composition of the heme channel, yet some important changes in this region may drive different substrate preferences and selectivity, which could favor their future engineering for specific applications.


Since the discovery of AaeUPO in 2004 (34), several other fungal peroxygenases have been described, but only a few have been produced in heterologous hosts. The PaDa-I mutant is one of these, the product of five rounds of directed evolution to improve secretion by yeast, and it was used here as a template to improve the expression of two new long-type UPOs. Taking lessons from our previous evolutionary campaigns, we expressed two unusual acidic PabUPOs at very high levels in yeast. In particular, we harnessed the evolved signal peptide from PaDa-I, together with secretion mutations, to produce these PabUPOs in a bioreactor with the highest volume activity reported so far. These UPOs are active and stable over a broad pH range. Since long-type UPOs seem to be more reluctant to heterologous functional expression than their short-type counterparts (with only 2 long-type UPOs and 9 short-type UPOs expressed in recombinant forms [7, 8, 18, 19, 2123]), the strategy of adopting mutations from evolved secretion mutants may notably reduce the experimental effort required and allow us to consider new engineering workflows to achieve functional heterologous expression of other long-type UPOs in a quick and simple manner in the future.


Strains and chemicals.

The P. pastoris expression vector (pPICZ-B), the P. pastoris strain X-33, and the antibiotic zeocin were purchased from Invitrogen (USA). Escherichia coli strain XL2-Blue competent cells were obtained from Agilent Technologies (USA). The restriction endonucleases EcoRI, XbaI, PmeI, BamHI, and XhoI; the DNA ligation kit; Antarctic phosphatase; and peptide-N-glycosidase F (PNGase F) were purchased from New England BioLabs (USA). iProof high-fidelity DNA polymerase was purchased from Bio-Rad (USA). Oligonucleotide primers and UPO genes were acquired from Integrated DNA Technologies (USA). The NucleoSpin plasmid kit and NucleoSpin gel and PCR cleanup kit were purchased from Macherey-Nagel (Germany). ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] was purchased from Panreac AppliChem (Germany); DMP (2,6-dimethoxyphenol) and NBD (5-nitro-1,3-benzodioxole) were purchased from TCI Europe (Switzerland); and veratryl alcohol, benzyl alcohol, and H2O2 were purchased from Merck Life Science (USA). All chemicals and medium components were of the highest purity available.

Cloning of PabUPOs in S. cerevisiae.

The genes encoding PabUPO-I (c271g7491) and PabUPO-II (c327g8238) from Candolleomyces (Psathyrella) aberdarensis (MycoBank accession no. MB827350; GenBank accession no. MH880928) (24, 25) were ordered with codon optimization for S. cerevisiae. The synthesized genes (Nt-PI, Nt-PII, Ev-PI, and Ev-PII containing overhangs to promote homologous recombination) were cloned under the control of the GAL1 promoter of the pJRoC30 expression shuttle vector, using BamHI and XhoI restriction enzymes. The linearized vector was loaded onto a preparative agarose gel and purified with the NucleoSpin gel and PCR cleanup kit. The corresponding gene (200 ng each) was mixed with the linearized plasmid (100 ng) and transformed into S. cerevisiae for in vivo gene reassembly and cloning by IVOE (35).

Site-directed recombination and site-directed mutagenesis.

PCRs for site-directed recombination (SDR) were carried out according to methods in Fig. S6 in the supplemental material. The primers used were RMLN (5′-CCTCTATACTTTAACGTCAAGG-3′), S/A61 R (5′-CTCCTGAACAGCTTCAATTATCTGAGMCGGTGTGGCCACACC-3′), S/A61-L/I79 F (5′-GGTGTGGCCACACCGKCTCAGATAATTGAAGCTGTTCAGGAGGGTTTCAATATGGAGCACGCAACTGCTMTATTTG-3′), A/V252 R (5′-CAATGTCAATTCCCTCCGTAC-3′), A/V252 F (5′-CCATCGTGCGGCCCAACCCTCTAGTACGGAGGGAATTGACATTGTGGYTTCAGCTCAT-3′), and RMLC (5′-GGGAGGGCGTGAATGTAAGC-3′). Codon degeneration using XYZ codons (underlined) was explored with the amino acid calculator at according to a protocol described previously (28). The PCR mixtures of the first experiment (Fig. S6A) contained, in a 50-μl final volume, dimethyl sulfoxide (DMSO) (3%), deoxynucleotide triphosphates (dNTPs) (1 mM; 0.25 mM each), iProof high-fidelity DNA polymerase (0.02 U/ml), the template Ev-PI (10 ng), and the following primers, depending on the PCR: PCR1 primers RMLN (0.5 μM) and S/A61 R (0.5 μM), PCR2 primers S/A61-L/I79 F (0.5 μM) and A/V252 R (0.5 μM), and PCR3 primers A/V252 F (0.5 μM) and RMLC (0.5 μM). Site-directed mutagenesis (SDM) PCR mixtures to obtain the Ev-PI-A252L mutant (Fig. S6B) contained, in a 50-μl final volume, DMSO (3%), dNTPs (1 mM; 0.25 mM each), iProof high-fidelity DNA polymerase (0.02 U/ml), the template Ev-PI (10 ng), and S/A61 R (0.5 μM)-RMLN (0.5 μM) for fragment 1 (PCR4) and SDM F (5′-CCATCGTGCGGCCCAACCCTCTAGTACGGAGGGAATTGACATTGTGTTGTCAGCTCAT-3′) (0.5 μM)-RMLC (0.5 μM) for fragment 2 (the codon with the mutation is underlined) (PCR5). For the second experiment of SDR (Fig. S6C), the conditions were a 50-μl final volume containing DMSO (3%), dNTPs (1 mM; 0.25 mM each), iProof high-fidelity DNA polymerase (0.02 U/ml), the template Ev-PI-A252L (10 ng), and the following primers, depending on the PCR: PCR6 primers RMLN (0.5 μM) and S/A61 R (0.5 μM) and PCR7 primers S/A61-L/I79 F (0.5 μM) and RMLC (0.5 μM). PCRs were carried out on a gradient thermocycler (MyCycler; Bio-Rad, USA) using the following parameters: 98°C for 30 s (1 cycle); 98°C for 10 s, 48°C for 30 s, and 72°C for 30 s (28 cycles); and 72°C for 10 min (1 cycle). PCR products were loaded onto a preparative agarose gel and purified with the NucleoSpin gel and PCR cleanup kit. The recovered DNA fragments were cloned under the control of the GAL1 promoter of the pJRoC30 expression shuttle vector, with the use of BamHI and XhoI to linearize the plasmid and to remove the parent gene. The linearized vector was loaded onto a preparative agarose gel and purified with the NucleoSpin gel and PCR cleanup kit. The corresponding PCR products (200 ng each) were mixed with the linearized plasmid (100 ng) and transformed into S. cerevisiae for in vivo gene reassembly and cloning by in vivo overlap extension (IVOE) (35), for both the SDR experiments and the SDM experiment. A total of 176 clones were evaluated in each SDR library, and 80 clones were evaluated in the SDM experiment.

PabUPO expression. (i) Culture media.

Sterile minimal medium for the microplate contained 100 ml 6.7% filtered yeast nitrogen base, 100 ml 19.2 g/liter filtered yeast synthetic dropout medium supplement without uracil, 100 ml filtered 20% raffinose, 700 ml double-distilled water (ddH2O), and 1 ml 25 g/liter filtered chloramphenicol. Sterile minimal medium for flasks contained 100 ml 6.7% filtered yeast nitrogen base, 100 ml 19.2 g/liter filtered yeast synthetic dropout medium supplement without uracil, 25 ml filtered 20% glucose, 775 ml ddH2O, and 1 ml 25 g/liter filtered chloramphenicol. Synthetic complete (SC) dropout plates contained 100 ml 6.7% filtered yeast nitrogen base, 100 ml 19.2 g/liter filtered yeast synthetic dropout medium supplement without uracil, 20 g autoclaved Bacto agar, 100 ml 20% filtered glucose, 1 ml 25 g/liter filtered chloramphenicol, and ddH2O to 1,000 ml. Sterile expression medium contained 720 ml autoclaved yeast peptone (YP), 67 ml 1 M filtered KH2PO4 (pH 6.0) buffer, 111 ml 20% filtered galactose, 22 ml filtered MgSO4 at 0.1 M, 31.6 ml absolute ethanol (depending on the enzyme), 1 ml 25 g/liter filtered chloramphenicol, and ddH2O to 1,000 ml. YP medium contained 10 g yeast extract, 20 g peptone, and ddH2O to 650 ml. The yeast extract-peptone-dextrose (YPD) solution contained 10 g yeast extract, 20 g peptone, 100 ml 20% sterile glucose, 1 ml 25 g/liter chloramphenicol, and ddH2O to 1,000 ml. Luria-Bertani (LB) medium was prepared with 5 g yeast extract, 10 g peptone, 10 g NaCl (5g NaCl for zeocin selection), 100 mg ampicillin or 25 mg zeocin, and ddH2O to 1,000 ml. Buffered methanol-complex medium (BMMY) contained 100 mM potassium phosphate buffer (pH 6.0), 3.5 g/liter yeast nitrogen base without amino acids, 400 μg/liter biotin, and 0.5% (vol/vol) methanol.

(ii) Microtiter plate expression in S. cerevisiae.

Individual clones were picked and cultured in sterile 96-well plates containing 50 μl of minimal medium for microplates. In each plate, column 6 was inoculated with PaDa-I, and well H1 was not inoculated (culture medium control). Plates were sealed to prevent evaporation and incubated at 30°C at 225 rpm with 80% relative humidity in a humidity shaker (Minitron-Infors; Biogen, Spain). After 48 h, 150 μl of expression medium was added to each well, followed by culture for an additional 48 h at 25°C. The plates were centrifuged at 2,000 rpm (at 4°C), and finally, 20-μl portions of the supernatants were screened for activity with DMP. The plasmids from positive wells were recovered with Zymoprep yeast plasmid miniprep kit I. Since the product of the Zymoprep was impure, and the DNA extracted was very low concentrated, the shuttle vectors were transformed into supercompetent E. coli XL2-Blue cells and plated onto LB medium with ampicillin (LB-ampicillin medium). Single colonies were selected to inoculate 5 ml of LB-ampicillin medium and incubated overnight at 37°C at 225 rpm. The plasmids were extracted (NucleoSpin plasmid kit), sent for DNA sequencing (GATC Biotech-Eurofins, Luxembourg), and transformed into S. cerevisiae for flask production.

(iii) Small-scale fermentation in S. cerevisiae.

A single colony from the S. cerevisiae clone containing the gene of interest was picked from an SC dropout plate, inoculated in minimal medium for flasks (10 ml), and incubated for 48 h at 30°C at 230 rpm. An aliquot of cells was removed and used to inoculate minimal medium (100 ml) at 500 ml (optical density at 600 nm [OD600] = 0.25). The cells completed two growth phases (6 to 8 h), and expression medium (900 ml) was then inoculated with the preculture (100 ml) (OD600 of 0.1). After incubation for 72 h at 25°C at 230 rpm (maximal UPO activity at an OD600 of 25 to 30), the cells were recovered by centrifugation at 4,500 rpm (4°C), and the supernatant was double filtered (using both a glass membrane and a nitrocellulose membrane with a 0.45-μm pore size).

PabUPO expression in P. pastoris: cloning, selection, and small-scale flask fermentation.

The DNA sequences containing Grogu and recombinant PabUPO-II (both preceded by the evolved signal peptide from the PaDa-I mutant) were cloned into the expression vector pPICZ-B. Vector pJRoC30-Grogu, resulting from SDR experiments, and vector pJRoC30-PabUPO-II were used to amplify the evolved UPO with the primers psnUPO1DIR2 (5′-ccggaattcATGAAATATTTTCCCCTGTTCCCAA-3′) and PabUPOI R (5′-gctctagaTCATAGTTGACCGTAGGGGA-3′) or psnUPO1DIR2 and PabUPOII R (5′-gctctagaTCAAAGTTGACCGTACGGGA-3′), which included targets for EcoRI and XbaI restriction enzymes, respectively (in boldface type; capital letters correspond to each UPO sequence). PCRs were performed using a thermocycler (MyCycler; Bio-Rad, USA) in a final volume of 50 μl containing 0.5 μM each primer, 100 ng template, 1 mM dNTPs (0.25 mM each), DMSO (3%), and iProof high-fidelity DNA polymerase (0.02 U/ml). The PCR conditions were 98°C for 30 s (1 cycle); 98°C for 10 s, 55°C for 27 s, and 72°C for 30 s (28 cycles); and 72°C for 10 min (1 cycle). The pPICZ-B vector and the PCR product were digested with the restriction enzymes EcoRI and XbaI at 37°C for 20 min. The linearized pPICZ-B vector 5′ and 3′ ends were dephosphorylated using Antarctic phosphatase (1 U for every 200 ng of linearized vector) at 37°C for 1 h. The PCR product and the linearized vector were loaded onto a preparative agarose gel, purified using the NucleoSpin gel and PCR cleanup kit, and ligated with T4 DNA ligase at room temperature for 60 min. After the transformation of the pPICZ-B-Grogu and pPICZ-B-PabUPO-II constructs into chemically competent E. coli XL2-Blue cells, the plasmid was proliferated, linearized with the restriction enzyme PmeI at 37°C for 1 h, and transformed into electrocompetent P. pastoris X-33 cells. Electrocompetent P. pastoris cells were prepared and transformed with the construct as described previously (36), using 1,000 ng of the linearized vector and 50 μl of competent cells. Transformants were grown on YPD plates (10 g/liter yeast extract, 20 g/liter peptone, 4 g/liter glucose, and 15 g/liter agar) containing 100, 300, and 500 mg/liter zeocin to favor the selection of “high-copy-number” clones.
Five transformants of each enzyme were inoculated in 3 ml of liquid YPD medium at 30°C at 230 rpm. The culture was refreshed to an OD600 of 0.3 in 10 ml of YPD medium, grown until it reached an OD600 of 1 after 3 to 4 h, and inoculated in 40 ml of BMMY in 500-ml baffled flasks. The cultures were incubated at 30°C, and they were supplemented with 500 μl of methanol every 24 h. After 3 days of methanol addition, the best clone of each enzyme with the highest activity was selected for bioreactor fermentation.

Production in a 5-liter fed-batch bioreactor.

P. pastoris clones containing Grogu and PabUPO-II were cultivated in a 5-liter glass vessel bioreactor (Biostat A plus; Sartorius Stedim, Germany) adapted to the Pichia fermentation process guidelines of Invitrogen (version B 053002; Thermo). Basal salts medium (BMG) for the bioreactor with an initial volume of 1.7 liters contained 26.7 ml/liter 85% phosphoric acid, 0.93 g/liter CaSO4·2H2O, 14.9 g/liter MgSO4·7H2O,18.2 g/liter K2SO4, 4.13 g/liter KOH, and 40 g/liter glycerol. The Pichia trace metal (PTM1) trace salts solution was sterilized by filtration and consists of the following ingredients: 6 g/liter CuSO4·5H2O, NaI at 0.08 g/liter, 3 g/liter MnSO4·H2O, 0.2 g/liter Na2MoO4, 0.02 g/liter H3BO3, 0.5 g/liter CoCl2, 20 g/liter ZnCl2, 65 g/liter FeSO4·7H2O, 0.2 g/liter biotin, and 5 ml/liter sulfuric acid. After autoclaving the bioreactor with the medium, 4.35 ml/liter PTM1 trace salts and 1 ml antifoam B (Sigma-Aldrich, Germany) were added. The pH was adjusted to 5.0 with an ammonium hydroxide solution (28%) and was kept constant during cultivation. The fermentation process was started by adding 120 ml of a P. pastoris preculture grown on BMG medium in a 500-ml Erlenmeyer shaking flask at 180 rpm at 30°C for 18 h. The glycerol batch was run at 800 rpm at 30°C. After the glycerol had been completely consumed, the glycerol fed-batch phase was followed by the addition of a 50% (wt/vol) glycerol feed containing 12 ml/liter of PTM1 trace salts (the dissolved oxygen [DO] concentration should be kept above 20 to 30%). After the completion of the glycerol fed-batch phase, indicated by the DO peak after 18 to 24 h, the methanol feed started by adding 3.6 ml/h/liter methanol, containing 12 ml/liter PTM1 trace salts. Within the first 2 to 3 h, the addition of methanol was slowly increased so that the culture could adapt and the DO spike would stay above 80%. Samples were taken regularly. After methanol was consumed or no increase of enzyme activity could be noticed, the vessel was harvested. The culture of P. pastoris cells was clarified by centrifugation at 10,000 × g for 1 h at 4°C. Subsequently, the supernatant was concentrated by ultrafiltration using tangential-flow cassettes (Sartocon slice cassette, Hydrosart, cutoff of 10 kDa; Sartorius Stedim, Germany).

Purification of Grogu and PabUPO-II.

Ammonium sulfate was added slowly to the corresponding ultrafiltrate containing Grogu up to a 40% solution (4°C). The resulting suspension was centrifuged at 10,000 × g for 1 h at 4°C to precipitate nontarget proteins. Afterwards, hydrophobic-interaction chromatography (HIC) with phenyl Sepharose FF (GE Healthcare Europe GmbH, Germany) in an XK 26- by 200-mm column was performed. The bound proteins were eluted within a linear gradient from 40% to 0% ammonium sulfate in 20 mM Bis-Tris buffer (pH 7). The pooled active fraction was applied for anion-exchange chromatography on a Mono Q 10/100 GL column (GE Healthcare) using 10 mM Naacetate (pH 5.5) as the mobile phase eluting with a 0.67 M NaCl gradient within 15 column volumes. Finally, this preparation was further isolated via size exclusion chromatography (SEC) on a HiLoad Superdex 75-pg 26- by 600-mm column (GE Healthcare) using 100 mM NaCl in 50 mM Na acetate (pH 6.8) as the eluent.
The ultrafiltrate of PabUPO-II was applied to Q Sepharose FF (XK 26/20 column; GE Healthcare) with 10 mM Na acetate (pH 6.0) and a linear gradient of 0.67 M NaCl. Eventually, SEC on a HiLoad Superdex 75-pg 26- by 600-mm column was performed. The homogeneity of the final Grogu and PabUPO-II preparations was proven by SDS-PAGE using precast gels (Invitrogen NuPAGE Bis-Tris 10 or 12%). The UV-visible (UV-Vis) absorption spectra were recorded with a Cary 60 UV-Vis spectrophotometer (Agilent, Germany).

Biochemical characterization.

ABTS kinetic constants were estimated in sodium phosphate-citrate buffer (pH 4.0; 100 mM), DMP kinetic constants were estimated in phosphate buffer (pH 6.0; 100 mM), and veratryl alcohol, benzyl alcohol, NBD, and H2O2 constants were estimated in phosphate buffer (pH 7.0; 100 mM). All kinetics except for H2O2 used a fixed concentration of H2O2 (2 mM). H2O2 kinetic constants were estimated using veratryl alcohol (8 mM) as the reducing substrate. Reactions were performed in triplicate, and substrate oxidations were monitored by spectrophotometric changes (ε418 ABTS·+ = 36,000 M−1 cm−1, ε469 cerulignone = 27,500 M−1 cm−1, ε280 benzaldehyde = 1,400 M−1 cm−1, ε310 veratraldehyde = 9,300 M−1 cm−1, and ε425 4-nitrocatechol = 9,700 M−1 cm−1). To calculate the Km and kcat values, the average Vmax was represented against the substrate concentration and fitted to a single rectangular hyperbola function with SigmaPlot 10.0, where parameter a was equal to kcat and parameter b was equal to Km.
The pH activity profile with veratryl alcohol was calculated with the appropriate dilutions of enzyme samples, prepared in such a way that aliquots of 20 μl gave rise to a linear response in the kinetic mode. The optimum pH activity was determined using Britton-Robinson buffer (100 mM) at different pH values (from 2.0 to 9.0), veratryl alcohol (5 mM), and H2O2 (2 mM). The activities were measured in triplicate, and the relative activity (percent) is based on the maximum activity at a certain pH for each enzyme.
The pH stability profile was calculated with appropriate pure enzyme dilutions incubated at different times over a range of pH values in 20 mM Britton-Robinson buffer (pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0). Samples were removed at different times (0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h), and activity was measured in phosphate buffer (pH 7.0; 100 mM) with veratryl alcohol (5 mM) and H2O2 (2 mM). The experiments were performed in triplicate and measured in the kinetic mode, and the residual activity was related to the 100% initial activity.
The kinetic thermostability of PabUPOs was estimated by assessing their T50 values using 96-well gradient thermocyclers (MyCycler; Bio-Rad, USA). Appropriate UPO dilutions were prepared with the help of a robot in such a way that 20-μl aliquots gave rise to a linear response in the kinetic mode. Next, 50 μl was used for each point in the gradient scale, and a temperature gradient profile ranging from 20°C to 80°C was established as follows: 20.0°C, 30.0°C, 31.6°C, 34.6°C, 39.5°C, 45.3°C, 49.6°C, 52.8°C, 55.0°C, 56.9°C, 59.9°C, 64.3°C, 69.7°C, 75.0°C, 78.1°C, and 80.0°C. After a 10-min incubation, samples were chilled on ice for 10 min and further incubated at room temperature for 5 min. Afterwards, 20-μl samples were assayed in sodium phosphate buffer (pH 6.0; 100 mM) containing H2O2 (2 mM) and DMP (2 mM). Reactions were performed in triplicate, and substrate oxidations were monitored by spectrophotometric changes. The thermostability values were deduced from the ratio between the residual activities with incubation at different temperatures and the initial activity at room temperature.
Purified UPOs (8 μg each) were subjected to two-dimensional electrophoresis in order to determine the pI (isoelectric point) by the Protein Chemistry Service of the Centro de Investigaciones Biológicas (CIB) (CSIC, Spain), and to molecular weight determination by MALDI-TOF mass spectrometry at the Proteomics Facility at the Centro Nacional de Biotecnología (CNB) (CSIC, Spain).


This work was supported by Comunidad de Madrid Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM, Spanish Government projects BIO2016-79106-R-Lignolution and PID2019-106166RB-100-OXYWAVE, and CSIC project PIE-201580E042. P.G.D.S. is grateful to the Ministry of Science, Innovation, and Universities (Spain) for her FPI and POP contracts (BES-2017-080040).

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 87Number 1910 September 2021
eLocator: e00878-21
Editor: Emma R. Master, University of Toronto
PubMed: 34288703


Received: 6 May 2021
Accepted: 14 July 2021
Accepted manuscript posted online: 21 July 2021
Published online: 10 September 2021


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  1. unspecific peroxygenase
  2. heterologous functional expression
  3. Saccharomyces cerevisiae
  4. Pichia pastoris
  5. directed evolution



Patricia Gomez de Santos
Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain
EvoEnzyme S.L., Madrid, Spain
Manh Dat Hoang
Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain
Institute of Biochemical Engineering, Technical University of Munich, Garching, Germany
Jan Kiebist
JenaBios GmbH, Jena, Germany
Harald Kellner
Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Zittau, Germany
René Ullrich
Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Zittau, Germany
Katrin Scheibner
Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany
Martin Hofrichter
Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Zittau, Germany
Christiane Liers
Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Zittau, Germany
Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain


Emma R. Master
University of Toronto

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  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.


If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

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