dataesr/scientific-paragraphs-categorization huggingface dataset:
The PCR amplification program consisted of denaturation at 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The relative gene expression data were normalized against an internal reference gene, cyclophilin (CYP)  [23]. The relative expression levels were calculated by the 2 ÀDDCt method. The primers were designed using Primer3 (http://frodo. wi. mit. edu/primer3/). The primers used for RT-qPCR in this study are listed in Table  S35.
Acknowledgments This work was supported by the  CAMS Innovation Fund for Medical Sciences (CIFMS)  (Grant No. 2016-I2M-3-016 ) and the  National TCM Standardization Project  (Grant No. ZY13ZH-C-JL-24 ). Francis Martin  is funded by the  French National Research Agency  through the  Laboratory of Excellence Advanced Research on the Biology of Tree and Forest Ecosystems  (Grant No. ANR-11-LABX 0002 01 ) and  Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University. We thank the  Beijing Genomics Institute (BGI)  for the project management that generated the initial sequence and assembly. We also thank  Yongchao Niu  and  Jinmin Lian  ( Biozeron Co. Ltd, Shenzhen, China ) for the support in data analysis.
Data availability The raw data for the genome and transcriptome of the Chinese strain CGMCC5. 78 W. cocos have been deposited in the Genome Sequence Archive  [63]  at the National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation (GSA: CRA003688), and are publicly accessible at http://bigd. big. ac. cn/gsa.
Full XRF spectra were additionally collected in several points of interest with a 30 s count time (electronic supplementary material, figure  S1 ). Distributions of Ca, Mn, Fe, Ni, Cu, Zn, Ga, As, Br (Kα 1 ) and Pb (Lβ 1 ) XRF lines were mapped using an incident beam energy of 13. 5 keV. Note that fitting of the full XRF spectra using the PyMCA data-analysis software  [43]  indicates that (i) although largely dominated by As, the As distribution also includes signal arising from Pb (Lα 1 line, which falls in the same energy domain as the As Kα 1 line), (ii) the Pb distribution (Lβ 1 line) is actually dominated by pile-up signal from iron, and (iii) no Br was detected (electronic supplementary material, figure  S1 ). Distributions of Al, Si, P, S and Cl Kα 1 XRF lines were mapped using an incident royalsocietypublishing.
Acknowledgements. Portions of this research were carried out at the  Stanford Synchrotron Radiation Lightsource (CA, USA) , a national user facility operated by  Stanford University  on behalf of the  U. S. Department of Energy, Office of Basic Energy Sciences , and we thank the user support staff at SSRL. P. G. thanks  G. D. Edgecombe  for discussion. U. B. and L. B. acknowledge support from the  France -Stanford Center for Interdisciplinary Studies Program , and P. G. from the  Institute of Earth Sciences, University of Lausanne. We thank two anonymous referees for their reviews of the manuscript.
Glucose-methanol-choline (GMC) oxidoreductases are a large and diverse family of flavin-binding enzymes found in all kingdoms of life. Recently, a new related family of proteins has been discovered in algae named fatty acid photodecarboxylases (FAPs). These enzymes use the energy of light to convert fatty acids to the corresponding C n-1 alkanes or alkenes, and hold great potential for biotechnological application. In this work, we aimed at uncovering the natural diversity of FAPs and their relations with other GMC oxidoreductases. We reviewed the available GMC structures, assembled a large dataset of GMC sequences, and found that one active site amino acid, a histidine, is extremely well conserved among the GMC proteins but not among FAPs, where it is replaced with alanine. Using this criterion, we found several new potential FAP genes, both in genomic and metagenomic databases, and showed that related bacterial, archaeal and fungal genes are unlikely to be FAPs.
We also identified several uncharacterized clusters of GMC-like proteins as well as subfamilies of proteins that lack the conserved histidine but are not FAPs. Finally, the analysis of the collected dataset of potential photodecarboxylase sequences revealed the key active site residues that are strictly conserved, whereas other residues in the vicinity of the flavin adenine dinucleotide (FAD) cofactor and in the fatty acid-binding pocket are more variable. The identified variants may have different FAP activity and selectivity and consequently may prove useful for new biotechnological applications, thereby fostering the transition from a fossil carbon-based economy to a bio-economy by enabling the sustainable production of hydrocarbon fuels.
HAOx Hydroxy fatty acid oxidase Oxidation of long-chain ω-hydroxy fatty acids to ω-oxo fatty acids was ascribed to ACE/HTH  [76] , a 594 amino acid-long GMC family protein not related to other HAOxs. Arabidopsis thaliana  [76]. Perhaps the most interesting GMC family members at the moment are fatty acid photodecarboxylases (FAP), a recently discovered class of enzymes initially identified in the green algae Chlorella variabilis  [77] , which convert long chain fatty acids to the corresponding C n-1 alkanes/alkenes in the presence of blue light. FAP activity was also confirmed for proteins from Chlamydomonas reinhardtii  [77] , Galdieria sulfuraria, Chondrus crispus, Nannochloropsis gaditana and Ectocarpus siliculosus  [78]. Overall, FAP genes were identified in more than 30 algal species as well as in metagenomic datasets  [78].
These studies show the potential of FAPs to be used in industrial biotechnological processes. In this work, therefore, we set out to assess the diversity of GMC proteins and identify putative FAPs in genomic and metagenomic databases, with the goal of understanding their natural diversity, which could guide the discovery and design of new variants with potentially altered activity and specificity. Consequently, we used the available experimental data to develop criteria that would distinguish FAPs from other GMC family proteins, and applied these criteria to ~150,000 publicly available GMC sequences. We identified several previously unreported putative FAP sequences, and analyzed the variability of the FAP active site and substrate-binding pocket amino acids. The results obtained may be useful for the design of FAPs with improved or even new properties for biotechnological applications, and hence could foster the transition from a carbon-based economy to the sustainable production of hydrocarbon fuels in the framework of bioeconomy.
Fatty Acid Photodecarboxylases (FAP) Domain Annotation and Structure In order to identify putative FAP genes in genomic and metagenomic databases, we started with reviewing the domain annotation of the best characterized FAP protein, Chlorella variabilis FAP (CvFAP), and its crystallographic structure (Figure  1 ). The 654 amino acid-long protein is annotated in the Pfam protein families database  [87]  as having two domains, GMC_oxred_N (PF00732, residues 84 to 383) and GMC_oxred_C (PF05199, 492 to 632, Figure  1a ), corresponding to N-and C-terminal parts of glucose-methanol-choline oxidoreductases, respectively. Whereas definitions of protein domains often postulate a compact structure that folds relatively independently of the rest of the protein  [88] [89] [90] , this is not the case for GMC_oxred_N and GMC_oxred_C, as the respective parts of the protein are interspersed and clearly cannot function independently of each other (Figure  1b ).
Common Features of Known Glucose-Methanol-Choline (GMC) Proteins Currently, the Pfam database  [87]  lists experimentally determined structures of 26 different GMC proteins, including CvFAP, available in the protein data bank (PDB). The pyranose 2-oxidase genes, such as that of Trametes multicolor (UniProt ID Q7ZA32, PDB ID 1TT0) are recognized in Pfam as having the GMC_oxred_C domain but not the GMC_oxred_N domain, despite encoding proteins highly similar to other GMC family members. Crystallographic structures are available for all but four currently known enzyme classes (choline dehydrogenases, fructose dehydrogenases, compound K oxidases and hydroxy fatty acid oxidases). For some of the proteins, more than one structure has been determined, and for some of the enzyme classes, more than one protein has been characterized structurally.
Ligand-Binding Pockets of GMC Family Proteins Understanding the conserved and variable features of the GMC proteins is required for delineating the differences between the FAPs and other family members. The catalytic properties of an enzyme are defined by the active site amino acids and their geometry. Active site arrangements in representative crystallographic structures of GMC proteins are shown in Figure  3. Respective PDB IDs are 1COY, 1NAA, 1TT0, 3GDN, 3Q9T, 3QVP, 4H7U, 4HA6, 4MJW, 4UDP, 4YNU, 4Z24, 5HSA, 5OC1 and 6A2U (Table  2 ). Whereas the obtained set of sequences did not contain duplicates, some of the sequences were highly similar. Analysis of this number of sequences is computationally prohibitive. Consequently, we clustered the sequences with the idea that the clusters of interest can then be re-analyzed in more detail.
Clustering at the level of 40% sequence identity produced 5660 clusters. Representative sequences (centroid sequences) from each cluster were used in downstream analyses. Respective PDB IDs are 1COY, 1NAA, 1TT0, 3GDN, 3Q9T, 3QVP, 4H7U, 4HA6, 4MJW, 4UDP, 4YNU, 4Z24, 5HSA, 5OC1 and 6A2U (Table  2 ). The structures reveal several cases where the FAD molecule is covalently bound to the protein or is autocatalytically modified. In pyranose dehydrogenase from Agaricus maleagris, FAD is modified by a covalent mono-or di-atomic species at the C(4a) position (Figure  3f, [96] ). In formate oxidase of Aspergillus oryzae, FAD is formylated at the C8α position (Figure  3n ,  [104] ); the modification is autocatalytic and enhances the enzyme activity  [107]. Finally, in some GMC family members FAD is covalently attached to the protein via C8α-His bonds (Figure  3a ,i,m), similar to VAO-type proteins  [31].
Phylogenetic Analysis of GMC Proteins Having established the main features of FAPs and other GMC oxidoreductases, we performed the phylogenetic analysis of publicly available sequences. To obtain the full coverage of the GMC family, we assembled several sets of sequences (Table  3 ), which were then clustered and analyzed. First, we retrieved the representative GMC_oxred_N and GMC_oxred_C sequences (seed sequences) from Pfam  [87]. These sequences were used to perform the PSI-BLAST search against the non-redundant set of sequences in the NCBI database. In total, 147,949 GMC_oxred_N-containing sequences and 150,593 GMC_oxred_C-containing sequences have been identified. Of these, 135,174 sequences contained both domains, and 163,368 sequences contained at least one of the two domains GMC_oxred_N or GMC_oxred_C.
The latter set is the most extensive one and should contain all potential proteins of interest. We note that the Pfam  [87]  and InterPro  [108]  databases contained around 30,000 and 100,000 GMC sequences, respectively, at the moment of the writing of this article. Whereas the obtained set of sequences did not contain duplicates, some of the sequences were highly similar. Analysis of this number of sequences is computationally prohibitive. Consequently, we clustered the sequences with the idea that the clusters of interest can then be re-analyzed in more detail. Clustering at the level of 40% sequence identity produced 5660 clusters. Representative sequences (centroid sequences) from each cluster were used in downstream analyses. Besides the representative sequences of those found in the NCBI database (labeled B1 in the Table  3 ), we also wanted to explicitly include several other sets of sequences in the analysis (Table  3 ).
Finally, the set B8 included the sequences of GMC proteins whose crystallographic structures have been determined, as listed in the Pfam database  [87]. All sequences were put into the joint dataset B_unique; if duplicate sequences were retrieved from different sources, the corresponding records were merged. The information about the sequences in the datasets A6 and B_unique may be found in Supplementary Datasets 1, 2 and 3. Next, we prepared a phylogenetic tree showing the relations between the sequences from B_unique (Figure  4 ; the data may be found in Supplementary Datasets  4  and 5 ). The tree reveals a number of distinct sequence clusters. Some of them contain sequences that have been characterized experimentally, whereas others do not. Whereas GOx and GDH group together, other enzymes acting on the same substrates -POx and PDH, COx and CHDH -belong to different branches.
Interestingly, the FAPs are not the only proteins lacking the conserved histidine. Around 20 genes in the dataset B_unique encode the proteins where the histidine is replaced with asparagine; examples are RKF24846. 1 from actinomycete Micromonospora globbae, XP_009009580. 1 from the leech Helobdella robusta, and XP_011557351. 1 from the moth Plutella xylostella. There are also around 20 proteins with His→Asp replacement, such as WP_090799066. 1 from the Gram-positive bacterium Asanoa ishikariensis, XP_007396379. 1 from the fungus Phanerochaete carnosa, OXA50702. 1 from the springtail Folsomia candida and RWS11943. 1 from the bug Dinothrombium tinctorium. Around 10 proteins possess the His→Gln replacement, such as XP_026615050. 1 from the fungus Aspergillus thermomutatus, XP_003436606. 1 from the mosquito Anopheles gambiae or XP_011424488.
Phylogenetic Analysis of Putative FAP Proteins As a next step, we analyzed the FAP branch of the overall phylogenetic tree. We built a smaller phylogenetic tree (Figure  5 ; the data may be found in Supplementary Datasets 6 and 7), now containing only the putative FAP proteins and three outgroup sequences (choline oxidase, pyridoxine 4-oxidase and 5-(hydroxymethyl)furfural oxidase). This tree reveals several sequence clusters (1-9), which roughly correspond to evolutionary relations between the host organisms  [78].
Phylogenetic Analysis of Putative FAP Proteins As a next step, we analyzed the FAP branch of the overall phylogenetic tree. We built a smaller phylogenetic tree (Figure  5 ; the data may be found in Supplementary Datasets 6 and 7), now containing only the putative FAP proteins and three outgroup sequences (choline oxidase, pyridoxine 4-oxidase and 5-(hydroxymethyl)furfural oxidase). This tree reveals several sequence clusters (1-9), which roughly correspond to evolutionary relations between the host organisms  [78]. The most prominent cluster (#1) contains several genomic sequences and is centered around the proteins from Chlorella variabilis and Chlamydomonas reinhardtii. Metagenomic sequences 1, 3 and 7 from the dataset B7 are progressively less similar to the sequences from cluster #1. The cluster #2 contains proteins from Nannochloropsis gaditana, Nannochloropsis salina and Ectocarpus siliculosus, but not, surprisingly, the other heterokonts Aureococcus anophagefferens (outside of recognizable clusters) or Bacillariophyta such as Fragilaria radians, Fragilariopsis cylindrus, Phaeodactylum tricornutum or Pseudo-nitzschia multistriata (cluster #7).
Cluster #3 contains sequences from Emiliania huxleyi (Ehu1, XP_005785285. 1) and Chrysochromulina tobinii, whereas second putative FAP from Emiliania huxleyi (Ehu2, XP_005757666. 1) clusters with the sequence 7 from the dataset B7 in between the clusters #1 and #2. Interestingly, clusters 4-6 feature multiple metagenomic sequences identified by Moulin et al. [78]  but lack any currently available genomic sequences. Two metagenomic sequences from the cluster 6, 52837172 and 97429747, are annotated as belonging to Dinophyta Neoceratium fusus and Heterocapsa, respectively, by Moulin et al. [78]. Cluster #7 contains Bacillariophyta proteins as well as a number of metagenomic sequences. Cluster #8 contains Rhodophyta proteins. Finally, cluster #9 contains a number of metagenomic sequences and a single genomic one, that from the recently sequenced tiny marine green alga, Chlorophyta Chloropicon primus  [114].
Natural Diversity of FAP Active Sites Having assembled a dataset of putative FAP sequences, we were interested to analyze the diversity of the active site and other residues in FAPs. We calculated the frequencies of observing a particular amino acid at a particular position in the multiple sequence alignment of FAP sequences (Supplementary Datasets 6 and 7). Some genomic sequences, such as Phaeodactylum tricornutum sequence with the GenBank ID XP_002178042. 1, and most, if not all, metagenomic sequences are partial. Still, these sequences provide important information and were included in the calculations. The results for the active site and fatty acid-binding pocket are shown in Figures  6 7 8. The multiple sequence alignment is provided as Supplementary Dataset 8. Frequencies of different amino acids are provided as Supplementary Dataset 9.
Sequence Analysis The sets of sequences mentioned and analyzed in this work are listed in Table  3. The seed Pfam sequences for GMC_oxred_N (PF00732) and GMC_oxred_C (PF05199) domains have been accessed on 12. 04. 2020 (Pfam version 32. 0). The seed sequences were used to perform PSI-BLAST searches  [113]  against the non-redundant set of sequences in the NCBI database (all non-redundant GenBank coding sequence translations, PDB, SwissProt, PIR and PRF excluding environmental samples from whole genome sequencing projects), also on 12. 04. 2020. The cutoff E-value was chosen at 0. 001 and 0. 003 for GMC_oxred_N and GMC_oxred_C, respectively. The results of the PSI-BLAST searches were combined into a single set of sequences. Full-length sequences were clustered using UCLUST  [117]  at the 40% identity level.
Multiple sequence alignment of the sequences in the dataset comprising the centroid sequences from clustering as well as other sequences of interest has been performed using the MAFFT FFT-NS-2 algorithm  [118]. Multiple sequence alignment of the putative FAP sequences has been performed using the MAFFT L-INS-i algorithm  [119]. For both tasks, MAFFT v. 7. 402  [120]  was used. In both alignments, columns containing more than 50% gaps were removed using trimAl  [121]. The phylogenetic tree of representative GMC sequences was calculated using FastTree2  [122]. JTT+CAT amino acid substitution model was used, with 20 rate categories, 10 rounds Variation of the amino acids surrounding the isoalloxazine ring of FAD is shown in Figure  6. Besides the absolutely conserved Ala576, which discriminates FAPs from related GMC proteins, two other proximal amino acids are strictly conserved as well and may be important for catalysis: Asn575 and Gln620.
Multiple sequence alignment of the sequences in the dataset comprising the centroid sequences from clustering as well as other sequences of interest has been performed using the MAFFT FFT-NS-2 algorithm  [118]. Multiple sequence alignment of the putative FAP sequences has been performed using the MAFFT L-INS-i algorithm  [119]. For both tasks, MAFFT v. 7. 402  [120]  was used. In both alignments, columns containing more than 50% gaps were removed using trimAl  [121]. The phylogenetic tree of representative GMC sequences was calculated using FastTree2  [122]. JTT+CAT amino acid substitution model was used, with 20 rate categories, 10 rounds of nearest-neighbor interchanges, 2 rounds of optimization, 10 rounds of subtree-prune-regraft moves and the maximum length of a move of 10. The phylogenetic tree of putative FAP sequences was calculated using RAxML v.
Conclusions GMC proteins are a large family of FAD-binding enzymes with great biotechnological potential. While some of them are characterized in detail and successfully employed in biotechnology, our work shows that many more may be found in nature. Some groups of putative GMC oxidoreductases are particularly interesting since they lack characterized representatives and may even employ new catalytic mechanisms since they lack the established catalytic residues. One recently discovered subfamily, FAPs, presents a fascinating type of enzymes using the energy of light for catalysis. We show that genomic and metagenomic databases harbor more than 200 putative FAP genes, which may be more stable, more efficient, or have different substrate specificities compared to the already characterized FAPs.
Catalysts 2020 , 22 Figure 3. 2020223 Figure 3. Representative active sites structures of different GMC family proteins. FAD is shown in magenta, the conserved histidine is in light blue, and the ligand (substrate) is in green (where present). Respective PDB IDs are 1COY, 1NAA, 1TT0, 3GDN, 3Q9T, 3QVP, 4H7U, 4HA6, 4MJW, 4UDP, 4YNU, 4Z24, 5HSA, 5OC1 and 6A2U (Table2).
4  and 5 ). Unexpectedly, the construct adopts a dual-domain fold with an apparent N-proximal module connecting via an interstitial helix (␣ 6 ) to a C-terminal domain, in which the last ϳ40 residues (C-terminal tail) remain unresolved in the structure (Fig. 4A  and B ). A search for three-dimensional (3D) structural homologs using the fold recognition server DALI  (25)  revealed that the N-proximal domain adopts a receiver (REC) domainlike (␤␣) 5 fold (26), where the central five-stranded parallel ␤-sheet is flanked by 4 ␣-helices, while the canonical ␣ 1 is mostly unfolded in an extended conformation by a stretch of proline and other small uncharged amino acids (Fig. 4C ). Canonical REC domains are typically found in tandem with DNA-binding modules in response regulator proteins, which use phosphoryl transfer from upstream kinases as input signals for transcription regulation  (26).
Structural and sequence alignments of BcsE REC * with phosphorylation-competent receiver domains, however, show significant deviation from the amino acid consensus of key functional motifs, indicating that the module is unlikely to function in phosphotransfer-dependent signal transduction (Fig. 4D ). Similar DALI search using the resolved C-proximal domain as an input revealed the closest structural homolog as the cytosolic C-terminal domain of Pseudomonas aeruginosa PelD. Interestingly, the latter is itself a c-di-GMP-binding protein responsible for the activation of synthase-dependent exopolysaccharide secretion, the Pel system in pseudomonads, and has been characterized as a degenerate GGDEF domaincontaining protein where c-di-GMP sensing is carried out by the conserved I-site motif  (27) (28) (29).
Crystallization, data collection, and structure determination. Crystals were obtained by sitting or hanging-drop vapor diffusion by mixing equal volumes of protein (1. 5 to 6 mg/ml) and reservoir solution followed by incubation at 4°C. BcsE 217Ϫ523 crystals also appeared within 3 to 14 days under multiple conditions, with diffracting data sets collected on crystals grown in 100 mM morpholineethanesulfonic acid (MES; pH 6. 0), 4% polyethylene glycol 4000 (PEG 4000), 200 mM MgCl 2 , 5% glycerol, and 50 M c-di-GMP. For cryoprotection, crystals were soaked in reservoir solution supplemented with 25% to 30% glycerol, 1 mM DTT, and 50 M c-di-GMP. Cryopreserved crystals were flash frozen and stored in liquid nitrogen. Data were collected on frozen crystals at 100 K using synchrotron radiation at beamlines PX1 and PX2 at the Soleil synchrotron.
Data reduction was carried out with the software package XDS  (42). Experimental phases were obtained by single-wavelength anomalous diffraction (SAD) experiments on crystals grown from selenomethionine-derivatized protein and with wavelengths corresponding to the experimentally determined selenium K-edge. Initial BcsE 217Ϫ523 models were obtained using the automated model building tools of PHENIX and Buccaneer  (43, 44). Reiterative refinements in PHENIX, COOT, and BUSTER yielded the final refined model  (43, 45, 46). Data collection and refinement statistics are summarized in Table  S2. For illustration purposes, all crystal structures were displayed with the PyMol Molecular Graphics System (Schrödinger, LLC) or UCSF Chimera  (47). The latter was also used for displaying the 3D reconstructions of the assembled Bcs macrocomplex.