The histone deacetylase clr3 regulates secondary metabolite production and growth under oxidative stress conditions in Penicillium brasilianum

Most of the biosynthetic gene clusters (BGCs) found in filamentous fungi are silent under standard laboratory cultivation conditions due to the lack of expression triggering stimuli, representing a considerable drawback in drug discovery. To access the full biosynthetic potential of these microbes, studies towards the activation of cryptic BGCs are essential. Histone acetylation status is an important regulator of chromatin structure which impacts in cell physiology and, therefore, expression of biosynthetic gene clusters in filamentous fungi. Histone deacetylases (HDACs) and histone acetyl-transferases (HATs) are responsible for maintaining and controlling this process under different cell conditions. In this study, clr3, a gene encoding a histone deacetylase in Penicillium brasilianum was deleted and associated phenotypic and metabolic changes evaluated. Results indicate reduced growth under oxidative stress conditions in the Δclr3 knockout strain. Also, the production of several secondary metabolites including austin-related meroterpenoids, brasiliamides, mycotoxins such as verruculogen and penicillic acid, as well as cyclodepsipeptides was reduced in the Δclr3 strain when compared to wild-type strain. Accordingly, addition of epigenetic modulators responsible for HDAC inhibition such as suberoylanilide hydroxamic acid (SAHA) and nicotinamide (NAA) to P. brasilianum growth media also culminated in reduction of secondary metabolite production. Mass Spectrometry Imaging (MSI) was applied to compare metabolite production and spatial distribution on the colony. Results suggest that Clr3 plays an important role in secondary metabolite biosynthesis in P. brasilianum, thus offering new strategies for regulation of natural product synthesis by assessing chromatin modification in P. brasilianum.


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To evaluate the impact of HDAC activity in P. brasilianum's secondary 219 metabolism and phenotype, the deletion of clr3 was performed. The clr3 gene was 220 chosen based on its sequence identity similarity with other known HDACs previously 221 deleted in A. fumigatus, A. nidulans and P. chrysogenum. The knockout strains for 222 these species presented different fungal development and metabolic profiles (23,24,25), 223 indicating that a Δclr3 P. brasilianum strain may also exhibit a different secondary 224 metabolism compared to the parental strain.

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Gene knockout was achieved through homologous recombination using a 226 deletion cassette constructed in vivo in S. cerevisiae using the hph gene, encoding 227 hygromycin B phosphotransferase, as a selection marker. Gene deletion strategy, as 228 well as diagnostic PCR agarose gel and Southern Blot analysis can be found in Fig 1   229 and confirmed the single copy integration of the deletion cassette at the clr3 locus 230 yielding a null mutant, which was further used for phenotypic and secondary metabolites 231 analyses.

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We observed no alteration in the chromatogram profile between the two strains 280 indicating that the deletion of clr3 did not induce the production of new metabolites or 281 repression of those constitutively formed in our culture conditions. On the other hand, 282 peak areas were significantly different for both strains, indicating that clr3 has an 283 important regulatory role in secondary metabolite production.

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To identify the molecules with different production levels in both strains, crude 14 286 was carried out by manually searching on Natural Products databases. Furthermore, 287 the obtained data was compared to HRMS data from previous studies related to P.

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The lack of novel induced secondary metabolites due to clr3 deletion was not 308 expected based on its transcription role to suppress gene expression and may be highly

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Since the deletion of clr3 did not induce the production of any new metabolites, 316 different approaches to modulate chromatin structure were sought. Besides gene 317 deletion, HDAC inhibition can be achieved through chemical epigenetic modulation (1).
318 In Aspergillus niger, growth in media supplemented by suberoylanilide hydroxamic acid 319 (SAHA), a HDAC inhibitor, was able to alter its secondary metabolism and induce a new 320 pyridone (38). In the Penicillium genera, the same strategy was applied in Penicillium 321 mallochii, resulting in two new natural sclerotioramine derivatives (39).

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In this study, two chemical epigenetic modulators were used: nicotinamide (NAA)   To better understand the magnitude of HDACs regulation of the secondary 331 metabolism in P. brasilianum, as well as to compare HDAC inhibition through clr3 332 deletion and chemical epigenetic modulation, metabolite quantification was performed.
333 Metabolites 1-15 were quantified, although data is presented only for those with