Genome Sequence of Aspergillus Flavus A7, a Marine- Derived Fungus with Antibacterial Activity

Genome

Genome sequence of Aspergillus flavus A7, a marine- derived fungus with antibacterial activity

Journal: Genome

Manuscript ID gen-2020-0066.R3

Manuscript Type: Note

Date Submitted by the 27-Nov-2020 Author:

Complete List of Authors: Gao, Yaru; China Pharmaceutical University College of Life Science and Technology, Du, Xinyang; China Pharmaceutical University College of Life Science and Technology Li, Huanhuan;Draft China Pharmaceutical University College of Life Science and Technology Wang, Ying; China Pharmaceutical University College of Life Science and Technology

Keyword: genome sequence, secondary metabolism, BGCs, Aspergillus flavus

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

1 Genome sequence of Aspergillus flavus A7, a marine-derived fungus with

2 antibacterial activity

3 Yaru Gao, Xinyang Du, Huanhuan Li, Ying Wang*

4 School of Life Science and Technology, China Pharmaceutical University, Nanjing 211198, PR

5 China.

6 ———————————————

7 *Corresponding author. Tel: +86-25-86185219. fax: +86-25-8321249. E-mail: [email protected]

Draft

9 Abstract

10 Due to the specific properties of the marine environment, marine microorganisms

11 have exclusive physicochemical characteristics that are different from those of

12 terrestrial microorganisms, which can produce various secondary metabolites (SMs)

13 with considerable structural diversity and biological activity. In this study, three

14 strains of coepiphytic Aspergillus with potential antibacterial activities, A7

15 (Aspergillus flavus), B27 (Aspergillus flavipes) and R12 (Aspergillus sydowii), were

16 isolated from the South China Sea. Via the Illumina MiSeq sequencing platform, the

17 genomes of the three strains were sequenced, and genome comparison showed the 18 highest diversity of the biosyntheticDraft gene clusters (BGCs) in A7. Meanwhile, a 19 comparison of physiological and genomic characteristics between A7 and other

20 Aspergillus flavus strains demonstrated the superior environmental adaptability of A7,

21 which is apparently consistent with the genetic richness of BGCs. By assigning reads

22 to known BGCs, putative BGCs were allocated in A7 that corresponded to various

23 SMs, including naphthopyrone, pyranonigrin E, cyclopiazonic acids, etc. Based on

24 gene homology analysis, we surmise that a region is involved in the biosynthesis of

25 ustiloxin-like RiPPs, a less thoroughly studied SM in fungi. Our results provide

26 genetic information for the investigation of marine Aspergillus sp., which may help to

27 elucidate their chemical diversity and adaptive strategies.

28 Keywords: Aspergillus flavus; genome sequence; secondary metabolism; BGCs

30 Abbreviation List:

31 BGCs: biosynthetic gene clusters

32 SMs: secondary metabolites

33 MH: Mueller-Hinton

34 MEA: Malt Extract Agar

35 MeOH: Methanol

36 LPCB: lactophenol cotton blue

37 ITS: internal transcribed spacer

38 BenA: β-tubulin 39 CaM: calmodulin Draft 40 ML: maximum-likelihood

41 GO: Gene Ontology

42 KEGG: Kyoto Encyclopedia of Genes and Genomes

43 KOG: euKaryotic Orthologous Groups

44 NRPS: nonribosomal peptide synthase

45 PKS: polyketide synthase

46 RiPP: ribosomally synthesized and posttranslationally modified peptide

48 Introduction

49 The prevailing extreme conditions of the marine environment have induced marine

50 microorganisms to develop exclusive adaptations in their genetic and physiological

51 characteristics, resulting in changes in metabolic patterns (Lindequist 2016), which

52 produce abundant secondary metabolites (SMs) (Rateb and Ebel 2011). Accumulating

53 evidence has highlighted the potential of marine-derived fungi and their biologically

54 active metabolites in drug discovery as sustainable sources of therapeutic agents;

55 special promise is exhibited by symbiotic microorganisms with marine invertebrates,

56 e.g., sponges, corals and ascidians (Lee et al. 2013; Skropeta and Wei 2014). A wide 57 range of chemically diverse compounds,Draft exhibiting antibacterial, antiviral and 58 antitumor properties, have been obtained from marine fungi. Among those fungal

59 strains, Aspergillus, a genus of well-defined asexual spore-forming fungi, is known as

60 a pathogen in view of its detrimental mycotoxins; however, Aspergillus can

61 prolifically produce valuable compounds, such as degraded starches, polysaccharides

62 and enzymes, which have been exploited on an industrial scale for the production of

63 biochemical and pharmaceutical agents (Lee et al. 2016). In previous work, a series of

64 fungi, including Aspergillus sp., were isolated from sponges and corals in the South

65 China Sea to screen potentially useful strains with antimicrobial activities. Among

66 these fungi, three Aspergillus sp., A7 (Aspergillus flavus), B27 (Aspergillus flavipes)

67 and R12 (Aspergillus sydowii), displayed broad bacteriostatic actions, underlining the

68 necessity of further research.

70 On the other hand, with the rapid development of sequencing technologies, the body

71 of knowledge concerning marine microorganisms and their natural products is in

72 transition. Notably, genomic sequencing is being utilized increasingly frequently as a

73 tool in microbial taxonomy, as well as in phylogenetic analysis and functional genome

74 mining (Nielsen and Nielsen 2017). In addition, this method could provide genetic

75 insights into the biosynthetic pathways and structural analysis of SMs, which are may

76 be employed to facilitate combinatorial biosynthesis of bioactive natural products

77 (Kjærbølling et al. 2018). In recent years, the genomes of various Aspergillus flavus

78 strains have been extensively studied and compared with those of other strains (Yin et 79 al. 2018), but marine-derived AspergillusDraft flavus strains have rarely been reported. 80

81 In this study, we present the draft genomes of the abovementioned Aspergillus strains

82 (A7, B27, and R12). Following the sequencing of the genomes of these strains,

83 fragment assembly, functional annotation and comparative genomic analysis were

84 performed, with an emphasis being placed on the prediction of SM gene clusters

85 present in A7, which exhibited compelling effects against bacteria. Moreover, certain

86 biosynthesis pathways were verified by chemical separation and identification in A7,

87 which may help to elucidate the ecological and biochemical profiles of

88 marine-derived Aspergillus.

90 Materials and methods

91 Preliminary Screening for Antibacterial strains

92 Strains A7 and R12 were isolated from corals (A7: 17° 6'12.4"N, 111° 29'57.8"E; B27:

93 16° 50'6.15"N, 112° 21'10.58"E) in the South China Sea. Strain B27 was isolated

94 from sponges in a nearby maritime location (17° 5'0.70"N, 111° 31'1.39"E). The

95 purified strains were activated and inoculated onto rice medium (rice 100 g, MgSO4

96 0.2 g, sea salt 1.5 g, H2O 100 mL) to culture at 28 °C for 15 days. The fermentation

97 products were extracted by methanol (MeOH) followed by decompressing distillation

98 to yield a crude extract. The crude extract was dissolved in 2 mL MeOH and

99 subsequently screened for antibacterial activity by the agar diffusion method.

100 101 Escherichia coli ATCC 25922, KlebsiellaDraft aerogenes ATCC 700603, Pseudomonas 102 aeruginosa ATCC 27853, methicillin-resistant Staphylococcus aureus (MRSA)

103 ATCC 43300), methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC

104 35984, Micrococcus luteus ACCC11001, and Acinetobacter baumannii ATCC 19606

105 were utilized as control strains. All the above strains were stored in the Marine

106 Pharmaceutical Laboratory of China Pharmaceutical University.

107

108 In brief, single colonies of control strains were inoculated into Mueller-Hinton (MH,

109 Solarbio, China) broth and cultured at 37 °C and 200 rpm for 18 h. The bacterial

110 inoculum was spread onto MH agar (Solarbio, China). Wells measuring 6 mm in

111 diameter were punched onto the surface of the agar using a sterile hole puncher. Next,

112 30 µL crude extract was added to the wells and incubated for 24 h at 28 °C. Each

113 assessment was developed in triplicate. MeOH was used as a vehicle control, while a

114 standard antibiotics (0.1 mM chloramphenicol) were used as a positive control. The

115 diameters (in mm) of the inhibition zone were recorded to estimate antimicrobial

116 activities, which were expressed by the ratio of the inhibition zone relative to that of

117 the positive control. The bacteriostatic activities were considered strong if the ratio

118 was greater than 1.0, moderate when the scale was between 0.5 and 1, and weak if it

119 was less than 0.5.

120

121 Morphological analysis

122 The spore suspensions of three strains were inoculated and glowed on Malt Extract 123 Agar (MEA, OXOID, UK) containingDraft 3% sea salt at 28 ℃ for 5 days. The colony 124 morphology was observed and characterized regarding parameters including size,

125 texture, color, soluble pigments, and exudates. Next, microscopic examination was

126 performed on spores and hyphae followed by lactophenol cotton blue (LPCB) staining

127 (Leck 1999) under a BA210 light microscope (Motic, Xiamen, China).

128

129 Molecular identification and phylogenetic analysis

130 For molecular identification, fungal mycelium was scratched by a sterile blade and

131 used for DNA extraction. The nuclear ribosomal internal transcribed spacer region

132 (ITS), partial β-tubulin (BenA) and calmodulin (CaM) genes were amplified by

133 primers ITS1 and ITS4 (White et al. 1990), Bt2a and Bt2b (Glass and Donaldson

134 1995), CMD5 and CMD6 (Hong et al. 2005), respectively. The primers sequences are

135 shown in Table S1. PCR amplification was performed using the following program:

136 95 °C for 10 min, 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min

137 followed by a final extension at 72 °C for 10 min (Visagie et al. 2014). Next, the PCR

138 products were subcloned for sequencing to establish the Basic Local Alignment

139 Search Tool (BLAST) search engine. Homologous ITS, BenA and CaM sequences

140 from various species were downloaded from the GenBank database (Table S2), and

141 multiple sequence alignment was conducted using ClustalW. Phylogenetic trees were

142 constructed by dataset of ITS, BenA and CaM sequences and concatenated sequences

143 using maximum-likelihood (ML) analysis in MEGA7 (Kumar et al. 2016).

144 145 Genome sequencing and assemblyDraft 146 Based on the bioactive screening, three strains (A7, B27, and R12) were believed to

147 be potentially antibacterial strains, for which genetic analysis is essential and effective.

148 Hence, the whole genomes of A7, B27 and R12 were sequenced by Genewiz Co.

149 (Suzhou, China) on the Illumina MiSeq sequencing platform.

150

151 In brief, the construction of DNA libraries was performed using 100 ng genomic

152 DNA, which was randomly fragmented to 500 bp by sonication (Covaris S220, USA).

153 Sequencing was subsequently performed using a 2×150-bp paired-end (PE)

154 configuration; image analysis and base calling were performed using HiSeq Control

155 Software. The adapter and low-quality sequences were removed from the raw

156 sequencing data by cutadapt (v1.9.1). The ideal reads were assembled and gap-filled

157 using Velvet (Zerbino and Birney 2008), SSPACE (Boetzer et al. 2011) and GapFiller

158 (Boetzer and Pirovano 2012), respectively. All genome sequencing data have been

159 submitted to the NCBI SRA database. The SRA accession numbers of A7, B27 and

160 R12 are PRJNA627810, PRJNA628551 and PRJNA628557, respectively.

161

162 Genome comparison of A7, B27 and R12

163 Cd-hit (Version 4.6) was used to cluster the cds sequences of A7, B27 and R12.

164 Clustering parameters were set as follows: the minimal sequence similarity was 70%,

165 and only hits with > 60% identity over 60% of the length of the query sequences were

166 considered. 167 Draft 168 The genomic sequences of three Aspergillus genomes were analyzed with the

169 antiSMASH (Blin et al. 2019) online server with the ClusterFinder algorithm to

170 identify the potential biosynthetic gene clusters (BGCs) based on homology analysis.

171

172 Intraspecies comparison of Aspergillus flavus

173 Comparison of tolerance of pH level and salinity

174 Aspergillus flavus ATCC 11492, the reference strain derived from terrestrial

175 ecosystems, was purchased from the Guangdong Microbial Culture Collection Center

176 (GDMCC, China).

177

178 Both strains, A7 and ATCC 11492, were cultured under the same photomixotrophic

179 conditions. Briefly, frozen stocks were inoculated into potato dextrose broth (PDB)

180 and incubated at 28 °C with shaking for 3 days. Subsequently, 0.1 mL of the activated

181 fungal cultures were coated onto MEA solid medium plates for 3 days at 28 °C. Next,

182 the spores were harvested with sterilized water and adjusted to 1.0×107 spores/mL.

183 Finally, 2.5 µL dilutions of spores were plated on different MEA media. The pH

184 range (4, 5, 6, 7, 8, 9, and 10) for growth and NaCl tolerance (0, 1, 3, 5, 7, and 10%

185 NaCl, w/v) was examined on MEA at 28℃ for 5 days. Colony diameter was measured

186 daily for five days.

187

188 Genome comparison of different Aspergillus flavus 189 A total of 9 genomic sequences fromDraft terrestrial Aspergillus flavus were downloaded 190 from NCBI GenBank for comparative analyses (Table 1). Next, the downloaded

191 genomic sequences were uploaded to antiSMASH to predict the SM BGCs.

192

193 Gene prediction and functional annotation

194 The software Augustus (version 3.3) (Stanke et al. 2006) was used to predict coding

195 genes and high-GC regions. Through a homology-based approach, the gene structures

196 were mapped to the reference genome Aspergillus flavus NRRL 3357 (Payne et al.

197 2009). Next, the coding genes were annotated with the NCBI nr database by BLAST,

198 and the functions of genes were annotated by the Gene Ontology (GO)(Harris et al.

199 2004) and Kyoto Encyclopedia of Genes and Genomes (KEGG)(Kanehisa and Goto

200 2000) databases. In addition, the predicted proteins were classified by the euKaryotic

201 Orthologous Groups (KOG) database (Tatusov et al. 2000).

202

203 Results

204 Antibacterial activities screening

205 Four kinds of G- bacteria and 3 kinds of G+ bacteria were employed to determine the

206 spectrum of antibacterial activity of the isolated strains (Table 2). Specifically, the

207 potent effect was visible in A7, which exhibited extensive inhibitory effects against 4

208 kinds of G- bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella

209 aerogenes and Acinetobacter baumannii), as well as 2 kinds of G+ bacteria (MRSA

210 and MRSE). B27 also actively inhibited the G- strain (Acinetobacter baumannii) and 211 G+ bacteria (MRSA, MRSE and MicrococcusDraft luteus). However, a modest action was 212 observed for R12 with weak efficiency on G+ bacteria (Micrococcus luteus). The

213 antibacterial properties of these strains stressed the necessity of studying them further.

214

215 Morphological analysis

216 Generally, morphology analysis provides a preliminary approach for microorganism

217 taxonomy. Morphological features, such as colony appearance, pigments, hyphae, and

218 conidiophores, are shown in Fig. 1. Apparently, A7 grows on MEA media with a

219 powdery appearance that is caused by numerous condiophores, exhibiting

220 yellow-brown mycelial areas but not soluble pigments and exudate, while fertile

221 vesicles are visible under microscope, exhibiting globose to subglobose morphology

222 (Fig. 1A). B27 grows poorly on MEA media, its colony surface is flocculent, its

223 sporulation and mycelial areas are yellowish white to pale yellow, exhibiting no

224 soluble pigment and no exudate, its spores are brownish orange without soluble

225 pigment, and its conidia are globose to subglobose (Fig. 1B). R12 grows poorly on

226 MEA media, its colony surface is velutinous when sporulating, it is mainly composed

227 of white sterile mycelia and becomes grayish green when spores are formed, soluble

228 pigment is mostly absent from this strain, exudate is mostly absent, the conidia head is

229 broom-shaped, similar to Penicillium, the phialides is ampulliform and the conidia are

230 globose to subglobose (Fig. 1C). The three strains conformed to the morphological

231 characteristics of Aspergillus in terms of colony texture, colors of mycelia, and

232 conidia; however, molecular species identification was warranted. 233 Draft 234 Molecular identification and phylogenetic analysis

235 Molecular markers have been widely accepted as crucial for the taxonomic

236 identification of fungi, among which ITS and some protein-coding genes, such as

237 ribosomal polymerase B2, BenA, and CaM, are popular barcode‐like molecules

238 (Schoch et al. 2012). In this study, the phylogenetic status of the bioactive strains (A7,

239 B27, and R12) was analyzed based on the ITS, CaM and BenA sequences. As shown

240 in the phylogenetic tree (Fig. 2), Aspergillus and Talaromyces are divided into

241 apparently separated genera. More specifically, A7, B27 and R12 cluster into the

242 Aspergillus clades supported by a 100% bootstrap value, which is consistent with the

243 highest identity scores. More specifically, A7 and Aspergillus flavus cluster into the

244 same branch as a sister taxon with B27, which appears in the same branch as

245 Aspergillus flavipes. R12 is clustered into a clade with Aspergillus sydowii, close to

246 Aspergillus creber, Aspergillus cvjetkovicii and Aspergillus puulaauensis.

247

248 Genome sequencing

249 In view of the potent bactericidal activity of A7, this strain is clearly the most distinct

250 one, which underscores the vital role played by SMs and the generation process.

251 Clearly, genomic analysis could demonstrate the inventory of all SM gene clusters

252 and the underlying mechanisms governing their production. Hence, the genome of A7

253 was sequenced with a coverage of 157.8 X. The draft genome was assembled into a 254 total size of 36.73 Mb, with a G+CDraft content of 48.39% composing 338 scaffolds. The 255 average length of consensus contigs was 108660 bp with an N50 of 634952 bp.

256 Augustus software was employed to predict the protein-coding genes, resulting in a

257 total of 11,711 protein-coding genes with an average length of 1575 bp. The resulting

258 genome assembly of B27 had a length of 32.33 Mb divided into 158 scaffolds. The

259 N50 scaffold length was 1,367,452 bp, the GC content was 52.42%, and the genome

260 coverage was 162.97X. The draft genome of R12 was observed to contain 1857

261 scaffolds with a total size of 35.39 Mb (50.59% G+C content). The N50 value and the

262 average scaffold size were 563,844 bp and 19,987.64 bp, respectively. Prediction of

263 protein-coding genes was performed using AUGUSTUS, and a total of 13,035 genes

264 were predicted. The general genomic characteristics of strains A7, B27 and R12 are

265 listed in Table 3.

266

267 Genome comparison of A7, B27 and R12

268 The core and unique genes represent phylogenetic conservation and species

269 specificity, respectively, to a certain extent. Comparative genomics could provide

270 useful information, such as effective identification of orthologous or diverse genes,

271 which could facilitate the understanding of those bioactive strains. As shown in the

272 Venn diagram (Fig. 3A), there were 2369 genes in common among A7, B27 and R12,

273 which accounted for 20.48%, 21.26%, and 18.79% of the total coding genes of A7,

274 B27, and R12, respectively. In addition, pairwise comparisons identified 3547 coding

275 genes shared between A7 and B27, accounting for 30.66% of the total coding genes in 276 A7. When these strains were comparedDraft with R12, less commonality was discernible, 277 demonstrated by 2988 genes in the intersection and an overlap rate of 24.98%, which

278 coincided with the phylogenetic relationship.

279

280 The genes responsible for SM production are usually arranged in multigene BGCs in

281 fungi (Marcet-Houben et al. 2012). In this study, the potential for SM production by

282 the A7 strain was genetically profiled by searching for BGCs in the antiSMASH

283 database. As shown in Fig. 3B, 58 gene clusters were involved in SM biosynthesis in

284 A7, including 26 nonribosomal peptide synthase (NRPS) clusters, 13 polyketide

285 synthase (PKS) clusters, 5 terpene clusters, 4 indole clusters, 2 NRPS-PKS hybrid

286 clusters, 1 fungal-ribosomally synthesized and posttranslationally modified peptide

287 (RiPP) cluster, and 7 other clusters. Interestingly, the SM gene clusters of A7,

288 especially hybrid gene clusters, are more diverse than those of B27 and R12,

289 indicating the diversity of SMs as well as the abundance of chemical skeletons.

290

291 Intraspecies comparison of Aspergillus flavus

292 Comparison of tolerance of pH and salinity

293 As the reference strain, ATCC 11492 was used to estimate the growth adaptability to

294 environmental stress, such as salinity and pH (Fig. 4). As shown in Fig. 4A, both

295 strains had variable growth in the MEA medium at all salinity levels (1-10%),

296 although with apparent growth restriction induced by increasing salinity. However, 297 A7 exhibited a robust pattern in Draft contrast to its terrestrial counterpart at the same 298 salinity, indicating an extensive tolerance to salt stresses. In terms of ambient pH (Fig.

299 4B), both strains were more tolerant to alkalinity than acidic media. Even so, an

300 adaptive advantage was more obvious in A7, at least within the detection range of this

301 work (pH 4-10). From this plot, A7 exhibited a tendency to survive in harsh

302 environments, which might be attributed to a higher genetic richness and metabolic

303 robustness.

304

305 Genome comparison of different Aspergillus flavus

306 Based on the above observations, we attempted to obtain further structural and

307 functional clues from the genomic information. Therefore, a genome-wide

308 comparison was performed among A7 and all available Aspergillus flavus genomes (9

309 available in GenBank at the time of this study). Consistent with its environmental

310 adaptive plasticity, the genome of A7 exhibits substantial richness and relative

311 abundance, characterized by the most significant diversity in BGCs, which may be an

312 exploitable genetic resource, possibly to produce a variety of SMs (Fig. 5).

313

314 Functional annotation

315 In terms of the structural-functional correlation, the gene functional annotations may

316 be inferred from primary structure homology. The genome of A7 yielded similar

317 annotation results in the GO, KEGG and KOG analyses: a large number of genes are

318 involved in metabolic pathways. Using KOG functional classification, we assigned 319 7467 proteins based on sequenceDraft similarity, accounting for 63.76% of the total 320 protein-coding genes (Fig. 6). For functional classification, the proteins were

321 categorized into 4 main KOG groups: intracellular processes (22.37%), metabolism

322 (39.91%), information storage processing (15.36%) and poorly characterized function

323 (22.37%). Notably, the most functional category of related protein-coding genes is

324 metabolism, such as amino acids (458), carbohydrates (453), lipids (529) or secondary

325 metabolites (506). The significant proportion (16.98%) of metabolism-related

326 gene-associated SMs might contribute to their potential antibacterial activities.

327

328 Secondary metabolite gene clusters of A7

329 The analysis of the BGCs of A7 highlighted 7 gene clusters showing 100% similarity

330 to known BGCs, corresponding to a variety of SMs, such as naphthopyrone, aflavarin,

331 6-methylsalicyclic acid, (-)-mellein, asperlactone, aculeacin A, leporin B,

332 cyclopiazonic acid (CPA) and pyranonigrin E (Fig. 7). As symbolic SMs of

333 filamentous fungi, these compounds have been assumed to have various bioactivities

334 by accumulating studies, including insecticidal (Tepaske et al. 1992), antifeedant

335 (Cary et al. 2015) and antifungal (Iwata et al. 1982) effects. In terms of the

336 biosynthetic pattern, the PKS (Fig. 7A-7D), NRPS (Fig. 7E) and NRPS-PKS hybrid

337 (Fig. 7F-7H) pathways are all involved, which may imply a more diverse toolkit, as

338 well as their derived compounds, as determined from the current limited

339 genome-mining scope.

340 341 For instance, a gene cluster presentDraft in Region 55.1 is presumed to synthesize aflatoxin 342 and CPA, which are important fungal metabolites and major concerns from a safety

343 perspective, as symbolic toxins are mainly produced by Aspergillus flavus (Frisvad et

344 al. 2019). The gene cluster of CPA is reported to be located in the subtelomeric region

345 close to the aflatoxin cluster in the Aspergillus flavus and Aspergillus oryzae genomes

346 (Chang et al. 2009). As shown in Fig. 8, the CPA BGC of A7 shares relatively high

347 amino acid homology with that of Aspergillus flavus NRRL 3357 (> 98%) and

348 Aspergillus oryzae NBRC 4177 (> 84%), despite the insertion of cpaH and cpaM in

349 Aspergillus oryzae NBRC 4177. To some extent, the hypotoxicity of Aspergillus

350 oryzae could be attributed to the detoxifying properties of cpaH (Kato et al. 2011),

351 which has been modified into as a relatively safe strain in food processing (Machida

352 et al. 2005). Essentially, mycotoxins are a very limited group of fungal SMs that are

353 produced more commonly for species competition or nutrient stress than for growth

354 (Frisvad et al. 2018). Therefore, it is not difficult to understand the conservation of

355 gene clusters of mycotoxins in marine-derived Aspergillus sp., especially considering

356 the intricate and dystrophic marine environment.

357

358 For another example, a genome mining approach demonstrated a high degree of

359 homology between A7 and Aspergillus flavus NRRL3357 for the BGC of ustiloxin B

360 (Fig. 9B): among the verified biosynthetic genes of the ustiloxin B BGC, there is

361 considerable similarity (over 60% amino acid identity), despite the lack of ustA, ustP1,

362 ustM and ustS. Notably, ustiloxin B is a secondary metabolite originally identified in 363 the rice pathogen UstilaginoideaDraft virens, which represents a class of macrocyclic 364 peptides (Fig. 9A) with strong potential to suppress microtubule assembly and mitosis

365 (Umemura et al. 2013). Recently, based on concurrent expression of contiguous genes

366 in the genome of Aspergillus flavus, a BGC termed ust has been identified, producing

367 ustiloxin B constructed by oxidative cyclization between the amino acid side chains,

368 which is classified into RiPPs. Among the 3 genes missing in A7, utsA is of great

369 importance, since it encodes the putative precursor peptide of ustiloxin B. It is

370 possible that numerous unidentified RiPP genes exist in fungi (Tsukui et al. 2015;

371 Umemura et al. 2014), which implies the presence of miscellaneous ustiloxin-like

372 products due to the potentially morphed precursor scaffolds. Recently, dozens of

373 ustiloxin-like ribosomal peptide precursor genes have been identified from Aspergilli,

374 computationally supporting this possibility (Nagano et al. 2016). The precursor

375 peptide UstA is highly unique in its characteristic that its core peptide repeats 16

376 times, which strongly contributes to identifying the gene in the complete sequences

377 (Umemura et al. 2014). Therefore, regarding current bioinformatics, the utsA

378 deficiency in the A7 genome may be attributable to insufficient knowledge of

379 homologous biosynthesis and precursor peptides, which warrants further study.

380

381 Discussion

382 Marine fungi, an important marine microorganism, have become potentially prolific

383 sources of highly bioactive SMs (Rateb and Ebel 2011). The first clinical applications

384 of SMs derived from marine fungi date back to the discovery of cephalosporin C in 385 the 1960s, which led to the developmentDraft of a series of valuable antibiotics (Fischbach 386 2009). During the following decades, an increasing number of marine fungi have been

387 characterized as sources of novel and potential therapeutic agents (Gomes et al. 2015).

388

389 In this study, we isolated a range of strains from corals and sponges in the South

390 China Sea and screened three bioactive strains, which were identified as Aspergillus

391 flavus (A7), Aspergillus flavipes (B27) and Aspergillus sydowii (R12), through

392 morphological and phylogenetic analysis. The fermentation products exhibited

393 promising bacteriostatic effects against both G+ and G- bacteria, with superior efficacy

394 being demonstrated in A7. The antibacterial properties called for consequent study on

395 the strains, for which the availability of a complete genome sequence would clearly be

396 practical and supportive.

397

398 Next, the whole genomes of A7, B27 and R12 were sequenced followed by

399 comparative genomics analysis and functional prediction for A7. Comparative

400 genomics labeled 2369 core genes, while the pattern of pairwise comparison appeared

401 consistent with the phylogenetic relationship, which paved the way for subsequent

402 genome analysis and mining. Thereafter, referring to a terrestrial counterpart,

403 Aspergillus flavus ATCC 11492, the stress tolerance of A7 to salinity or pH was

404 investigated for integrative characterization from phylogenetic and phenotypic aspects.

405 The results of this analysis showed that A7 possessed a broader adaptive capacity to

406 harsh environmental conditions, which was consistent with its oceanic origin. Clearly, 407 intraspecies comparison of AspergillusDraft flavus genomes could help to elucidate the 408 acclimation and adaptation of A7 to harsh marine environments, genetically

409 highlighting the mechanisms governing ecological survivability and adaptive

410 plasticity.

411

412 These potent bioactivities underscored the necessity for the follow-up investigation,

413 especially for A7, of which the classification is confirmed at both the 16S rRNA and

414 genomic levels to Aspergillus flavus. Known as a saprophytic and pathogenic fungus

415 with a cosmopolitan distribution, Aspergillus flavus produces significant quantities of

416 SMs, such as sterigmatocystin, CPA, kojic acid, and β-nitropropionic acid, to cope

417 with environmental or ecological stress (e.g., UV radiation, survival competition and

418 fungivorous predators) (Hedayati et al. 2007; Reverberi et al. 2010). In the case of A7,

419 kojic acid and CPA were isolated and characterized from the rice fermentation extract

420 through chemical separation (presented in another paper), to which the bacteriostatic

421 efficacy of fermentation production could be at least partly attributed. Therefore, from

422 a genetic perspective, it is reasonable to characterize the highly homologous CPA

423 BGC in A7, and it remains to be further investigated.

424

425 Early studies have affirmed that the Aspergillus flavus genome contains hallmark

426 enzymatic genes involved in SM synthesis, which are far from fully characterized

427 (Amaike and Keller 2011). Consistently, a total of 57 putative BGCs were predicted

428 in the A7 genome. Except for the BGCs mentioned above, there are a variety of gene 429 clusters that show low similaritiesDraft to the BGCs characterized in the database (Table 430 S3), hinting that a wider range of BGCs might designate putative products from

431 Aspergillus sp. to a large chemodiversity.

432

433 Following spurting reports on the bioactive metabolites from microorganisms

434 associated with marine invertebrates, these microbes represent an important reservoir

435 of untapped chemical diversity that enriches therapeutic arsenal (Haygood et al. 1999).

436 Conceivably, the utilization of microbial resources provides a possibility for scale-up

437 production to hurdle the limited supply and unravel their full potential through

438 fermentation or suitable biotechnology approaches (Gomes et al. 2016; Newman

439 2018). From a structural-functional perspective, sequencing-based genomics assays

440 could present a large amount of biological information for the clarification of the

441 biosynthetic pathways of some potent biological agents. As far as Aspergillus sp. is

442 still somewhat difficult to obtain enough natural or semisynthetic compounds due to

443 limitations in current approaches, such as culturable conditions, purification

444 techniques and biosynthesis schemes. In this sense, the current study adds to our

445 knowledge of the genetic profiles and architectures of marine Aspergillus, although

446 further investigation is necessary.

447

448 Conclusion

449 In this study, we performed whole-genome sequencing, comprehensive phylogenetic

450 analysis and genome comparisons on several bioactive strains of Aspergillus 451 previously derived from South China.Draft The subsequent analysis of the physiological 452 characteristics and biosynthetic potential of A7, which was the most potent strain

453 among them, presented a range of putative BGCs for representative fungal SMs,

454 including naphthopyrone, aflavarin, 6-methylsalicyclic acid, (-)-mellein, asperlactone,

455 aculeacin A, leporin B, CPA and pyranonigrin E. Additionally, a gene cluster

456 responsible for the production of ustiloxin-like RiPP was also predicted. Moreover,

457 there are a variety of uncharacterized gene clusters, which awaits comprehensive

458 explorations of their biological functions and putative products. The results of this

459 study may provide a foundation for further research investigating marine Aspergillus

460 sp., facilitating the understanding and exploitation of those fungal species.

461

462 Acknowledgments

463 This work was supported by funds from the National Key R&D Program of China

464 (2018YFC0311001), the Priority Academic Program Development of the Jiangsu

465 Higher Education Institutions (PAPD) and the Fundamental Research Funds for the

466 Central Universities (SM20190116021).

467

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Draft

614 Table 1. Genome overview of 9 Aspergillus flavus from the terrestrial ecosystem.

615 Table 2. Assessment of antimicrobial activities of fermentation products

616 E. coli: Escherichia coli; P. aeruginosa: Pseudomonas aeruginosa; K. aerogenes:

617 Klebsiella aerogenes; MRSA: methicillin-resistant Staphylococcus aureus; MRSE:

618 methicillin-resistant Staphylococcus epidermidis; M. luteus: Micrococcus luteus; A.

619 baumannii: Acinetobacter baumannii. The antimicrobial activities were estimated by

620 measuring the diameters of the inhibition zones and expressed on a scale relative to

621 the standard antibiotics (0.1 mM chloramphenicol). The activities were considered

622 weak (+), moderate (++), or strong (+++) when the value was < 0.5, between 0.5 and 623 1.0 or ≥ 1.0. Draft 624 Table 3. General features of the A7, B27 and R12 genomes

625 Fig. 1. Morphology of characterized strains. A-C: Colony and microscopic

626 morphology after 7 days of incubation. From left to right: obverse colonies on MEA,

627 reverse on MEA, conidiophores and conidiogenous at 40× magnification (scale bar 10

628 µm).

629 Fig. 2. Maximum likelihood tree of A7, B27, and R12 based on the concatenated

630 sequences of ITS, BenA and CaM. Multiple sequence alignment was conducted

631 using Clustal W (default settings), and phylogenetic relationships were based on ML

632 analysis with 1000 bootstrap replications in MEGA7. Numbers above branches

633 indicate bootstrap values. The sequence accession numbers and detailed strain

634 information for the phylogenetic tree are provided in supplemental Table S2.

635 Fig. 3. Genome comparison of A7, B27 and R12. A. Venn diagram of core and

636 specific genes in each strain (A7, B27, R12) through cluster analysis of cds sequence;

637 B. Comparative analysis of types of BGCs among the three Aspergillus species (A7,

638 B27, R12). NRPS: nonribosomal peptide synthase; PKS: polyketide synthase; RiPP:

639 ribosomally synthesized and posttranslationally modified peptide.

640 Fig. 4. Comparison of the physiological characteristics between A7 and ATCC

641 11492. A. Effect of different NaCl concentrations on colony growth of Aspergillus

642 flavus (A7 and ATCC 11492) at 5 days; B. Effect of pH values on the growth of

643 Aspergillus flavus colonies (A7 and ATCC 11492) at 5 days after inoculation. 644 Fig. 5. Comparison of A7 with otherDraft Aspergillus flavus. Different colors represent 645 different BGC types.

646 Fig. 6. KOG classifications of putative proteins in the genome of A7. I:

647 intracellular processes; II: metabolism; III: information storage/processing; IV: poorly

648 characterized function.

649 Fig. 7. Schematic representation of A7 putative BGCs showing high similarity

650 with genes from characterized BGCs. A-G. The upper part represents the BGC in

651 A7, followed by the known BGCs in the MIBiG database.

652 Fig. 8. Schematic comparison of the CPA BGC between A7 and reference strains.

653 The homologous genes in region 55.1 and known CPA BGCs from Aspergillus oryzae

654 NBRC 4177 or Aspergillus flavus NRRL 3357 are marked with the same color, and

655 missing genes are marked with a red frame. BGC descriptions and amino acid

656 homology (query cover and identity) are listed.

657 Fig. 9. Putative ustiloxin-like BGC in A7. A. ustiloxin B structure; B. The putative

658 ustiloxin-like BGC of A7 and the comparison of this cluster with the ustiloxin B

659 cluster reported for Aspergillus flavus NRRL3357. Compared with the ustiloxin B

660 BGC of Aspergillus flavus NRRL3357, ustA, ustP1, ustM and ustS were missing in

661 region 46.1, which is marked with a read frame. The homologous genes are marked

662 with the same color. Known BGC descriptions and amino acid homologies (query

663 cover and identity) are listed below.

Draft

Table 1. Genome overview of 9 Aspergillus flavus from the terrestrial ecosystem

Strain number NRRL 3357 AF12 E1404 CS1137 WRRL 1519 CA14 NRRL TERIBRI SU16

21882

Isolation source moldy peanuts Cotton field Peanut Cotton seed almond nut Pistacia vera Zea mays muddy water Wheat Qu

soil Total sequence 36.00 36.27 35.94 Draft35.68 36.28 35.96 35.30 34.10 35.82 length (Mb)

Assembly level Complete Scaffold Scaffold Scaffold Scaffold Scaffold Scaffold Contig Chromosome

Genome

Genome coverage 600.0x 40.0x 124.0x 123.0x 248.0x 121.0x 100.0x 200.0x 570.0x

Sequencing Illumina Illumina Illumina Illumina Illumina Illumina Illumina Illumina Illumina technology HiSeq HiSeq HiSeq HiSeq MiSeq MiSeq HiSeq HiSeq HiSeq

GenBank assembly GCA_009017 GCA_00371 GCA_013146 GCA_003711 GCA_00286 GCA_00370 GCA_00244 GCA_00415 GCA_00985 accession 415.1 1345.1 025.1 285.1 4195.1 9025.1 3195.2 0275.1 6665.1

Draft

Table 2. Assessment of Antimicrobial activities of fermentation products

E. coli P. aeruginosa K. aerogenes MRSA MRSE M. luteus A. baumannii

A7 + + ++ + ++ − +

B27 − − − ++ +++ ++ +

R12 − − − − − + −

E. coli: Escherichia coli; P. aeruginosa: Pseudomonas aeruginosa; K. aerogenes: Klebsiella aerogenes;

MRSA: methicillin-resistant Staphylococcus aureus; MRSE: methicillin-resistant Staphylococcus

epidermidis; M. luteus: Micrococcus luteus; A. baumannii: Acinetobacter baumannii. The antimicrobial

activities were estimated by measuring the diameters of the inhibition zones and expressed on a scale

relative to the standard antibiotics (0.1 mM chloramphenicol). The activities were considered weak (+), moderate (++), or strong (+++) when the Draftvalue was < 0.5, between 0.5 and 1.0 or ≥ 1.0.

Table 3. General features of the A7, B27 and R12 genomes

Genome A7 B27 R12

Assembly size (Mp) 35.02 32.33 35.39

G+C (%) 48.39 52.42 50.59

Assembled scaffolds 338 158 1857

N50 length (bp) 634952 1367452 563844

verage length(bp) 108659.86 210934.2 19987.64

Predicted Protein-Coding Genes 11711 11307 13035

Average length of Predicted Protein-Coding Genes (bp) 1575.27 1662.22 1548.37 Average depth of reads cover Draft157.8 162.97 218.12 Sequencing Method Illumina HiSeq Illumina HiSeq Illumina HiSeq

Draft

Fig.1. Morphology of characterized strains. A-C: Colony and microscopic morphology after 7 days of incubation. From left to right: obverse colonies on MEA, reverse on MEA, conidiophores and conidiogenous at 40× magnification (scale bar 10 µm).

Draft

Fig. 2. Maximum likelihood tree of A7, B27, and R12 based on the concatenated sequences of ITS, BenA and CaM. Multiple sequence alignment was conducted using Clustal W (default settings), and phylogenetic relationships were based on ML analysis with 1000 bootstrap replications in MEGA7. Numbers above branches indicate bootstrap values. The sequence accession numbers and detailed strain information for the phylogenetic tree are provided in supplemental Table S2.

Draft

Fig. 3. Genome comparison of A7, B27 and R12. A. Venn diagram of core and specific genes in each strain (A7, B27, R12) through cluster analysis of cds sequence; B. Comparative analysis of types of BGCs among the three Aspergillus species (A7, B27, R12). NRPS: nonribosomal peptide synthase; PKS: polyketide synthase; RiPP: ribosomally synthesized and posttranslationally modified peptide.

Draft

Fig. 4. Comparison of the physiological characteristics between A7 and ATCC 11492. A. Effect of different NaCl concentrations on colony growth of Aspergillus flavus (A7 and ATCC 11492) at 5 days; B. Effect of pH values on the growth of Aspergillus flavus colonies (A7 and ATCC 11492) at 5 days after inoculation.

Draft Fig. 5. The comparison of A7 with other Aspergillus flavus. Different colors represent different BGC types.

Draft

Fig. 6. KOG classifications of putative proteins in the genome of A7. I: intracellular processes; II: metabolism; III: information storage/processing; IV: poorly characterized function.

Draft

Fig. 7. Schematic representation of A7 putative BGCs showing high similarity with genes from characterized BGCs. A-G. The upper part represents the BGC in A7, followed by the known BGCs in the MIBiG database.

Draft

Fig. 8. Schematic comparison of the CPA BGC between A7 and reference strains. The homologous genes in region 55.1 and known CPA BGCs from Aspergillus oryzae NBRC 4177 or Aspergillus flavus NRRL 3357 are marked with the same color, and missing genes are marked with a red frame. BGC descriptions and amino acid homology (query cover and identity) are listed.

Draft

Fig. 9. Putative ustiloxin-like BGC in A7. A. ustiloxin B structure; B. The putative ustiloxin-like BGC of A7 and the comparison of this cluster with the ustiloxin B cluster reported for Aspergillus flavus NRRL3357. Compared with the ustiloxin B BGC of Aspergillus flavus NRRL3357, ustA, ustP1, ustM and ustS were missing in region 46.1, which is marked with a read frame. The homologous genes are marked with the same color. Known BGC descriptions and amino acid homologies (query cover and identity) are listed below.