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Article

Conversion of Unsaturated Short- to Medium-Chain Fatty Acids by Unspecific Peroxygenases (UPOs)

by
Alexander Karich
1,*,
Fabian Salzsieder
1,
Martin Kluge
1,
Miguel Alcalde
2,
René Ullrich
1 and
Martin Hofrichter
1
1
Unit of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Markt 23, 02763 Zittau, Germany
2
Institute of Catalysis ICP-CSIC, 28049 Madrid, Spain
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2023, 3(3), 826-840; https://doi.org/10.3390/applmicrobiol3030057
Submission received: 21 June 2023 / Revised: 11 July 2023 / Accepted: 13 July 2023 / Published: 19 July 2023
(This article belongs to the Special Issue Exclusive Papers Collection of Editorial Board Members and Invited Scholars in Applied Microbiology)
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Abstract

:
Eighteen short- to medium-chain monounsaturated fatty acids were screened for hydroxylation and epoxidation using eleven different peroxygenase preparations. Most of these unspecific peroxygenases (UPOs) are secreted by fungal species of the dark-spored basidiomycetous families Psathyrellaceae and Strophariaceae, two belonged to the white-spored genus Marasmius (Marasmiaceae), and one belonged to the ascomycetous family Chaetomiaceae. The fatty acids (FAs) studied were categorized into three groups based on the position of the double bond: (i) terminal unsaturated FAs (between ω and ω-1), (ii) α-β-unsaturated FAs (between C2 and C3), and (iii) β-γ-unsaturated FAs (between C3 and C4). Their chain lengths ranged from three to nine carbon atoms. FAs with a terminal double bond were significantly oxidized by only two UPOs, namely CglUPO and CraUPO (peroxygenases from Chaetomium globosum and Coprinellus radians, respectively), producing different products. FAs with internal double bonds were converted by all tested UPOs. While epoxides were observed as products in the case of α-β-unsaturated fatty acids, only CglUPO formed β-γ-epoxides from the corresponding FAs. The product pattern of the other UPOs for β-γ-unsaturated FAs was quite similar. On the other hand, the product pattern for oxidized α-β-unsaturated FAs was more variable and, in some cases, specific to a particular UPO. For example, in the reaction with trans-2-nonenoic acid, the enzymes clustered into six groups based on the formed products.

1. Introduction

Fungal unspecific peroxygenases (UPOs, EC 1.11.2.1) belong to the superfamily of heme-thiolate proteins and utilize H2O2 as a co-substrate (i.e., as electron acceptor and oxygen donor) in diverse oxyfunctionalization reactions [1,2,3]. Although UPOs are not structurally and phylogenetically closely related to cytochrome P450 monooxygenases (P450s), they share with them a cysteine-ligated heme in the active site, which appears to be a prerequisite for efficient oxygen transfer [2,4,5]. They are ubiquitous within the kingdom of fungi (Eumycota and a few pseudofungal Stramenopiles), and over 4000 UPO genes have been identified in fungal genomes so far, but not in other organisms [2].
Functionally, UPOs are hybrids of (per)oxygenases and peroxidases and can perform typical monooxygenase reactions (introduction of a single peroxide-borne oxygen atom into the substrate) or classic peroxidase reactions (one-electron oxidations, for example, of phenolic substrates). Their substrate spectrum varies from monoaromatic to polyaromatic compounds, heterocyclic, cycloaliphatic and aliphatic hydrocarbons, as well as alcohols, aldehydes, amines, and halides. The first described enzyme of this type, the peroxygenase of Cyclocybe (Agrocybe) aegerita (AaeUPO), has been reported to have no less than 300 substrates [1,2,3,6].
The limitations of UPO-catalyzed reactions can be summarized as follows: (i) Large or bulky substrate molecules (>400 Da), such as cholesteryl caprylate, nitrophenyl-terminated PEG oligomers, and coronene, cannot be oxygenated as they cannot pass through the heme channel to reach the active site of UPOs (steric hindrance). (ii) Strongly deactivated substrates, like nitrobenzenes, cannot be oxidized. (iii) Low solubility generally limits the amount of substrate that reaches the active site, often in conjunction with (i) or (ii). (iv) Hydroxylations catalyzed by UPOs require an abstractable hydrogen at an appropriate carbon (C-H), and epoxidations require hydrogen atoms flanking a double bond (HC=CH); therefore, certain compounds lacking these prerequisites are not subject to oxygenation reactions (e.g., the scission of biphenyl ethers or the epoxidation/hydroxylation of hexachlorobenzene). (v) Lastly, drastic physical conditions such as high temperatures (>60 °C), extreme pH (>10, <3), or the presence of organic solvents (>60%) can lead to enzyme inactivation [3,6]. Additionally, UPOs can be inactivated by excess amounts of H2O2, although the extent of this “co-substrate inhibition” varies among different UPOs [7].
Among the various aliphatic substrates tested thus far, several long-chain fatty acids (FAs) have proven to be suitable substrates for UPOs regardless of whether they were saturated or unsaturated. UPOs hydroxylate such FAs, with a chain length of 16 carbon atoms or longer (up to C20), at the terminal and subterminal positions, i.e., at ω-2/3 [8,9]. When double bonds are present, their epoxidation was found to be the preferred reaction [10,11,12,13]. Furthermore, two pathways for chain-shortening reactions have recently been postulated, where C1-moieties (formate or formaldehyde) are likely released in an α-oxidation of the FA [14,15]. Most of these reactions catalyzed by UPOs are also known to be performed by P450 monooxygenases [16]. However, these reactions occur intracellularly, require NAD(P)H as a co-substrate, and usually involve a complex electron transport chain. In contrast, UPO-catalyzed reactions occur outside the hyphae/cells and only require inexpensive and readily available H2O2 [3].
From a biotechnological standpoint, unsaturated FAs are of particular interest, especially in terms of producing chiral building blocks [17]. The majority of related studies have focused on long-chained fatty acids and fatty acid methyl esters, while little is known about the oxidizability of short- and medium-chain FAs (S/MCFAs) [18,19,20]. Therefore, in this study, we investigated the peroxygenation of various monounsaturated S/MCFAs using a representative set of UPOs. The positions of the double bonds within the studied FAs included C2/3 (α-β), C3/4 (β-γ), and terminal positions, while the chain length ranged from C3 to C9.

2. Materials and Methods

2.1. Enzyme Preparations

All UPOs used in this study were purified enzyme preparations; their specific activities and the references describing their production and purification procedures are listed in Table 1.
Candolleomyces eurysporus and C. aberdarensis (family Psathyrellaceae) were cultivated and the secreted UPOs purified as described by [23] to yield the two so far undescribed isoenzymes CeuMS III and CabMS II, respectively [23].

2.2. Conversion of Medium Chained Fatty Acids (MCFAs)

The following setup was used in a total volume of 0.5 mL to evaluate the relative (%) and absolute (µM) conversion of 18 unsaturated short- to medium-chain fatty acids (S/MCFAs, Table 2) by the different UPOs listed in Table 1: 1 mM FA, 20 mM potassium phosphate buffer (KPi, pH 6.0), 1 unit (UVA) enzyme (UPO) per mL (this amount oxidizes 1 µmol veratryl alcohol (VA) according to the standard assay to 1 µmol veratraldehyde [5]) and 10% acetone (to improve the solubility of the hydrophobic FAs). In this way, the enzymatic activity (and the amount) of each individual UPO was standardized in the reactions based on the substrate VA to address any concerns to that effect, regarding possible variations in activity from enzyme batch to enzyme batch.
The reactions were performed in HPLC vials and started by adding 1 mM H2O2 (50 µL with a pipette), which was followed by immediate vigorous stirring. After an incubation time of 15 min, H2O2 was completely consumed, and the samples were analyzed by HPLC (details see below).
In the case of 2-nonenoic and 2-octenoic acid, in addition to the above experimental setup, H2O2 was added stepwise in several portions to the reaction mixtures to check whether active UPO remained. Thus, peroxide was added eight times (250 µM each) to give a final volume of 0.5 mL; all other reaction components were the same as above.
One problem that needs to be highlighted is the absence of commercially available, authentic product standards for hydroxylated and epoxidized FAs. As a result, the identification of products was solely based on mass fragmentation patterns obtained from LC/MS, UV spectra (when available and indicative), and physicochemical properties, such as retention times on a C18 column. The conclusions drawn were supported by the use of chemically prepared Prileshajev epoxides. The products were then quantified accordingly.

2.3. Preparation of Epoxides via the Prilezhaev Reaction

All unsaturated S/MCFAs used as UPO substrates were also chemically oxidized at the double bond to obtain the corresponding epoxides (Prilezhaev products). These products were then analyzed by HPLC (LC/MS) and compared with the products obtained from enzymatic reactions, serving as authentic standards. The reactions were carried out as follows: A 1.5 mL solution of 0.5 M m-chloroperbenzoic acid (mCPBA) in dichloromethane was prepared on ice. To this solution, an equimolar amount of unsaturated S/MCFA (0.75 mmol) was added, and the initial reaction mixture was kept cool to prevent excessive heat generation. The reaction mixture was allowed to stand in the thawing ice bath until it reached room temperature. This process resulted in the formation of a precipitate, which was believed to be unreacted mCPBA. The reaction duration varied, typically taking a few hours for most cases, but extending up to several weeks for certain trans-isomers. The residual reaction mixture, containing dichloromethane and epoxy-FAs, was stored under cold conditions and subsequently diluted in water prior to analysis using HPLC. The authenticity of the epoxide standards synthesized in this way was confirmed by LC-MS analyses.

2.4. Analyses

The enzymatic and chemical reaction mixtures were analyzed using a Phenomenex Kinetex® column (2.6 µm C18 100 Å 100 × 2.1 mm) and an Agilent 1200 System under isocratic conditions. For the separation, substrate-specific isocratic mixtures of acetonitrile (5–55%) and aqueous formic acid (0.01%) were used as solvent, depending on the length of the aliphatic chain of the FA. The HPLC system was coupled with a diode-array detector (DAD, Agilent, Waldbronn, Germany) and an Ion Trap mass spectrometer (MS) detector (Bruker, Billerica, MA, USA). The MS was run in the negative ESI ionization mode.
Most FA substrates exhibited limited and weak UV spectra, and their products often lacked a discernible absorption spectrum (due to the conversion of the double bond through epoxidation). As a result, chromatograms on a mass basis (such as total ion count/TIC or specific mass fragments) were used to calculate the amount of products formed during UPO catalysis. It is clear that this is not a “true” but an “apparent” quantification, but the ionization of any compound, substrate or product, in the negative ionization mode is based on the negative charge of the carboxylic group. Therefore, ionization intensity is a good basis for estimating product amounts, although it is not an exact quantification method.

2.5. Chemicals

All chemicals were purchased from Sigma-Aldrich (Merck, Germany) at the highest purity grade available.

3. Results and Discussion

3.1. Terminally Unsaturated FAs

The conversion (µM) of seven terminally unsaturated S/MCFAs (from C3 to C9) by eleven UPOs is summarized in Figure 1. CglUPO was by far the most active enzyme and reached the highest conversion rates (about 500 µM, i.e., 50%) for both 8-nonenoic and 7-octenoic acid, while 6-heptenoic acid was only converted by about 20% and shorter FAs were converted by less than 5% (Figure 1). CraUPO exhibited the second-best performance and oxidized about 25% and 30% of 8-nonenoic and 7-octenoic acid, respectively. Consequently, its effectiveness was roughly half that of CglUPO. The percentage conversion of 6-heptenoic acid was even in the same range (with 20%) as that of CglUPO. However, in contrast to the latter, CraUPO oxidized also 5-hexenoic acid and 4-pentenoic acid to some extent (at 15% and 10%, respectively). Other UPOs’ conversion rates for these S/MCFAs did not exceed 5%. In summary, we conclude that terminally unsaturated FAs consisting of less than six carbon atoms do not serve as a proper substrate for UPOs with the exception of CraUPO.
Authentic standards of potential oxygenation products (i.e., hydroxy and/or keto FAs) are not commercially available. Thus, to identify the products formed in reactions with conversion rates ≥ 25% (i.e., metabolites of 8-nonenoic, 7-octenoic acid and 6-heptenoic acid), we used chemically prepared epoxides and compared them with the compounds formed during UPO catalysis. The single or double hydroxylated products were tentatively determined according to their MS fragmentation patterns, indicative mass increases (+16, +14 m/z) and retention times on the C18 column (HPLC).
Both CglUPO and CraUPO oxidized the terminal unsaturated FAs to form the corresponding epoxides. In addition, CraUPO hydroxylated the FAs at two other positions. According to the retention times and the fragmentation patterns, these two positions may correspond to the allylic ω-2 and ω-3 positions. In contrast, CglUPO, in addition to the epoxides, formed no less than six, five and four monohydroxylated products in the course of the oxidation of 8-nonenoic, 7-octenoic and 6-heptenoic acids, respectively. Thus, CglUPO undoubtedly attacked any possible carbon atom of these substrates (Figure 2).
Interestingly, the epoxide of 8-nonenoic acid was found in the enzymatic reaction mixtures of all UPOs, albeit sometimes only in trace amounts, indicating nonetheless that the C8 chain fits best into the heme access channels. On the other hand, the molecule of 7-octenoic acid, which is only one carbon atom smaller (one CH2), was converted into the corresponding epoxide only by AaeUPO and the two Marasmius UPOs. This suggests that even small changes in the size of FA molecules have strong effects on heme channel passage and proper binding near the oxygen-bearing heme iron. Similar observations were made by Martínez and coworkers when studying the specificity of rAaeUPO mutants in the oxidation of long FAs [10].

3.2. Unsaturated FAs with the Double Bond at the β,γ-Position

FAs with an internal double bond between Cβ and Cγ (positions C3 and C4; inset Figure 3) are, in contrast to terminally unsaturated FAs, arranged as cis- or trans-isomers. Such diastereomers are compounds with significantly different properties. Most FAs of this type are only available as a mixture of both isomeric forms; only 3-hexenoic acid could be purchased as pure cis- and trans-isomers. Thus, we used in most cases the diastereomeric mixtures in our enzymatic conversions.
CglUPO again was by far the best performing enzyme in these experiments, reaching total conversion values of 827 µM and 716 µM for the oxidation of isomeric mixtures of 3-nonenoic and 3-octenoic acid, respectively (compare Figure 3). This implies that about 83% and 72% of the applied H2O2 was utilized by the enzyme to oxygenate the target compounds. Another group of UPOs (AaeUPO, CeuMQ V and CeuMS III as well as CabMS II, CabMQ II and rCab II) was about three times less effective and reached maximum conversion rates between 20% and 30% only. A third group of UPOs (rCab I, MroUPO and MweUPO) was rather ineffective and converted merely 10% of the substrates at best.
Two conclusions can be drawn from the results of the conversion of cis- and trans-3-hexenoic acid: (i) Most UPOs do not substantially oxidize cis-3-hexenoic acid (only in traces at best, i.e., <2%), but they can oxidize trans-3-hexenoic acid at least to some extent (up to 20%), and (ii) two enzymes (CglUPO and CraUPO) apparently do not “discriminate” between cis- and trans-isomers with respect to their oxidation preference. Unfortunately, whether this conclusion can be applied to other cis/trans S/MCFA pairs must remain unclear, since this was the only available pair of pure cis/trans isomers (similar behavior of other diastereomers, however, seems plausible).
Conversion levels (µM) of CglUPO for cis- and trans-3-hexenoic acid were almost equal, but it is worth mentioning that the former isomer was epoxidized in substantial amounts, whereas the latter was epoxidized only in traces (the majority were hydroxylated products). This might be caused by a favorable positioning and binding of cis-configured double bonds in the heme cavity. At this point, it is worth mentioning that trans-Prileshaev products of S/MCFAs were more difficult to synthesize chemically than the corresponding cis-isomers. While the former were formed in complete conversions within a few hours, the complete chemical oxidation of the latter required several days to weeks.
When 3-nonenoic acid was used as substrate, only CglUPO formed the epoxide, and the conversion rate was 55%. In addition, CglUPO formed some minor products indicative of hydroxylation at C2, C4 and C5 positions (Figure 3 and Figure 4, Figure S1). All other tested UPOs favored hydroxylation at C3 (sp2-hybridized carbon, as C4) with rates of 55–80% and interestingly did not form the 3,4-epoxy derivative. Both 3- and 4-hydroxy-3-nonenoic acids were present in their oxo-forms (ketones), as indicated by a significant decrease in absorbance in the UV spectrum. Unsaturated FAs show a non-specific UV-spectrum with an absorption shoulder in the UV region (around 220 nm) caused by the aliphatic double bond (alkenyl) and the carbonyl function of the carboxyl group [28,29]. Thus, when the aliphatic double bond is lost, e.g., after epoxidation, the product loses its UV absorbance between 200 and 240 nm. Since oxo-fatty acids are subject to keto-enol tautomerism, they tend to attenuate or even lose their UV spectrum when the equilibrium is shifted toward the oxo-form [30].
Applmicrobiol 03 00057 g004 550
Figure 4. LC-MS elution profiles of enzymatic reactions with several UPOs and 3-nonenoic acid as substrate. Peak labeling (C7, C8, etc.) corresponds to the position of hydroxylation in the aliphatic chain; the arrow indicates epoxidation between C3 (Cβ) and C4 (Cγ) (compare Figure 5 and Figure 6).
Figure 4. LC-MS elution profiles of enzymatic reactions with several UPOs and 3-nonenoic acid as substrate. Peak labeling (C7, C8, etc.) corresponds to the position of hydroxylation in the aliphatic chain; the arrow indicates epoxidation between C3 (Cβ) and C4 (Cγ) (compare Figure 5 and Figure 6).
Applmicrobiol 03 00057 g004
It should be noted that the percentage conversions refer to the total amount of products formed (i.e., roughly the substrate converted); therefore, high percentages of the products may not correspond to large total amounts of product. Thus, in the case of CglUPO and 3-noneoic acid, 827 µM of the substrate was converted, of which 55% (=455 µM) corresponded to the epoxide. In contrast, MroUPO converted merely 90 µM of substrate, of which 80% (=72 µM) was 3-hydroxy-3-nonenoic acid.
Product formation with the substrates 3-octenoic and 3-heptenoic acid was followed using three representative UPOs (Figures S2 and S3): (i) CglUPO—ascomycetous short enzyme (UPO protein family I), (ii) AaeUPO—basidiomycetous long enzyme (UPO protein family II) and (iii) MroUPO—basidiomycetous short enzyme (family I) [2,3]. When testing these substrates and UPOs, the distribution of products was comparable to 3-nonenoic acid. Thus, CglUPO formed the corresponding epoxides as major products (72% and 92% for 3-octenoic and 3-heptenoic acid, respectively). The other two UPOs, AaeUPO and MroUPO, oxidized the FAs at the C3 position, yielding 80% and 89% 3-hydroxy-3-octenoic acid, as well as 41% and 92% 3-hydroxy-3-heptenoic acid, respectively, all being present in their corresponding oxo-form (see Figure 7 and Figure 8; additional data are given in the Supplement Tables S1–S6). In conclusion, the percentage proportion of the major product increased with the decreasing chain length of the FA when CglUPO and MroUPO were used as biocatalysts.
AaeUPO, in contrast, showed a switch of major product distribution between 3-octenoic and 3-heptenoic acid. When longer chained substrates (C8 and C9) were applied, the corresponding C3-hydroxylation product was the major one, but when 3-heptenoic acid was used, the C3 and C5 (ω-2) hydroxylation products were roughly equally distributed. With a total conversion of approx. 30%, these values correspond to about 150 µM of each product.

3.3. Unsaturated FAs with the Double Bond at α,β-Position

The third group of substrates studied comprised unsaturated S/MCFAs with chain lengths of five to nine carbon atoms (C5-C9) and a double bond at α,β-position (between C2 and C3; see Figure 9). Of the five tested compounds, four were trans-isomers and one was a mixture of respective diastereomers (6-heptenoic acid).
As already shown in the case of other substrates, CglUPO was the most effective enzyme in the conversion of trans-2-nonenoic acid and trans-2-octenoic acid. With 60% and 49%, the conversion rates were somewhat less but comparable to those of FAs with the double bond at the β,γ-position (C3-C4). The other UPOs formed three clusters: (i) Three UPOs, all originating from the agaric family Psathyrellaceae (Basidiomycota) converted the two substrates at substantial levels, giving rates of 45–48% and 20–27%, respectively. (ii) Four UPOs, comprising members of the agaric families Psathyrellaceae and Strophariaceae, were less efficient and gave rates of about 25–30% and 10–15%, respectively. (iii) Three other UPOs, which included enzymes from the Psathyrellaceae and the bright-spored Marasmiaceae, converted the two FAs only to a small extent (<5%).
The results for 2-heptenoic acid have not been easy to interpret, since it was only available as a diastereomeric mixture and it remained unclear in which ratio the individual isomers were present in the commercial preparation. Nevertheless, CglUPO converted also this mixture to a substantial extent (37%). CeuMQ V was the only other UPO that oxidized this FA at a rate greater than 10%. FAs with chain lengths of less than seven carbon atoms were not suitable substrates, and the tested UPOs reached conversion rates of 10–20% at best.
Interestingly, CglUPO hardly converted trans-2-hexenenoic acid. Thus, 2-heptenoic acid apparently represents the lower limit for the substantial conversion of unsaturated S/MCFAs with the double bond in the α,β-position.
The product pattern for trans-2-nonenoic acid was more diverse than for any other substrate tested (Figure 10, Figure 11 and Figure 12). Accordingly, the tested UPOs clustered into six groups:
  • UPOs mainly forming the epoxide between C2 and C3 (α,β-position) with yields of about 80% and producing minor hydroxylation products at different positions (C2, C3, C7 and C8). This group included wild-type and recombinant UPOs deriving from dark-spored agaric Basidiomycota (genera Candolleomyces, Psathyrellaceae), i.e., CeuMQ V, CabMQ II and rCab II.
  • One UPO forming the epoxide with yields around 25% but favoring hydroxylation at the C4 position (about 45%); furthermore, the enzyme formed a so far unidentified oxygenation product (C?) and a keto-product (4-oxo-trans-2-nonenoic acid, which was probably deriving from a second hydroxylation at the C4 position (second hydroxylation at the C4 position gives a gem-diol that is in equilibrium with the chemically favored oxo-FA, 14%). CglUPO was the only enzyme in this group.
  • UPOs favoring subterminal hydroxylation at C8 (26–36%), C7 (25–29%), and C6 (14%) and additionally, forming the epoxide (5–18%) and other hydroxylation products at C2/3 (about 20% and 3%, respectively). This group included UPOs from dark-spored agaric Basidiomycota, AaeUPO and CraUPO.
  • Enzymes yielding the epoxide (about 55%) to some extent as well as hydroxylating subterminally and at the C2 position (about 20%). Two wild-type UPOs, CeuMS III and CabMS II (both from the genus Candolleomyces).
  • A UPO hydroxylating at C2 (34%), C3 (18%) and favorably at the C8 (ω-1; 37%) position. One recombinant enzyme, rCab I (from the genus Candolleomyces).
  • Two enzymes favorably hydroxylating close to the carboxylic group at C2 (28–35%), C3 (25–28%), and C4 (4–5%) positions, and yielding the epoxide to minor extent; in addition, they catalyze a second hydroxylation yielding dihydroxylated products. This group includes UPOs from bright-spored agaric Basidiomycota (genus Marasmius), MroUPO and MweUPO.
Substrate conversion in relation to different peroxide dosages was evaluated with trans-2-nonenoic and trans-2-octenoic acid as model compounds (Supplementary Figures S4–S8). The results indicate complete enzyme inactivation during the course of the reactions (after the addition of 1 µmol H2O2 corresponding to 2 mM) for most UPOs, with the exception of AaeUPO, CraUPO and rCab I. In particular, inactivation was strong in the case of MroUPO, MweUPO, CabMS II and CeuMS III. Interestingly, other UPOs (CeuMQ V, rCab II, CabMQ II and CglUPO) were not inactivated when trans-2-nonenoic acid was used as a substrate, but they were inactivated with trans-2-octenoic acid. This suggests that the more compact a cis-form is, the more effectively/tightly it is bound in the active site, reducing catalase-like side reactions that can lead to the formation of unproductive intermediates (UPO-compound III) and aggressive oxygen radicals [7].
Analogous to the unsaturated FAs with the double bond at β,γ-position, the product spectra for the shorter acids (trans-2-octenoic acid and 2-heptenoic acid; Figure 13 and Figure 14, respectively; Figures S10 and S11) were evaluated using only three representative UPOs. Again, a similar picture emerged: CglUPO preferred epoxidation and hydroxylation at the C4 position, AaeUPO preferred epoxidation and hydroxylation at the C2 position, and MroUPO hydroxylated at different positions (C2, C3, C4) and, in the case of trans-2-octenoic acid, it promoted “overoxidation”, leading to the formation of keto products via the corresponding gem-diols.

4. Concluding Remarks

In the present study, one UPO stood out in terms of the oxidation of S/MFAs, namely CglUPO. However, it should be noted that the enzyme dosage was calculated based on the standard UPO assay with veratryl alcohol (VA) as substrate (i.e., the same amount of VA units/UVA was used in all tests). Since CglUPO does not perform well with this substrate, it is possible that comparatively too high enzyme amounts were applied; consequently, the performance of this enzyme may have been overestimated. Other methods for comparing and standardizing the amount of UPO that needs to be added to reaction mixtures are based on either protein concentration or heme absorbance, but they have their own shortcomings and should be tested in future comparative studies [6,7].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/applmicrobiol3030057/s1, Figure S1. MS-fragmentation patterns of products deriving from 3-nonenoic acid; Figure S2. (a) MS-Chromatograms of reaction setups including 3-heptenoic acid and three different UPOs (CglUPO, AaeUPO and MroUPO), (b) MS-fragmentation patterns of products deriving from 3-heptenoic acid; Figure S3. (a) MS-Chromatograms of reaction setups including 3-octenoic acid and three different UPOs (CglUPO, AaeUPO and MroUPO), (b) MS-fragmentation patterns of products deriving from 3-octenoic acid; Figure S4: Amount of residual trans-2-nonenoic acid (t-C9-2-en, left, green) or trans-2-octenoic acid (t-C8-2-en, right, green), as well as peak areas (relative for each product only) of the corresponding ω-1 (dark blue) and ω-2 (light blue) hydroxy products or further UV-absorbing products after different amount of H2O2 dosage (orange represents the sum of both products); Figure S5. Amount of residual trans-2-nonenoic acid (t-C9-2-en, left, green) or trans-2-octenoic acid (t-C8-2-en, right green), as well as peak areas (relative for each product only) of the corresponding ω-1 (dark blue) and ω-2(light lue) hydroxy products or further UV-absorbing products after different amount of H2O2 dosage (orange represents the sum of both products); Figure S6. Amount of residual trans-2-nonenoic acid (t-C9-2-en, left green) or trans-2-octenoic acid (t-C8-2-en, right, green), as well as peak areas (relative for each product only) of the corresponding ω-1 (dark blue) and ω-2 (light blue) hydroxy products or further UV-absorbing products after different amount of H2O2 dosage (orange represents the sum of both products); Figure S7. Amount of residual trans-2-nonenoic acid (t-C9-2-en, left, green) or trans-2-octenoic acid (t-C8-2-en, right, green), as well as peak areas (relative for each product only) of the corresponding tentatively identified products after different amount of H2O2 dosage (δ-hydroxy violet, γ-hydroxy pink, δ –keto green, sum of products, orange); Figure S8. Amount of residual trans-2-nonenoic acid (t-C9-2-en, left, green) or trans-2-octenoic acid (t-C8-2-en, right, green), as well as peak areas (relative for each product only) of the corresponding ω-1 product (dark blue) after different amount of H2O2 dosage; Figure S9. MS-fragmentation patterns of products deriving from 2-nonenoic acid; Figure S10. (a) MS-Chromatograms of reaction setups including 2-heptenoic acid and three different UPOs (CglUPO, AaeUPO and MroUPO), (b) MS-fragmentation patterns of products deriving from 2-heptenoic acid; Figure S11. (a)MS-Chromatograms of reaction setups including 2-octenoic acid and three different UPOs (CglUPO, AaeUPO and MroUPO), (b) MS-fragmentation patterns of products deriving from 2-octenoic acid; Table S1. additional, numeric data for Figure 6, rel. product yield with 3-nonenoic acid and various UPOs—product in %, apparently identified according to MS-ionization; Table S2. additional, numeric data for Figure 7, rel. product yield with 3-octenoic acid and various UPOs—product in %, apparently identified according to MS-ionization; Table S3. additional, numeric data for Figure 8, rel. product yield with 3-heptenoic and various UPOs—product in %, apparently identified according to MS-ionization; Table S4. additional, numeric data for Figure 10, rel. product yield with trans-2-nonenoic and various UPOs—product in %, apparently identified according to MS-ionization. * refers to a product that has been hydroxylated at an unknown position.; Table S5. additional, numeric data for Figure 13, rel. product yield with trans-2-octenoic acid and various UPOs—product in %, apparently identified according to MS-ionization. * refers to a product that has been hydroxylated at an unknown position; Table S6. additional, numeric data for Figure 14, rel. product yield with 2-heptenoic acid acid and various UPOs—product in %, apparently identified according to MS-ionization. * refers to a product that has been hydroxylated at an unknown position.

Author Contributions

Conceptualization, A.K. and R.U.; formal analysis, M.K. and F.S.; methodology, R.U. and M.A.; writing—original draft preparation, A.K. and M.H.; writing—review and editing, A.K., R.U., M.H. and M.A.; funding acquisition, M.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by BioBased Industries Joint Undertaking under the European Union’s Horizon 2020 Research and Innovation Programme, grant number 792063 (SusBind project; https://susbind.eu, accessed on 1 January 2023).

Data Availability Statement

All additional data is available in the supplementary section.

Conflicts of Interest

The authors declare no conflict of interest.

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Applmicrobiol 03 00057 g001 550
Figure 1. Conversion of different terminally unsaturated FAs by a selection of UPO preparations from different fungi. Cgl—Chaetomium globosum, Cra—Coprinellus radians, Aae—Agrocybe aegerita, Ceu—Candolleomyces eurysporus, Cab—Candolleomyces aberdarensis, Mwe—Marasmius wettsteinii, Mro—Marasmius rotula.
Figure 1. Conversion of different terminally unsaturated FAs by a selection of UPO preparations from different fungi. Cgl—Chaetomium globosum, Cra—Coprinellus radians, Aae—Agrocybe aegerita, Ceu—Candolleomyces eurysporus, Cab—Candolleomyces aberdarensis, Mwe—Marasmius wettsteinii, Mro—Marasmius rotula.
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Figure 2. Formula scheme illustrating the formation of hydroxylation products in the reaction of UPOs with terminally unsaturated S/MCFAs using 8-nonenoic acid as an example.
Figure 2. Formula scheme illustrating the formation of hydroxylation products in the reaction of UPOs with terminally unsaturated S/MCFAs using 8-nonenoic acid as an example.
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Figure 3. Conversion of FAs with the double bond between Cβ and Cγ (C3 and C4) by a selection of UPOs from different fungi (for abbreviations, see the legend of Figure 1).
Figure 3. Conversion of FAs with the double bond between Cβ and Cγ (C3 and C4) by a selection of UPOs from different fungi (for abbreviations, see the legend of Figure 1).
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Figure 5. Formula scheme illustrating the formation of hydroxylation products in the reaction of UPOs with unsaturated FAs bearing the double bond in the β, γ-position, using 3-nonenoic acid as an example.
Figure 5. Formula scheme illustrating the formation of hydroxylation products in the reaction of UPOs with unsaturated FAs bearing the double bond in the β, γ-position, using 3-nonenoic acid as an example.
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Figure 6. Relative product yields (%, visualised by red bars) and product distribution with 3-nonenoic acid and several UPOs.
Figure 6. Relative product yields (%, visualised by red bars) and product distribution with 3-nonenoic acid and several UPOs.
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Figure 7. Relative product yields (%, visualised by red bars) and product distribution using 3-octenoic acid as substrate for several UPOs.
Figure 7. Relative product yields (%, visualised by red bars) and product distribution using 3-octenoic acid as substrate for several UPOs.
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Figure 8. Relative product yields (%, visualised by red bars) and product distribution with 3-heptenoic acid and several UPOs.
Figure 8. Relative product yields (%, visualised by red bars) and product distribution with 3-heptenoic acid and several UPOs.
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Figure 9. Conversion of S/MCFAs with the double bond at α,β-position by different UPOs (for abbreviations see legend of Figure 1).
Figure 9. Conversion of S/MCFAs with the double bond at α,β-position by different UPOs (for abbreviations see legend of Figure 1).
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Figure 10. Relative product yields (%, visualised by red bars) in the reaction of different UPOs with trans-2-nonenoic acid. * refers to a product that has been hydroxylated at an unknown position.
Figure 10. Relative product yields (%, visualised by red bars) in the reaction of different UPOs with trans-2-nonenoic acid. * refers to a product that has been hydroxylated at an unknown position.
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Figure 11. LC-MS elution profiles of reactions with various UPOs and trans-2-nonenoic acid as substrate. Peak markings correspond to the site of the hydroxylation in the aliphatic chain (compare Figure 10 and Figure 12, Figure S9).
Figure 11. LC-MS elution profiles of reactions with various UPOs and trans-2-nonenoic acid as substrate. Peak markings correspond to the site of the hydroxylation in the aliphatic chain (compare Figure 10 and Figure 12, Figure S9).
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Figure 12. Formula scheme illustrating the formation of hydroxylation products in the reaction of UPOs with unsaturated FAs bearing the double bond at the α,β-position, using 2-nonenoic acid as an example.
Figure 12. Formula scheme illustrating the formation of hydroxylation products in the reaction of UPOs with unsaturated FAs bearing the double bond at the α,β-position, using 2-nonenoic acid as an example.
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Figure 13. Relative product yields (%, visualised by red bars) in the reaction of different UPOs with trans-2-octenoic acid. * refers to a product that has been hydroxylated at an unknown position.
Figure 13. Relative product yields (%, visualised by red bars) in the reaction of different UPOs with trans-2-octenoic acid. * refers to a product that has been hydroxylated at an unknown position.
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Figure 14. Relative product yields (%, visualised by red bars) in the reaction of different UPOs with 2-heptenoic acid.
Figure 14. Relative product yields (%, visualised by red bars) in the reaction of different UPOs with 2-heptenoic acid.
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Table
Table 1. Unspecific peroxygenases (UPOs, EC 1.11.2.1) used in this study.
Table 1. Unspecific peroxygenases (UPOs, EC 1.11.2.1) used in this study.
Enzyme (UPO Abbreviation)Fungal SpeciesSpecific Activity (UVA/mg) *Isoelectric Point (pI)Reference
CglUPOChaetomium globosum3.94.3–4.7 (5.59 *)[21]
CraUPOCoprinellus radians72.03.8–4.2[22]
AaeUPOCyclocybe (Agrocybe) aegerita82.04.9–6.1[5]
CeuMQ VCandolleomyces eurysporus12.54.25[23]
CeuMS IIIC. eurysporus11.07.6herein
CabMS IIC. aberdarensis72.05.1herein
CabMQ IIC. aberdarensis172.04.1[23]
rCab IIC. aberdarensis (heterologously expressed in Pichia pastoris)21.05.1[24]
rCab IC. aberdarensis (heterologously expressed in in Pichia pastoris)21.0n.d.[24]
MweUPOMarasmius wettsteinii50.05.1 **[2,25]
MroUPOM. rotula25.45.0–5.3[26]
* theoretical pI [27], ** UVA—activity units related to the oxidation of 1 µmol veratryl alcohol into vertraldehyde per minute [5].
Table
Table 2. List of substrates (unsaturated S/MCFAs) and acetonitrile concentrations applied for their isocratic separation on a C18-column (for more details, see HPLC methods below); t—trans, c—cis, m—not further defined mixture of conformational isomers.
Table 2. List of substrates (unsaturated S/MCFAs) and acetonitrile concentrations applied for their isocratic separation on a C18-column (for more details, see HPLC methods below); t—trans, c—cis, m—not further defined mixture of conformational isomers.
Number of Carbon AtomsSubstrates (Unsaturated Short- and Medium-Chain Fatty Acids)Abbreviation and Double Bond Position (according to IUPAC Nomenclature)Acetonitrile Concentration
9trans-2-nonenoic acidt-C9:1(2)55%
3-nonenoic acidm-C9:1(3)
8-nonenoic acidC9:1(8)
8trans-2-octenoic acidt-C8:1(2)50%
3-octenoic acidm-C8:1(3)
7-octenoic acidC8:1(7)
72-heptenoic acidm-C7:1(2)40%
3-heptenoic acidm-C7:1(3)
6-heptenoic acidC7:1(6)
6cis-2-hexenoic acidc-C6:1(2)30%
trans-2-hexenoic acidt-C6:1(2)
trans-3-hexenoic acidt-C6:1(3)
5-hexenoic acidC6:1(5)
5trans-2-pentenoic acidt-C5:1(2)15%
3-pentenoic acidm-C5:1(3)
4-pentenoic acidC5:1(4)
43-butenoic acidC4:1(3)7%
(vinylacetic acid)
32-propenoic acidC3:1(2)5%
(acrylic acid)
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Karich, A.; Salzsieder, F.; Kluge, M.; Alcalde, M.; Ullrich, R.; Hofrichter, M. Conversion of Unsaturated Short- to Medium-Chain Fatty Acids by Unspecific Peroxygenases (UPOs). Appl. Microbiol. 2023, 3, 826-840. https://doi.org/10.3390/applmicrobiol3030057

AMA Style

Karich A, Salzsieder F, Kluge M, Alcalde M, Ullrich R, Hofrichter M. Conversion of Unsaturated Short- to Medium-Chain Fatty Acids by Unspecific Peroxygenases (UPOs). Applied Microbiology. 2023; 3(3):826-840. https://doi.org/10.3390/applmicrobiol3030057

Chicago/Turabian Style

Karich, Alexander, Fabian Salzsieder, Martin Kluge, Miguel Alcalde, René Ullrich, and Martin Hofrichter. 2023. "Conversion of Unsaturated Short- to Medium-Chain Fatty Acids by Unspecific Peroxygenases (UPOs)" Applied Microbiology 3, no. 3: 826-840. https://doi.org/10.3390/applmicrobiol3030057

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