Expression, Purification, and Characterization of a Well-Adapted Tyrosinase from Peatlands Identified by Partial Community Analysis

In peatlands, bacterial tyrosinases (TYRs) are proposed to act as key regulators of carbon storage by removing phenolic compounds, which inhibit the degradation of organic carbon. Historically, TYR activity has been blocked by anoxia resulting from persistent waterlogging; however, recent events of prolonged summer drought have boosted TYR activity and, consequently, the release of carbon stored in the form of organic compounds from peatlands. Since 30% of the global soil carbon stock is stored in peatlands, a profound understanding of the production and activity of TYRs is essential to assess the impact of carbon dioxide emitted from peatlands on climate change. TYR partial sequences identified by degenerated primers suggest a versatile TYR enzyme community naturally present in peatlands, which is produced by a phylogenetically diverse spectrum of bacteria, including Proteobacteria and Actinobacteria. One full-length sequence of an extracellular TYR (SzTYR) identified from a soda-rich inland salt marsh has been heterologously expressed and purified. SzTYR exhibits a molecular mass of 30 891.8 Da and shows a pH optimum of 9.0. Spectroscopic studies and kinetic investigations characterized SzTYR as a tyrosinase and proved its activity toward monophenols (coumaric acid), diphenols (caffeic acid, protocatechuic acid), and triphenols (gallic acid) naturally present in peatlands.


Supporting data
Development of a metagenomic DNA extraction protocol and quality assessment of the DNA extract. Prior to cell lysis and the extraction of DNA, the samples were washed with a humic substance removal solution adjusted to pH 9.0 (see Materials and methods) to efficiently remove fulvic acids and low molecular weight humic acids. Polyvinylpyrrolidone (PVP), which forms hydrogen bonds with phenolic compounds 1 , was added to the humic substance removal solution, resulting in an increased efficiency of phenolic compound removal (compared to humic substance removal solution without PVP). The washed peat samples were subjected to different forms of cetyltrimethylammonium bromide (CTAB) assisted cell lysis, namely cell lysis via Ultra-Turrax, glass beads, and repetitive freeze-thaw cycles ( Figure S4). Freezing samples in liquid nitrogen followed by incubation at 65 °C proved most efficient in terms of quality and quantity of the metagenomic DNA extract ( Figure S4). To further minimize the co-extraction of humic substances a cell lysis buffer adjusted to pH 7.9 was used. At pH 7.9 high molecular weight humic substances (humins and high molecular weight humic acids) remain insoluble while low molecular weight humic substances (fulvic acids and low-molecular-weight humic acids) have been removed previously by the humic substance removal solution (pH 9.0) 2 .
The purification of the crude metagenomic DNA extract was performed by phenol:chloroform:isoamyl alcohol extraction. Phenol, which is partially soluble in the aqueous phase and inhibits downstream applicability of the extracted metagenomic DNA (e.g. for PCR), was removed by extracting the aqueous layer with chloroform prior to the precipitation of DNA.
Co-extraction of humic substances is a major concern in the extraction of DNA from soil s showing high organic matter content since they impede downstream applications 3,4 . Thus, the metagenomic DNA extracts were assessed in terms of PCR inhibition potential using Taqpolymerase and Q5 High-Fidelity DNA polymerase (see supporting Materials and methods).
The DNA extracts showed limited DNA inhibition potential with Q5 High -Fidelity DNA polymerase tolerating higher DNA extract concentrations (corresponding to 150 ng metagenomic DNA per 10 µl PCR reaction) compared to Taq-polymerase (corresponding to 30 ng metagenomic DNA per 10 µl PCR reaction). Thus, all further PCR experiments were performed using Q5 High-Fidelity DNA polymerase.
Thermofluor assay of SzTYR. To test the thermal stability of SzTYR a thermofluor assay was performed. Since its sampling site exhibits a pH value of 9.0 -9.5, SzTYR (which shows a pH Michael-1,6-addition, redox exchange, and intramolecular cyclization 7 . Thereby, the concentration of phenolic compounds is reduced, which potentially boosts the activity of soil organic matter degrading enzymes. To experimentally prove the co-polymerization of phenolic compounds naturally present in peatlands 8,9 a solution containing coumaric acid, caffeic acid, protocatechuic acid, and gallic acid (in the following referred to as "phenol-mix"; see supporting Materials and methods, Figure S7) was incubated with SzTYR (in 50 mM TRIS-HCl pH 8.5).
After 72 hours a dark precipitate indicated the formation of insoluble, high molecular weight polymers ( Figure S12), which were separated from the supernatant by centrifugation. The phenolic content of the supernatant (phenol-mix after incubation with SzTYR without insoluble, high molecular weight polymers) was determined using the folin-ciocalteu method 10 and revealed that after incubation with SzTYR for 72 hours, the phenol content has been reduced by 55 % (compared to the phenol content of the phenol-mix prior to the addition of SzTYR).
Thus, the results prove the formation of high-molecular weight polymers from a complex mixture of phenolic compounds naturally present in peatlands.  (Table S1). Q5 High-Fidelity DNA polymerase (NEB, Ipswich, USA) was used according to the PCR setup recommended by the supplier with 150 ng metagenomic DNA and 25 % (v/v) high GC enhancer (NEB, Ipswich, USA) added to the PCR reaction. Amplicons were cloned into the pENTRY-IBA51 vector using SapI as the restriction endonuclease. After cloning into E. coli TOP 10 cells (Thermo Fisher, Waltham, USA) single colonies were selected, plasmids were isolated and analyzed by Sanger sequencing.

Investigation of the co-polymerization of various phenolic substrates.
To test the copolymerization of various phenolic compounds a solution containing 2 mM coumaric acid, 2 mM caffeic acid, 2 mM protocatechuic acid, and 2 mM gallic acid was incubated with 20 µg SzTYR in 50 mM TRIS -HCl (pH 8.5; to reduce autooxidation 15 ) in a total volume of 200 µl.
The mixture was incubated for 72 hours at room temperature.
For the determination of the total phenolic content 100 µg SzTYR were added to 1 ml of a solution containing 2 mM coumaric acid, 2 mM caffeic acid, 2 mM protocatechuic acid, and 2 mM gallic acid ( Figure S7) in 50 mM TRIS -HCl (pH 8.5). After incubation at room temperature for 72 hours, the mixture was centrifuged at 20,000 g for 10 minutes. The supernatant was separated from the pellet. To determine the phenolic content of the supernatant, 100 µl of the supernatant were mixed with 200 µl 10 % folin-ciocalteu reagent (Merck, Darmstadt, Germany) and 800 µl of an aqueous 700 mM Na2CO3 solution. The mixture was incubated at room temperature for 2 hours and the absorption was then measured on a Shimadzu UV-1800 spectrophotometer at 765 nm 10 . As a reference, the phenolic content of a solution containing 2 mM coumaric acid, 2 mM caffeic acid, 2 mM protocatechuic acid, and 2 mM gallic acid (without SzTYR added) in 50 mM TRIS -HCl (pH 8.5) was determined according to the protocol described above. Photometric measurements were performed in triplicates.   Table S4. Partial TYR amino acid sequences. Amino acid sequences identified from peat samples covering the sequence between the two degenerated primers (Table S1). HisB1 and HisB2 are highlighted in brown, the 1 st activity controller (HisB1+1) is highlighted in cyan and the 2 nd activity controller (HisB2+1) is highlighted in magenta.   -ATGTCGCGCATTACGCGTCGTCATGCCCTGGGTGCAGCCG  CAGCAACCGCTCTGACGGGTTTGGCGCTTGCGGGCTTAGCG  CGTACTGCGGGTGCGACTTCAGCCCCACGTGCGACAGGCCA  TCAGCCTCCGGAAGGCCATGCTGGGCATGATGGACCGCAGC  CGTTTAGCGAAACCTTTCAGGGTCGCCGCATTGAAGGCGCTC  CCAGTCACGCGGATGGCCACCATGGAGGCTATGCGGTTCGC  ATCGATGGGGAGGAACTGCACGTGATGCGCAATGCCGACTC  TACGTGGGTGTCCGTCATCAACCACTACGAAACCTTCACCAC  CCCACGGGCAGTTGCGCGCGCTGCCGTGATTGAGCTGCAAG  GTGCACGTCTGGTACCGCTCGCCTAA-3'  Table S6. Full-length sequences of MelC2 (SzTYR), MelC1 (caddie protein) and MelC1 codon-optimized (caddie protein). TGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAA  TACTCTGTTCCGCATGGAACGGGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGG  CCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTG  AGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG  CAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGAT  GACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCT  GCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAG  CGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGA  AAGCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGG  GGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGG  TGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGC  GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCG  ACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACG  GCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCMCAAGCAGCGGAGCATGT  GGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATWTACCGGAAAGCAT  CMGAGATGGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTC  GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCA  GCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACCGCCGGGGTCAACTCGGAGGA  AGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACA  ATGGCCGGTACAATGAGCTGCGATACCGTGAGGTGGAGCGAATCTCAAAAAGCCGGTC  TCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCA  GATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCA  CGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCGTAAGGGAGGGAG-3' 16S RNA from Streptomyces sp. ZL-24 (GenBank Accession number: MH700447.1) GGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAAT  ACTCTGTTCCGCATGGAACGGGGTTGAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGC  CTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGA  GAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC  AGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATG  ACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGC  AGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCG  TTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGATGTGAAA  GCCCGGGGCTTAACCCCGGGTCTGCATTCGATACGGGCTAGCTAGAGTGTGGTAGGGG  AGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGG  CGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAAC  AGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACAT  TCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCG  CAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCT  TAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGA  GATGGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGT  CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCAT  GCCTTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGT  GGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGG  CCGGTACAATGAGCTGCGATGCCGTGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAG  TTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATC  AGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAA  AGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAG-3'  Table S7  The caddie protein and the TYR both start with ATG (start codon) and end with TAG (stop codon). The sequence has been identified within the scope of this work. S23 Figure S5. Phylogenetic tree of the 19 identified nucleotide sequences (Table S2). The tree was rooted to the characterized TYR sequence from Agaricus bisporus (AbPPO3, UniProt Identifier: C7FF04) which represents a valuable outgroup due to its phylogenetic localization in a different kingdom (fungi). Sequences of TYR enzymes identified by the BLAST search (identity level > 75 %) and their respective host organisms were included (Table S3) to offer additional information about the localization of the identified partial TYR sequences in the phylogenetic spectrum (Table S2,   Melting temperatures (Tm) are reported in Table S9. Measurements were performed in triplicates for each pH condition. For clarity, averaged fluorescence curves are presented.