bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 Title 2 3 Domain-centric dissection and classification of prokaryotic poly(3-hydroxyalkanoate) 4 synthases 5 6 Running Title 7 8 Domain organizations and interactions in prokaryotic PHA synthases 9 10 Authorship 11 12 Zhanzhong Liu1#, Zuobin Zhu2#, Jianye Yang3, Sheng Wu3, Qinghua Liu4,5, Mengmeng 13 Wang4,5, Huiling Cheng3, Jiawei Yan1, Liang Wang3,4* 14 15 Affiliation 16 17 1Xuzhou Infectious Diseases Hospital, Xuzhou Jiangsu, 221000, China 18 2Department of Genetics, School of Biological Sciences and Technology, Xuzhou Medical 19 University, Xuzhou Jiangsu, 221000, China 20 3Department of Bioinformatics, School of Medical Informatics, Xuzhou Medical University, 21 Xuzhou Jiangsu, 221000, China 22 4Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, School of Pharmacy, 23 Xuzhou Medical University, Xuzhou Jiangsu, 221000, China 24 5Department of Pharmaceutical Analysis, School of Pharmacy, Xuzhou Medical University, 25 Xuzhou Jiangsu, 221000, China 26 27 #These authors contribute equally to the study. 28 29 *Correspondence author: for all correspondence, please refer to Dr. Liang Wang 30 [email protected] 31 32 33 34 bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
35 Abstract 36 37 Although many enzymes and multiple pathways involve in Polyhydroxyalkanoates (PHAs) 38 synthesis, PHA synthases play a determinant role in the process, which include three subunits 39 of PhaC, PhaE, and PhaR. Currently, PHA synthases are categorized into four classes 40 according to its primary sequences, substrate specificity, and subunit composition. However, 41 theoretical analysis of PHA synthases from the domain perspective has not been performed. 42 In this study, we dissected PHA synthases thoroughly through analysis of domain 43 organization. Both referenced bacterial and archaeal proteomes were then screened for the 44 presence and absence of different PHA synthases along NCBI taxonomy ID-based 45 phylogenetic tree. In addition, sequences annotated as bacterial and archaeal PhaCs in 46 UniProt database were also analyzed for domain organizations and interactions. In sum, the 47 in-silico study provided a better understanding of the domain features of PHA synthases in 48 prokaryotes, which also assisted in the production of PHA polymers with optimized chemical 49 properties. 50 51 Keywords 52 53 Polyhydroxyalkanoate, PHA synthase, PhaC, Domain organization, Co-occurrence 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
69 70 Introduction 71 72 Polyhydroxyalkanoates (PHAs) are a family of biodegradable and biocompatible polyesters 73 that are synthesized intracellularly by a wide range of bacteria and archaea [1]. It naturally 74 serves as carbon and energy resources for prokaryotes and provides important functions for 75 their physiological activities, such as high salinity tolerance and desiccation resistance, etc., 76 although the specific molecular mechanisms are still not solved. There are three types of
77 PHAs according to their side chains, short-chain length (SCL) PHA (PHASCL), medium-chain
78 length (MCL) PHA (PHAMCL), and the mixture of both SCL and MCL PHAs (PHASCL-MCL) 79 [2]. Previous studies confirm that there are three enzymes involved in PHA synthesis, which 80 include Acetyl-CoA acetyltransferase (PhaA), Acetoacetyl-CoA reductase (PhaB), and 81 Poly(3-hydroxyalkanoate) polymerase subunit PhaC (PhaC) [3]. There are another two 82 subunits, PhaE and PhaR, for Poly(3-hydroxyalkanoate) polymerase, which integrate with 83 subunit PhaC to form active PHA synthase, respectively [4]. A heterogeneous group of small- 84 sized proteins with no catalytic functions, Phasin (PhaP), was also tightly involved in granule 85 structure formation and PHA metabolism [5]. As for PHA utilization, two enzymes, 86 intracellular PhaZ and extracellular PhaZ, play important roles in this process [4]. Except for 87 the above-mentioned classical pathway, enzymes from several other pathways are also 88 indirectly involved in PHA synthesis, such as 3-oxoacyl-[acyl-carrier-protein] reductase 89 (FabG), (R)-specific enoyl-CoA hydratase (PhaJ), Malonyl CoA-acyl carrier protein 90 transacylase (FabD), Succinate-CoA ligase [ADP-forming] subunit alpha (SucD), NAD- 91 dependent 4-hydroxybutyrate dehydrogenase (4HbD), and 4-hydroxybutyrate-CoA:CoA 92 transferase (OrzF), etc [4]. Currently, more than 150 different hydroxyalkanoic acids are 93 known to occur as constituents of PHAs [6] and a total of 14 PHA synthesis pathways have 94 been identified with many enzymes involved in these processes [7]. 95 96 The key enzymes involved in PHA synthesis are PHA synthases, which are a group of 97 heterogeneous enzymes and include PhaC, PhaE, and PhaR that form homodimers, 98 heterodimers, or work concordantly in their active states. These enzymes are currently 99 divided into four categories, Class I, Class II, Class III and Class IV [8]. Class I, III and IV 100 prefer SCL) carbon chain monomers (C3-5) while class II utilizes MCL PHA monomers 101 (C6–14) [8]. In some exceptions, Class I PHA synthase could produce mixed PHAs 102 incorporated with both SCL and MCL hydroxyalkanoates that show better functional bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
103 properties compared with homo-polymers [2,7]. PhaC plays a key role in PHA synthesis and 104 is the core subunit of PHA synthase. It has been extensively studied through metabolic 105 engineering so as to optimize the properties of PHAs for industrial application. Active sites in 106 different types of PhaCs belonging to a variety of bacteria were also reported [9]. However, 107 these studies are only sporadic and cannot give an overview of PhaC features in prokaryotes. 108 In addition, diversity and widespread of PhaC in bacterial species were only studied with a 109 small set of genomes involved. PhaCs in archaea were recently investigated systematically. A 110 novel type of Class III PhaC was widely identified with an elongated C terminus in halophilic 111 archaea, which is indispensable for enzymatic activity [10]. Another study systematically 112 investigated the distribution patterns of PHA synthesis pathway (PhaA, PhaB, and PhaC) and 113 degradation pathway (PhaZ) in archaeal species, according to which PHA accumulation is 114 skewedly associated with halophilic archaeal species [3]. 115 116 Currently, the classification of PHA synthases is mainly through experimental studies based 117 on primary sequences, substrate specificity, and subunit composition of the enzymes. In 118 addition, PHA classes and corresponding descriptions recorded in the database are diverse 119 with no consistent standards. One of the features of protein primary sequences used for 120 identifying PhaCs is a lipase box of G-X-S-X-G [8]. In addition, a characteristic secondary 121 structure containing α/β hydrolase fold was also highlighted [8]. However, these features are 122 not sufficient to distinguish PhaCs in different classes. Due to the widespread accumulation 123 of PHAs in prokaryotes, there is a large number of sequences of PHA synthases present in 124 public database. However, there are no systematic studies looking at these sequences from 125 domain-centric perspectives, which could provide a much clearer classification of and a 126 better understanding about PHA synthases. For details of PHA synthases, please refer to 127 Table 1. 128 129 From the domain-centric view, proteins can be considered as a combination of functionally 130 and structurally independent domains [15]. Domains normally have independent evolutionary 131 roadmaps, which can re-arrange and/or recombine with each other, leading to protein 132 functional diversification and organism adaptation to new niches [15,16]. In addition, domain 133 associations (co-occurrence) in a set of homologous sequences also reflect how proteins form 134 and evolve at secondary structural level [15]. In this study, we first performed a systematic 135 analysis of PHA synthases reported in literature via HMM models sourced from Pfam 136 database (version 32.0, 17929 entries) and identified representative domains present in bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
137 different classes. Their distributions in referenced prokaryotic proteomes were then studied in 138 order to look for biologically meaningful patterns from evolutionary point of view. In 139 addition, for sequences annotated as PhaCs that were retrieved from UniProt database, their 140 domain interactions were also calculated and visualized to reflect its significance in different 141 types of PhaCs. 142 143 In sum, this study looks into the diversity of PHA synthases from protein domain-based 144 standpoint. This provides a novel angle for PHA synthase classification. In addition, we also 145 overviewed the domain changes along taxonomy identifier based phylogenetic trees so as to 146 understand how domain organization evolves to contribute to PHA synthesis. The 147 conclusions would facilitate the understanding of the structures of PHA synthases and their 148 evolutions in prokaryotes. It is also able to assist in the engineering efforts for the production 149 of PHA polymers with optimized chemical properties. 150 151 Methods and Materials 152 153 Collections of sequences and proteomes in bacteria and archaea 154 Sequences of representative PHA synthases (PhaC, PhaE, and PhaR) were initially collected 155 from literature (Supplementary Table 1), which were well classified into different groups 156 based on corresponding phylogenetic studies [9,17-19] and were used as references to study 157 domain organizations of PHA synthases in protein database [20]. Since UniProt 158 Knowledgebase (UniProtKB) is the central-hub database for proteins with accurate, 159 consistent and rich annotation, we choose it as our source for collecting high-quality data [21]. 160 Two groups of PhaC-related sequences were selected from UniProt database that were 161 present in bacteria and archaea, respectively. The sequences were picked-up via advanced 162 searching of keywords “taxonomy:bacteria name:phac NOT name:phar NOT name:phae” and 163 “taxonomy:archaea name:phac NOT name:phar NOT name:phae”. A total of 2004 bacterial 164 proteins and 210 archaeal proteins were collected (Supplementary Table 2). As for 165 referenced prokaryotic proteomes, 5447 bacterial species (Supplementary Table 3) and 287 166 archaeal species (Supplementary Table 4) were downloaded together with metadata from 167 UniProt release 2019_02 [21]. 168 169 Domain organizations and interactions in PHA synthases bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
170 PHA synthases from literature were initially dissected via HMMER webserver with default 171 settings and the visualization of domain interactions were present in Figure 1 [22]. 17929 172 HMMs (version 32.0) were then downloaded from Pfam database for sorting domain 173 organization of selected PhaCs from UniProt database [20,21]. Criterion for significant hits 174 was set as E-value less than 1e-10 for all domains. Domain interactions in PhaCs selected 175 from UniProt database were visualized by CytoScape based on Pfam-sourced HMMs, which 176 gives the abundance of specific domains and also the associations among domains in a cluster 177 of homologous sequences [23]. Abundance of a single domain is reflected by its circle radius 178 while the strength of association between domains is displayed as line width. For domain 179 circles, we artificially set the limits of circle sizes based on the least and the largest times of 180 domain occurrence in collected sequences, while circle size of the rest domains are 181 adaptively generated according to their ranks in the sort. In terms of domain associations, 182 both the distances between domains and the times of domain co-occurrence were considered 183 when calculating the connecting strength (Figure 2). 184 185 Homologs of PHA synthases in prokaryotic reference proteomes 186 According to preliminary analysis, PHA synthases (PhaC, PhaE, and PhaR) in literature 187 exhibit a variety of domain organizations. Five subtypes of PhaC exist, which include type 1 188 with PhaC_N domain (PF07167), type 2 with Abhydrolase_1 domain (PF00561) in a long 189 sequence, type 3 with PhaC_N and Abhydrolase_1 domains, type 4 with Abhydrolase_1 and 190 HHH_5 domains (PF14520), type 5 with Abhydrolase_1 domain in a short sequence. As for 191 subunit PhaE, it contains PHA_synth_III_E domain (PF09712). Subunit PhaR has two 192 subtypes, type 1 with no domain and type 2 with PHB_acc_N domain (PF07879) and 193 PHB_acc domain (PF05233). There is another group of PHA synthesis enzyme exclusively 194 for PHB production that includes PHBC_N domain (PF12551) and PhaC_N domain, which is 195 identified during HMM analysis. In order to identify homologs of PHA synthases in 196 prokaryotic reference proteomes, 17929 HMMs were used to search selected bacterial and 197 archaeal proteomes with E-value set to less than 1e-10. Due to the lack of known Pfam 198 domain for some PhaR, local BLAST was performed by using PhaR (UniProt ID 199 A0A386BX77) from Bacillus cereus as the query sequence with following criteria: E- 200 value<1e-10, 30% 204 then used for evolutionary analysis of PHA synthases by incorporating into phylogenetic 205 trees. 206 207 Phylogenetic analysis of PHA synthases 208 NCBI Taxonomy ID based phylogenetic trees, which were generated by phyloT 209 https://phylot.biobyte.de/, were constructed to explore the distribution patterns of all different 210 types of PHA synthases in collected bacterial and archaeal proteomes [24]. A maximum- 211 likelihood (ML) phylogenetic tree based on a group of selected full length of PhaC sequences 212 were constructed via fasttree, which were accompanied with domain organizations by in- 213 house python scripts (available under request). 214 215 Statistical analysis 216 217 All statistical analyses were performed by using R package. Student’s t-test was performed 218 through two-tailed unequal variance with P-value less than 0.05 as significant difference. 219 220 Results and discussion 221 222 Domain-based classification of PHA synthases 223 224 It has been reported that class I PHA synthase is a homodimer of PhaC [8]. Class II PHA 225 synthase is mainly found in Pseudomonas spp. and consists of two different PhaCs, PhaC1 226 and PhaC2, with PhaC1 as the active form [8]. In this study, based on the initial domain 227 organizations of PHA synthases from literature, three sub-types of PhaCs in class I and II 228 PHA synthases were identified and reported, that is, type 1 PhaC with only Abhydrolase_1 229 domain, type 2 PhaC with only PhaC_N domain, and type 3 PhaC with both PhaC_N and 230 Abhydrolase_1 domains (Supplementary Table 1). According to the result, Class I PHA 231 synthase has all three subtypes of PhaCs while Class 2 PHA synthase only harbours PhaC_N 232 domain (type 2), which is not necessarily a general rule due to the small number of sequence 233 samples. All three types of PhaCs have comparatively similar length of around 600 amino 234 acids (AA). It would be interesting to find the unique features for the three sub-types of 235 PhaCs. However, it is difficult to distinguish the two classes of PHA synthases just at the 236 sequence level. Class III PHA synthase consists of PhaC and PhaE while class IV PHA 237 synthase consists of PhaC and PhaR. PhaC in these two classes is shorter and around half bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 238 length of PhaC in class I and II PHA synthases. Based on the domain analysis of the curated 239 sequences from literature, PhaC in class III PHA synthases has two sub-types, one is with 240 Abhydrolase_1 domain only and the other one is comprised of Abhydrolase_1 and HHH_5 241 domains. Interestingly, all the PhaCs with HHH_5 domain shared a high similarity with 242 PhaCs with Abhydrolase_1 domain only and were found exclusively in halophilic archaea 243 species such as Haloarcula hispanica and Haloferax mediterranei, etc. [18]. It has been 244 proved that the extended C-terminus (HHH_5) of PhaC is essential for PHA production in 245 halophilic archaea and could also play important roles in environment adaptation [10]. Please 246 see Figure 1 for the illustrated classification of the four classes of PHA synthases. 247 248 A couple of other domains were also found in PHA synthases, which include DUF3141 and 249 PHBC_N. DUF3141 resembles the Abhydrolase_1 domain and basically exists as a single 250 domain protein that is mainly present in Proteobacteria [25,26]. It is also rarely associated 251 with PhaC_N domain in several protein sequences according to Pfam database [20]. As for 252 PHBC_N, it was also found to concatenate with PhaC_N to form a novel type of PhaCs, 253 which does not fall into the four classes of PHA synthase and might have different functions 254 in the PHB metabolism [26]. Unlike the sequence diversity of PhaC at the domain level, 255 PhaE is comparatively conservative. All analyzed PhaE sequences consistently possess the 256 PHA_synth_III_E domain. In contrast, there is no domain information for PhaR sequences 257 sourced from Bacillus related species, such as Bacillus cereus and Bacillus megaterium. 258 However, a novel type of PhaR from other bacteria such as Chromohalobacter salexigens 259 and Bradyrhizobium japonicum consists of PHB_acc_N (PHB/PHA accumulation regulator 260 DNA-binding domain) and PHB_acc (PHB accumulation regulatory domain), which 261 suggested that PhaR sequences could also be rather diverse and different types of PhaR might 262 arise multiple times in evolution with similar functions. 263 264 Distribution patterns of PHA synthases 265 266 Due to the diversification of PHA synthases and its subunits, understanding how these 267 enzymes distribute along phylogenetic trees is necessary to better interpret their functions, 268 domain organizations and evolution. According to the domain-based classification of PHA 269 synthases, we systematically searched 5447 bacterial species and 287 archaeal species for 270 how PHA synthases distribute along phylogenetic trees, respectively. 271 bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 272 Bacteria 273 274 In total, only 5416 bacterial species were mapped in the phylogenetic tree due to the outdated 275 taxonomy IDs, which were mainly divided into 14 bacterial groups and classes. These 276 include (1) Firmicutes, (2) Bacilli, (3) Clostridia, (4) Actinobacteria, (5) Micrococcales, (6) 277 Streptomycetales, (7) Corynebacteriales, (8) Cyanobacteria/Melainabacteria group, (9) 278 Proteobacteria, (10) Alphaproteobacteria, (11) Gammaproteobacteria, (12) 279 Betaproteobacteria, (13) Delta/Epsilon subdivisions and (14) the FCB group (Figure 2). 280 Based on the dissection of HMM domain models for all the sequences in those proteomes, a 281 total of 7 groups of PHA synthases and/or subunits, were present. PhaC has three subclasses, 282 sequences with only Abhydrolase_1 domain, only PhaC_N domain or both Abhydrolase_1 283 and PhaC_N domains. According to the result, the most abundant subtype of PhaC should be 284 those with Abhydrolase_1 domain. Abhydrolase_1 belongs to the superfamily of hydrolytic 285 enzymes that share a common fold, which has been identified in a large collection of 286 enzymes with different origins and functions [27]. Thus, Abhydrolase_1 is rather abundant in 287 bacterial proteomes. Sequences with only Abhydrolase_1 domain have two sub-types, long 288 and short. Long PhaC with only Abhydrolase_1 domain should belong to Class I and Class II 289 PHA synthases while short PhaCs with only Abhydrolase_1 domain belong to Class III and 290 Class IV PHA synthases. However, due to the difficulty to determine the boundaries between 291 long and short PhaCs, it is hard to show how the two subtypes distribute along phylogenetic 292 trees. In contrast, Class III and Class IV PHA synthases are determined by the presence of 293 PhaE and PhaR. For the other two types of PhaCs with domains of PhaC_N or 294 PhaC_N/Abhydrolase_1, they are mainly clustered in the phylum of Actinobacteria and 295 Proteobacteria while species in other groups or classes only sporadically harbour the 296 enzymes. Thus, the two types of PhaCs might serve special functions in these two phylums 297 and requires further experimental investigation for their roles 298 299 PhaE is an independent subunit of Class III PHA synthases. According to our study, PhaE is 300 sporadically distributed along the phylogenetic tree, showing no preferred association with 301 bacterial species in any phylums or classes. However, it is noteworthy that a protein (UniProt 302 ID A0A252E8H1) with both Abhydrolase_1 and PHA_synth_III_E domains was found in 303 Nostoc sp. T09, a genus of Cyanobacteria that has been found in various environments. This 304 novel type of enzyme has never been reported before and could be caused by a gene fusion 305 event in the bacteria or could also be a sequencing error during genome assembly. Further bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 306 investigation is required to address this issue. If indeed a novel protein exists that harbors 307 functions of both PhaC and PhaE, it would be interesting to see what the properties and 308 kinetics of this enzyme are for PHA synthesis when compared with the PhaC and PhaE 309 heterodimer. 310 311 PhaR is the subunit of Class IV PHA synthases. It was found to be specifically associated 312 with Bacilli class in the Firmicutes Group, as previous studies suggested [14]. According to 313 the domain analysis, another two species, Desulfallas gibsoniae DSM 7213 (E-value=1.86E- 314 17) and Thermosyntropha lipolytica DSM 11003 (E-value=2.39e-14), also possess PhaR. 315 Thus, this enzyme might play important roles in these bacterial species. By figuring out how 316 and why they acquire PhaR genes, it could be helpful for us to better understand PHA 317 metabolism in bacteria. Interestingly, a novel group of PhaR with two small domains, 318 PHB_acc_N and PHB_acc, were found to be widely and abundantly distributed in the 319 subdivisions of Proteobacteria class. In addition, a new PHB synthase, which has both 320 PHBC_N and PhaC_N domains, were mainly found in alpha- and beta-Proteobacteria 321 subdivisions. Their physiological functions and distribution patterns are worthy of further 322 exploration so as to better understand their unique roles. 323 324 Archaea 325 326 Previous bioinformatics analysis showed the complete metabolism pathway of PHA in 327 archaea by focusing on enzymes including PhaA, PhaB, PhaC, PhaE, PhaP, and PhaZ, 328 according to which it was confirmed that PHA accumulation is tightly related to halophilic 329 archaea with larger proteome size and higher GC contents [3]. In this study, we are only 330 interested in PHA synthases (PhaCs and its subunits) and explored their distribution patterns 331 in archaea. It was shown that PhaCs with single Abhydrolase_1 domain are most abundant in 332 archaeal species, similar to the pattern in bacteria, although some of the strains do not 333 harbour the enzyme, such as Nanoarchaeum equitans and Ignicoccus hospitalis, etc. 334 Interestingly, Ignicoccus hospitalis is the host organism for Nanoarchaeum equitans [28]. In 335 addition, species in unclassified Euryarchaeota such as candidate divison MSBL1 archaeon 336 seems to have very small genomes, most of which tend to lack PhaCs, losing PhaC 337 accumulation ability. Since the uncultured archaeal lineage MSBL1 strains are from brine 338 pool, they are also adapted to hypersaline environment [29]. Thus, PHA accumulation might 339 not be necessary for saline environment adaptation and survival of halophilic archaea. On the bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 340 other hand, Halobacteria shows to possess multiple copies of PhaCs with only a single 341 Abhydrolase_1 domain. As for the second type of PhaCs that consists of two domains, 342 PhaC_N and Abhydrolase_1, they are mainly spread in the Halobacteria phylum and TACK 343 group. Interestingly, there is no PhaC in archaea that has a single domain of PhaC_N. Thus, 344 PhaC with only PhaC_N domain could be a marker for PHA synthases in bacteria and might 345 play unique physiological features in PHA synthesis. A novel type of PhaC that is solely 346 identified in archaea have two domains, Abhydrolase_1 and HHH_5, as illustrated above 347 (Figure 1). The enzyme is mainly restricted to Halobacteria except for one strain uncultured 348 archaeon A07HB70. Class III PHA synthase is mainly found in Halobacteria and the TACK 349 group due to the presence of PhaE while Class IV PHA synthase is not found in archaea, 350 which reflects the unique evolutionary features of archaeal species when compared with 351 bacterial species. It is worth noting that both Thaumarchaeota phylum from the TACK group 352 and the Halobacteria class seem to have abundant and diverse PHA synthesis enzymes. 353 354 In order to better understand the distribution patterns of PHA synthases in both bacteria and 355 archaea, we summarized the corresponding presence and loss of PHA synthase types in 356 Table 2. For each domain, its Pfam ID is also shown so its functions could be easily retrieved 357 from Pfam database. 358 359 Domain interactions (co-occurrence) in PhaCs 360 361 Distribution patterns of PHA synthases give us a clear view of how different types of 362 enzymes evolve and cluster along phylogenetic trees. However, it cannot measure domain 363 abundance and co-occurrence quantitatively in a group of protein with multiple classes, 364 which could reflect core domains and domain relationships in a clustered sequence group 365 [15]. In this study, PhaC sequences were collected from UniProt database as long as their 366 protein names include PhaC and does not include PhaE and PhaR. Through a thorough 367 analysis of all selected sequences, we visualized the domain interactions in both archaeal 368 PhaCs (Figure 4) and bacterial PhaCs (Figure 5). 369 370 As for bacterial PhaCs, it is obvious that all domains generated through HMM dissection 371 were divided into four groups, among which three groups are independently clustered. The 372 fourth group include single domains with no co-occurrence with others, though they could 373 also be present in the clustered groups and interact with other domains (Figure 4A). The bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 374 dominant group includes domains of Abhydrolase_1, PhaC_N, DUF3141, PHBC_N, 375 Hydrolase_4, PHB_depo_C, Abhydrolase_6 and PHA_synth_III_E, among which 376 Hydrolase_4, PHB_depo_C, and Abhydrolase_6 were not identified in the four PHA 377 synthase classes. PHB_depo_C is the C-terminal domain of bacterial poly(3-hydroxybutyrate) 378 depolymerase that degrades PHB into oligomers and monomers of 3-hydroxy-butyric acid 379 [30]. Since PHB_depo_C is dominantly present in intracellular PHB depolymerase, it is 380 interesting to see that it also co-occurs with Abhydrolase_1 in sequences annotated as Poly- 381 beta-hydroxybutyrate polymerase or Polyhydroxyalkanoate synthase. In addition, association 382 between PHB_depo_C and DUF3141 (resembled to Abhydrolase_1) was observed only in a 383 protein named DUF3141 domain-containing protein (UniProt ID A0A3A4PYR6) in the 384 strictly anaerobic Desulfobacteraceae bacterium, which was further confirmed through 385 whole UniProt database search. As for PHB_depo_C (E-value= 9.8e-13), Abhydrolase_1 (E- 386 value=3.5e-16), and PhaC_N (E-value=8.8e-11), their associations were identified in a 387 protein titled Class III poly(R)-hydroxyalkanoic acid synthase subunit PhaC (UniProt ID 388 A0A1Q7IEF1) in Ktedonobacter sp. These associations deserve further investigation both 389 theoretically and experimentally in order to understand their origin and potential functions. 390 391 As for the other two clustered domains and some of the independent domains such as 392 Oxidored_q2 and Na_H_Exchanger, their existence in the domain interaction map largely 393 indicates mis-annotations that might be present in UniProt database. For example, the 394 adh_short, adh_short_2, and KR cluster is mainly found in 3-oxoacyl-ACP reductase that 395 catalyses the 3-oxoacyl-ACP reduction step in the fatty acid synthesis pathway [31]. 396 However, in Burkholderia puraquae and Burkholderia pyrrocinia, proteins (UniProt ID 397 A0A1D8BIL9 and A0A0N9MGN9) with adh_short domain were named Acetoacetyl-CoA 398 reductase and PhaC, respectively. As for 3CDH related domain cluster, it is mainly present in 399 3-hydroxyadipyl-CoA dehydrogenase. However, enzymes with the 3CDH domains in 400 Desmospora sp. (UniProt ID F5SKZ9), Pseudomonas entomophila (UniProt ID Q1I9V2), 401 Enterobacter hormaechei (UniProt ID F5RRQ1) were all annotated as 3-hydroxyacyl-CoA 402 dehydrogenase PhaC. 403 404 In terms of archaeal PhaCs, their domain organizations showed that a total of six domains, 405 PhaC_N, Abhydrolase_1, HHH_5, Hydrolase_4, PHB_depo_C and DUF3141, were present 406 and all connected together (Figure 5). In addition, PhaC_N and Abhydrolase_1 shows the 407 strongest connections. Except for Hydrolase_4, all domains have been observed in PHA bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 408 synthase and all the domains co-occurs with each other to form a single cluster, though 409 HHH_5, PhaC_N, and Abhydrolase_1 are also able to exist independently in single domain 410 form. Proteins (UniProt ID A0A062VD38 and A0A062V164) from archaea Candidatus 411 Methanoperedens nitroreducens harbour PHB_depo_C, Abhydrolase_1 and PhaC_N, which 412 also raised the question of why domains for synthesis and degradation co-occur. In addition, 413 it suggested that this type of enzyme is not restricted to bacteria and should be explored 414 further, especially for the functions that the PHB_depo_C domain might play in the enzyme. 415 416 Conclusion 417 418 PHAs are a group of biodegradable and biocompatible natural thermoplastics with good 419 mechanical properties. A variety of building blocks are available for PHA synthesis, which 420 leads to its structural polydispersity. PHA synthases play a central role in PHA biosynthesis. 421 Its activity and substrate specificity determine characteristics of the generated polymers. 422 Although PHA synthases are currently divided into four classes based on kinetics and 423 mechanisms of reactions, no studies attempt to classify and summarize PHA synthases via 424 domain-centric viewpoints. In this study, we re-defined five sub-types of PhaCs in the four 425 classes of PHA synthases according to domain organizations, together with the conservative 426 PhaE and the two types of PhaR. Another two rare types of PhaCs, one with 427 PHA_synth_III_E domain and the other one with PHB_depo_C domain, were also observed 428 and required further investigation for their existence and functions. In addition, distributions 429 of different types of PHA synthases along evolutionary trees were also explored. Interesting 430 patterns were identified with insights into how PHA synthases were preferred in different 431 bacterial and archaeal species, which seem to have different preferences for PHA synthases 432 and might be determined by unique evolutionary pathways and adaptation to specific niches. 433 In addition, we also investigated the domain co-occurrence in PhaC sequences collected from 434 UniProt database. Core domains and correlations among domains were studied. However, 435 misannotation of PhaC enzymes from UniProt database should draw some attention and 436 information from public database should be used with carefulness. In sum, through domain- 437 based analysis, we get a better understanding and an overview of how PHA synthase is 438 classified, which also give hints to further industrial engineering of the enzymes to develop 439 better bioplastic products. 440 441 Supplementary Materials bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 442 443 Table S1: Domain organizations of phylogenetically classified enzymes directly associated 444 with PHA synthesis that include PhaC, PhaE, and PhaR; Table S2: Collection of bacterial and 445 archaeal protein sequences annotated as PhaCs in UniProt database; Table S3: Distributions 446 of PHA synthase related domains in 5447 bacterial proteomes; Table S4: Distributions of 447 PHA synthase related domains in 287 archaeal proteomes. 448 449 Author Contributions 450 451 Conceptualization, Liang Wang; Data curation, Qinghua Liu and Mengmeng Wang; Formal 452 analysis, Zhanzhong Liu, Zuobin Zhu and Liang Wang; Funding acquisition, Liang Wang; 453 Methodology, Liang Wang; Project administration, Liang Wang; Validation, Jianye Yang, 454 Sheng Wu and Huiling Cheng; Visualization, Zhanzhong Liu and Zuobin Zhu; Writing – 455 original draft, Liang Wang; Writing – review & editing, Zhanzhong Liu, Zuobin Zhu and 456 Jiawei Yan. 457 458 Funding 459 460 This work was supported by the Excellent Researcher’s Startup Foundation at Xuzhou 461 Medical University (D2016007), Natural Science Foundation of Jiangsu Province 462 (BK20180997), and the Innovative and Entrepreneurial Talent Scheme in Jiangsu Province 463 (2017). 464 465 Conflict of interests 466 467 The authors declare no conflict of interests. 468 469 References 470 471 1. Chen G-Q. Plastics Completely Synthesized by Bacteria: Polyhydroxyalkanoates. In: 472 Plastics from Bacteria. (2010) 17-37. 473 2. Chek MF, Hiroe A, Hakoshima T, Sudesh K, Taguchi S. PHA synthase (PhaC): 474 interpreting the functions of bioplastic-producing enzyme from a structural 475 perspective. Applied Microbiology and Biotechnology, 103(3), 1131-1141 (2018). bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 476 3. Wang L, Liu Q, Wu X et al. Bioinformatics Analysis of Metabolism Pathways of 477 Archaeal Energy Reserves. Scientific Reports, 9(1) (2019). 478 4. Wang L, Yang J, Huang Y et al. Systematic Analysis of Metabolic Pathway 479 Distributions of Bacterial Energy Reserves. G3: Genes|Genomes|Genetics, 480 (2019). 481 5. Maestro B, Sanz JM. Polyhydroxyalkanoate-associated phasins as phylogenetically 482 heterogeneous, multipurpose proteins. Microbial Biotechnology, 10(6), 1323-1337 483 (2017). 484 6. Jia K, Cao R, Hua DH, Li P. Study of Class I and Class III Polyhydroxyalkanoate 485 (PHA) Synthases with Substrates Containing a Modified Side Chain. 486 Biomacromolecules, 17(4), 1477-1485 (2016). 487 7. Chek MF, Kim S-Y, Mori T et al. Structure of polyhydroxyalkanoate (PHA) synthase 488 PhaC from Chromobacterium sp. USM2, producing biodegradable plastics. Scientific 489 Reports, 7(1) (2017). 490 8. Mezzolla V, D’Urso O, Poltronieri P. Role of PhaC Type I and Type II Enzymes 491 during PHA Biosynthesis. Polymers, 10(8) (2018). 492 9. Rehm BHA. Polyester synthases: natural catalysts for plastics. Biochemical Journal, 493 376(1), 15-33 (2003). 494 10. Han J, Hou J, Liu H et al. Wide Distribution among Halophilic Archaea of a Novel 495 Polyhydroxyalkanoate Synthase Subtype with Homology to Bacterial Type III 496 Synthases. Applied and Environmental Microbiology, 76(23), 7811-7819 (2010). 497 11. Jia Y, Yuan W, Wodzinska J, Park C, Sinskey AJ, Stubbe J. Mechanistic studies on 498 class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III 499 synthases share a similar catalytic mechanism. Biochemistry, 40(4), 1011-1019 (2001). 500 12. Solaiman DKY, Ashby RD. Genetic Characterization of the Poly(hydroxyalkanoate) 501 Synthases of Various Pseudomonas oleovorans Strains. Current Microbiology, 50(6), 502 329-333 (2005). 503 13. Müh U, Sinskey AJ, Kirby DP, Lane WS, Stubbe J. PHA Synthase fromChromatium 504 vinosum: Cysteine 149 Is Involved in Covalent Catalysis†. Biochemistry, 38(2), 826- 505 837 (1999). 506 14. McCool GJ, Cannon MC. PhaC and PhaR Are Required for Polyhydroxyalkanoic 507 Acid Synthase Activity in Bacillus megaterium. Journal of Bacteriology, 183(14), 508 4235-4243 (2001). bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 509 15. Wang L, Yang J, Xu Y, Piao X, Lv J. Domain-based Comparative Analysis of 510 Bacterial Proteomes: Uniqueness, Interactions, and the Dark Matter. Current 511 Genomics, 20(2), 115-123 (2019). 512 16. Toll-Riera M, Albà MM. Emergence of novel domains in proteins. BMC Evolutionary 513 Biology, 13(1) (2013). 514 17. Lu Q, Han J, Zhou L, Zhou J, Xiang H. Genetic and Biochemical Characterization of 515 the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax 516 mediterranei. Journal of Bacteriology, 190(12), 4173-4180 (2008). 517 18. Cai L, Tan D, Aibaidula G et al. Comparative genomics study of 518 polyhydroxyalkanoates (PHA) and ectoine relevant genes from Halomonas sp. TD01 519 revealed extensive horizontal gene transfer events and co-evolutionary relationships. 520 Microbial Cell Factories, 10(1) (2011). 521 19. Hai T, Lange D, Rabus R, Steinbuchel A. Polyhydroxyalkanoate (PHA) 522 Accumulation in Sulfate-Reducing Bacteria and Identification of a Class III PHA 523 Synthase (PhaEC) in Desulfococcus multivorans. Applied and Environmental 524 Microbiology, 70(8), 4440-4448 (2004). 525 20. Finn RD, Bateman A, Clements J et al. Pfam: the protein families database. Nucleic 526 Acids Research, 42(D1), D222-D230 (2014). 527 21. Consortium TU. UniProt: the universal protein knowledgebase. Nucleic Acids 528 Research, 45(D1), D158-D169 (2017). 529 22. Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity 530 searching. Nucleic Acids Research, 39(suppl), W29-W37 (2011). 531 23. Shannon P, Markiel A, Ozier O et al. Cytoscape: a software environment for 532 integrated models of biomolecular interaction networks. Genome Res, 13(11), 2498- 533 2504 (2003). 534 24. Federhen S. The NCBI Taxonomy database. Nucleic Acids Research, 40(D1), D136- 535 D143 (2011). 536 25. Sznajder A, Pfeiffer D, Jendrossek D, Pettinari MJ. Comparative Proteome Analysis 537 Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in 538 Ralstonia eutropha H16. Applied and Environmental Microbiology, 81(5), 1847-1858 539 (2015). 540 26. Batista MB, Müller-Santos M, Pedrosa FdO, de Souza EM. Potentiality of 541 Herbaspirillum seropedicae as a Platform for Bioplastic Production. In: Microbial 542 Models: From Environmental to Industrial Sustainability. (2016) 23-39. bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 543 27. Ollis DL, Cheah E, Cygler M et al. The α/β hydrolase fold. "Protein Engineering, 544 Design and Selection", 5(3), 197-211 (1992). 545 28. Junglas B, Briegel A, Burghardt T et al. Ignicoccus hospitalis and Nanoarchaeum 546 equitans: ultrastructure, cell–cell interaction, and 3D reconstruction from serial 547 sections of freeze-substituted cells and by electron cryotomography. Archives of 548 Microbiology, 190(3), 395-408 (2008). 549 29. Mwirichia R, Alam I, Rashid M et al. Metabolic traits of an uncultured archaeal 550 lineage -MSBL1- from brine pools of the Red Sea. Scientific Reports, 6(1) (2016). 551 30. York GM, Lupberger J, Tian J, Lawrence AG, Stubbe J, Sinskey AJ. Ralstonia 552 eutropha H16 Encodes Two and Possibly Three Intracellular Poly[D-(-)-3- 553 Hydroxybutyrate] Depolymerase Genes. Journal of Bacteriology, 185(13), 3788-3794 554 (2003). 555 31. Guo Q-Q, Zhang W-B, Zhang C et al. Characterization of 3-Oxacyl-Acyl Carrier 556 Protein Reductase Homolog Genes in Pseudomonas aeruginosa PAO1. Frontiers in 557 Microbiology, 10 (2019). 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 577 578 579 580 581 582 583 584 Table 1 Summary of four classes of PHA synthases. PHA UniProt Length Active States Subunits Product Bacteria Ref Synthase ID (AA) PhaC1 Cupriavidus Class I Homodimer SCL P23608 589 [11] PhaC1 necator PhaC1 Pseudomonas P26494 559 Class II PhaC1 MCL [12] PhaC2 oleovorans P26496 560 PhaC3 Allochromatium P45370 355 Class III Heterodimer SCL [13] PhaE vinosum P45372 357 PhaC3 Bacillus Q9ZF92 362 Class IV Heterodimer SCL [14] PhaR megaterium Q9ZF94 199 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 600 601 602 603 604 605 606 607 Table 2 Summary of comparative distributions of PHA synthases in bacteria and archaea. PHA Synthase Domain Organization Pfam ID Bacteria Archaea PhaC Abhydrolase_1 PF00561 + + PhaC PhaC_N PF07167 + - PhaC PhaC_N/Abhydrolase_1 PF07167/ PF00561 + + PhaC Abhydrolase_1/HHH_5 PF00561/PF14520 - + PhaE PHA_synth_III_E PF09712 + + PhaR - - + - Novel PhaR PHB_acc_N/PHB_acc PF07879/PF05233 + - PHB Polymerase PHBC_N/PhaC_N PF12551/PF07167 + - 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 624 625 626 627 628 629 630 631 632 Figure 1 Illustration of four PHA synthase classification in prokaryotes. Class I PHA 633 synthase is a PhaC homodimer while Class II PHA synthase is a heterodimer formed by two 634 different PhaCs (e.g. combination 1, 2, 3 in dashed lines). Class III PHA synthase has two 635 subunits PhaC and PhaE. Class IV PHA synthase consists of PhaC and PhaR. Based on 636 domain organization and protein length, PhaC in four classes of PHA synthases is further 637 divided into five sub-types as specified in the illustration. 638 639 Figure 2 Distribution patterns of PHA synthases along NCBI taxonomy ID based 640 phylogenetic trees. 5416 bacterial species sourced from UniProt database were included in 641 the tree and belongs to 14 bacterial groups and classes, which are 1. Firmicutes (1239), 2. 642 Bacilli (91061), 3. Clostridia (186801), 4. Actinobacteria (201174), 5. Micrococcales 643 (85006), 6. Streptomycetales (85011), 7. Corynebacteriales (85007), 8. 644 Cyanobacteria/Melainabacteria group (1798711), 9. Proteobacteria (1224), 10. 645 Alphaproteobacteria (28211), 11. Gammaproteobacteria (1236), 12. Betaproteobacteria 646 (28216), 13. Delta/Epsilon subdivisions (68525) and 14. FCB Group (1783270). A total of 7 647 groups of PHA synthases or subunits, together with bacterial proteome sizes were labelled 648 and coloured accordingly in the phylogenetic tree. 649 650 Figure 3 Distribution patterns of PHA synthases along NCBI taxonomy ID based 651 phylogenetic trees in 287 archaeal species sourced from UniProt database. A total of 14 652 archaeal groups and classes were annotated, which include 1. Halobacteria (183963), 2. 653 Methanomicrobia (224756), 3. Candidatus Nanohaloarchaeota (1462430), 4. Unclassified 654 Euryarchaeota (33867), 5. Methanomada Group (2283794), 6. Diaforarchaea Group 655 (2283796), 7. Thermococci (183968), 8. Archaeoglobi (183980), 9. Methanonatronarchaeia 656 (171536), 10. Methanopyri (183988), 11. TACK group (1783275), 12. Unclassified Archaea 657 (29294), 13. Environmental Samples (48510), and 14. DPANN Group (1783276). PHA bioRxiv preprint doi: https://doi.org/10.1101/693432; this version posted July 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 658 synthases or subunits, together with archaeal proteome sizes, were shown in the phylogenetic 659 tree and were annotated accordingly. 660 661 Figure 4 Interactions of domains in 2004 annotated PhaC enzymes from bacteria. All 662 sequences were thoroughly collected from UniProt database. Radius of color-filled circles 663 reflects the comparative abundance of the domain in all analyzed sequences. Edge shows the 664 connection of domain pairs and its width indicates strength of the interactions between the 665 two domains. 666 667 Figure 5 Interactions of domains in 210 annotated PhaC enzymes from archaea. Six domains, 668 PhaC_N, Abhydrolase_1, HHH_5, Hydrolase_4, PHB_depo_C and DUF3141, were present. 669 Domain abundance and connection strength were illustrated through circle radius and line 670 width. ≈ 600 AA Class I PhaC PhaC Type 1 PhaC_N 1 2 PhaC Type 2 Abhydrolase_1 3 Class II PhaC PhaC Type 3 PhaC_N Abhydrolase_1 PHA Synthase Type 4 Abhydrolase_1 PhaC Class III PhaC Type 5 Abhydrolase_1 HHH5 PhaE PhaE PHA_synth_III_E PhaC Type 4 Abhydrolase_1 Class IV PhaC PhaR Type 1 PhaR PHB_a PHB_a Type 2 cc_N cc 2282123 1249933 1915063 1936080 1635173 1850252 1349785 1848903 1127692 2058306 1250198 1850246 1250059 1907397 1622118 1889779 1109740 1125876 1836467 1736289 1178778 1664318 1304281 1038014 1324352 1642818 754417 754409 262005 533325 521097 860228 447689 1317122 1121284 706436 754423 104268 888059 762486 1055723 669041 593133 1250005 584609 691689 1080225 510955 558152 429344 1855336 536441 2058137 313603 570521 1155689 420404 1137780 2008672 1150389 1336804 28189 516051 996801 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1427517 406817 1427518 351671 406818 14 1628855 36870 1005995 642227 224915 372461 107806 1076551 55209 706191 1891675 660596 1560201 69222 634500 252393 1736225 1619313 465817 65700 1912096 929813 630626 1115515 1005667 566551 1006004 1487935 1115512 515618 1224318 1367852 435910 903503 41514 99287 623 300267 61647 701347 83334 83333 Abhydrolase_1 1432557 1566259 61645 399742 1736699 637910 290338 572265 203907 693444 891974 1681196 1028307 574 39831 1125630 272620 1549748 637679 745714 2043170 156889 1434232 1519374 314260 2058213 684719 335992 1002672 1528098 357244 272947 292805 222891 205920 269484 212042 320483 1868589 991905 1220535 856793 1445552 909943 488538 1605283 2015570 331869 Proteome Size 1523432 1492281 2015572 1759059 565050 2056860 366602 69666 PhaC_N/Abhydrolase_1 11 1282363 1121022 573065 260084 715226 2201350 1914756 1813876 2170551 1736442 450851 1445034 1523422 1736443 871741 345167 1736353 633149 1848616 1963835 1380357 553217 2022747 382514 1150626 1288970 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1077972 1005945 1849032 574651 92442 1246476 1300347 2171979 402596 670078 2135677 2138300 1664 1736232 290399 1736488 1736431 1652545 521096 1528099 2056849 136273 1292020 679197 2029858 446860 640132 378753 341202 37923 1903117 1236550 1736349 680646 85336 1313067 1344003 762948 1223543 37927 1004901 1223542 1338436 1241906 1077974 2020377 465515 930171 1075090 574650 290340 1073574 288705 1108045 1739367 2170743 1112204 2024402 861360 556325 1121927 554083 1193518 526226 1618207 1223544 407159 1276920 1223545 1193181 1837282 1220583 1194083 1415166 173053 1823 1193182 37332 584657 455432 710696 279262 1386089 1127134 262209 1133849 313589 1110697 857417 247156 1758689 756689 767452 208602 1385521 1519492 1385518 1538463 587636 228006 443156 45979 2283195 38310 384677 1805827 1869167 191292 1619947 1829 2003551 260936 947969 1587522 593907 101510 988821 1564114 1386082 525370 1295626 398843 1855324 1435356 446466 1653478 590998 2018041 2173174 1273125 1736362 1813677 1672219 103808 948458 1990687 862422 168276 1736607 2023150 545619 679318 1138585 300028 2182385 858619 47847 187491 1903186 257309 2069310 161895 1331682 1451189 1704590 401472 446465 1287475 47845 196164 1249481 1125779 396014 1203568 1224163 548476 592308 156978 1581067 1705 478801 1274 662755 1224162 1641401 1544413 1631356 185761 571913 1581138 1807500 1487956 225845 681645 585530 1050174 1703 1384076 946573 1404245 199591 1035195 479117 1229781 161896 743718 558173 1972128 1881052 1203622 1300344 645127 88382 526729 441500 446471 585529 1401064 903523 2079535 372484 1737425 442709 553207 285351 1072256 446469 28028 1814289 1437875 1089455 1184609 1224164 1724 1863 1184607 1437874 203267 504474 525268 1945885 1431546 620910 196627 2135789 556530 1121357 35755 471853 156976 648782 1945888 525263 1121362 860221 306537 638953 1203562 471856 1581050 1043493 1703924 2055947 661399 2173852 571915 1798190 883169 1470557 136857 55952 2071994 1718998 1348662 100226 1834080 1577075 875328 284040 443218 310779 1299321 67350 1185650 700597 700508 1703944 1519489 1209984 280871 1739111 1078020 1172182 444597 680198 285473 350058 2080746 85968 146020 1609133 1793 463642 126673 1703920 36811 1469144 1226750 1415557 1122247 1415546 152142 2202516 710421 1855349 1609137 59750 258533 359131 246196 1716141 710685 1331668 1834076 566461 216594 2020326 1428644 1856861 2099693 1725411 1273687 465541 933944 486698 1834121 235986 1858794 2100817 1150591 1840095 1834073 1703937 169765 1839759 1552759 1305826 1778 953739 1609095 29311 1879023 996637 1260918 1535768 1545728 1838281 1284664 1834149 1841861 47716 1736457 1906740 56425 380248 28045 1621259 1856859 1906 1223307 398694 1856866 2080748 1834111 1761906 310782 1790 316280 1747766 272631 68570 1682113 443255 83332 36816 58340 262316 1834117 2036037 1299328 1839780 408015 1299327 154654 1882757 1609098 220927 1703927 1380770 2219663 1519495 235985 2080747 2126346 146536 1882771 67373 452652 477245 2067551 2018025 2064 67267 665007 1736486 1348663 943816 68209 1894 1609099 1827605 159449 68214 253839 285568 36818 698760 1081613 1940 2044307 1286094 2282652 1915 1355015 1616117 1352941 285458 1765722 39478 2109333 457429 2218666 565073 909626 1300267 1839762 591158 1109743 1735453 1819298 1727216 1240678 310780 1955065 1428628 76728 310781 371608 1078086 1609103 42234 1678637 47763 467200 67285 1737066 1839774 1857892 645465 1075402 936756 1576605 1262452 1048205 1703929 75293 352211 1958787 591167 1322334 563922 1609100 1510197 417292 1690221 2185284 1214101 227882 1938849 1003195 1114943 1415558 1440053 1519493 457427 698759 1938835 1718985 2058924 2020325 1907 444103 1827606 1428626 1415551 1223523 1214242 1703933 68231 934019 2099583 1915400 1804758 83291 1609134 40318 1885 1352936 1839763 2020324 1855348 66430 1736503 1571774 2053043 1428652 862751 2282738 749414 910347 53446 1938860 710705 403935 1196353 1519490 1305830 1650659 1365176 178306 768679 1550241 572478 397948 1160895 368408 933801 399549 671065 41673 273063 273057 1326980 330779 1056495 384616 1104324 694429 415426 2309 2027460 399550 633148 1163730666510 583356 490899 1465461 1184251 272557 1198115 453591 940295 1407055 190192 1903181 1410606 1927129 113653 1499975311458 589924 387631 1502295 224325 1001994 693661 572546 1343739 859192 186497 859350 604354 1898749436308 69014 582419 926571 523850 1237085 439481 1353260 1495144 2058097 1713725 1713724 414004 1295009 374847 1577791 2035441 1236689 2035439 1769296 1932700 263820 2035442 273075 2035437 312540 333146 2137776 1961136 2035438 1054217 2055893 261391 1412872 1198116 1412871 667135 1412874 1242866 1412873 667137 1920749 667138 8 1242690 1531428 9 456320 228908 12 267377 1294122 419665 880724 573063 243232 13 523846 187420 10 1379702 1860100 877455 118062 7 339860 1945579 11 1945580 6 1945632 1945577 1789762 1945578 230361 PhaE 49547 224719 1300164 190976 14 5 47311 634498 55758 2006182 66851 1849261 1698263 _N/Abhydrolase_1 1698273 1698270 1698285 1698288 Abhydrolase_1 1698282 Proteome Size 1698257 1698271 4 1698261 1698276 Ahhydrolase_1/HHH_5 1698277 1383850 1698258 1227481 1698280 PhaC 1230454 1698275 1383851 1698269 1483399 1698265 1698281 1173487 1698272 1483400 63740 1698284 1698283 1238428 29283 1698268 2248 1698287 1698279 1853690 1698278 699431 2 3 1698259 755307 1698264 43928 1 1698262 1073996 1698266 1267564 1698274 1698267 1070774 1698260 1324957 699433 1698286 660517 1072681 889962 469382 889948 1227487 2056316 523841 304371 309800 351160 1238426 410358 362976 323259 555874 668570 521011 1126245 660521 593750 456442 1514971 937775 1236180 28892 756883 679926 64091 86622 1407499 1201294 355548 1392998 751944 349307 1110509 553468 990316 2056317 1679489890420 1839936 795797 1838285 867904 1873524 644295 679901 1261545996166 547558 558529 259564 1090322 1094980 1604004767519 1434100 188937 485914 192952 268739 269797 694430 348780 797302 797304 1333523 797114 1495067 1932360 797299 358396 1227454 1202768 797210 29540 547559 1230460 1114856 1033806 29295 1186196 1679096272569 1325472 1085028 1085029 931277 1132509 253108 1227455 745377 543526 1227499 588898 797303 2086290 373386 1230457 PhaC_N 8.21 Abhydrolase_1 1.6 3.0 0.8 3.04 Hydrolase_4 DUF3141 1.4 1.8 0.8 3.0 1.0 1.0 1.01 0.8 Abhydrolase_6 PHB_depo_C 1.0 PHBC_N adh_short_C2 1.0 0.8 PHA_synth_III_E adh_short 1.0 3HCDH KR 0.8 3HCDH 2.6 3HCDH_N 2.0 0.8 3HCDH_RFF DUF429 DUF3141 PhaC_N Abhydrolase_1 Oxidored_q2 TetR PHB_depo_C Na_H_Exchanger AMP-binding PhaC_N 1.72 8.87 Hydrolase_4 2.16 Abhydrolase_1 0.81 1.66 1.01 2.17 3.08 1 3.84 PHB_depo_C DUF3141 1.02 HHH_5 HHH_5 PhaC_N Abhydrolase_1