Penicillium subrubescens adapts its enzyme production to the composition of plant biomass

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Introduction
Plant biomass is one of the most abundant, valuable and sustainable polymeric substrates, and is currently the most suitable resource for renewable bioenergy and biochemical production.Plant biomass consists mainly of lignocellulose that is rich in polysaccharides (cellulose, hemicelluloses and pectin) and lignin (complex aromatic polymer) (Aguilar et al., 2013;Sánchez & Cardona, 2008).To be able to efficiently use such complex material, it needs to be degraded and/or modified.Different methods have been developed for the conversion of lignocellulosic biomass.However, the most environmentally sustainable way relies on the use of enzymes.An assortment of carbohydratehttps://doi.org/10.1016/j.biortech.2020.123477active and lignin-modifying enzymes is required to efficiently and completely depolymerize lignocellulosic biomass.Filamentous fungi are excellent producers of plant cell wall degrading enzymes, and some species can also fine-tune their enzyme production specifically to the target biomass (Mäkelä et al., 2014;Rytioja et al., 2014; van den Brink & de Vries, 2011).Fungal enzymes have been used in a variety of industries, e.g.biofuels, biochemicals, food, feed, pulp and paper, textile, pharmaceutical and cosmetic (Himmel et al., 2007).However, only a handful of species have been developed as fungal cell factories, e.g.Aspergillus niger, Aspergillus oryzae, Trichoderma reesei and Myceliophthora thermophila.This is partly due to the narrow substrate range, low enzyme production level, low secretion capacity, toxin production, and poor growth behavior under standard fermentation conditions that makes many other fungi unattractive for industrial applications (Benocci et al., 2017).
Penicillium subrubescens is a saprobic species belonging to section Lanata-diversicata of the genus Penicillium, which has several distinctive morphological features (Mansouri et al., 2013).The species showed significant promise as its plant cell wall-degrading enzyme production levels and saccharification abilities were similar to that of the industrial species A. niger (Mäkelä et al., 2016).Recently, genomic analysis of P. subrubescens revealed an expanded set of Carbohydrate Active enzyme (CAZyme)-encoding genes in comparison with related Penicillium species, but more similar in general numbers to A. niger and other aspergilli (Peng et al., 2017).This expansion does not occur across all CAZy families, but is rather specific to hemicellulose, pectin and inulin degrading enzymes, occurring particularly in glycoside hydrolase (GH) family GH1, GH11, GH12, GH29, GH32, GH36, GH43, GH51, GH54, GH62 and GH67, as well as carbohydrate esterase (CE) family CE8 (Peng et al., 2017).
Understanding how CAZymes are produced by P. subrubescens during growth on different plant biomass is critical to develop this fungus as a novel fungal cell factory.Previously, we reported the extracellular enzyme activity and saccharification abilities of P. subrubescens cultures grown on wheat bran and sugar beet pulp (Mäkelä et al., 2016).These two feedstocks differ in their carbohydrate composition.The major polysaccharides present in wheat bran are cellulose and (arabino)xylan, whereas those in sugar beet pulp are cellulose, pectin and xyloglucan (Benoit et al., 2015).To gain insight into how P. subrubescens degrades plant biomass, we report a comparative exoproteome and transcriptome analysis of P. subrubescens cultures grown on wheat bran and sugar beet pulp in this study.

Fungal culture conditions
To prepare the substrates for cultivation, wheat bran and sugar beet pulp were ground using Ultra Centrifugal Mill ZM 200 with 12-toothrotor (RETSCH GmbH, Haan, Germany).The final particle size is less than 40 µm.Penicillium subrubescens FBCC1632/CBS132785 was cultivated (10 6 spore/mL) in minimal medium (de Vries et al., 2004) supplemented with 1% wheat bran or 1% sugar beet pulp at 25 °C, 250 rpm.The culture broth was collected every 24 h over the course of three days and centrifuged at 14,000 rpm, 4 °C, 5-10 min, and stored at −20 °C prior to trichloroacetic acid (TCA)/acetone protein precipitation for exoproteome analysis.For transcriptomic analysis, the 3-day old mycelium was harvested as described in Dilokpimol et al., 2017, prior to RNA extraction.

RNA extraction
RNA was extracted from 3-day old cultures grown on wheat bran or sugar beet pulp as previously described in Peng et al., 2017, using TRIzol reagent (Invitrogen/Thermo Fischer Scientific, Carlsbad, CA) and purified by NucleoSpin RNAII (Macherey-Nagel, Düren, Germany).The quality of RNA samples was verified using Fragment Analyser (Advanced Analytical Technologies, Thermo Fisher Scientific, Ankeny, IA).

Trichloroacetic acid/acetone protein precipitation
All steps were performed at 4 °C unless specified otherwise.Two mL of culture broth was centrifuged at 2500 × g for 30 min.The supernatant was collected by centrifugation 37500 × g for 1 h.Two volumes of ice cold 20 mM dithiothreitol (DTT) in 20% TCA/80% acetone were mixed with the supernatant.The mixture was left on ice for 1 h prior to centrifugation at 3200 × g for 30 min.The precipitated protein pellet was washed with 15 mL iced cold 20 mM DTT in 80% acetone and incubated at −20 °C overnight.The solution mixture was centrifuged at 3200 × g for 30 min.The pellet was resuspended in 100 µL 0.25% Anionic Acid Labile Surfactant (AALS) (Protea Biosciences, Morgantown, WV) in 200 mM ammonium bicarbonate pH 7.8.The protein concentration was determined using the RCDC Protein Assay (Bio-Rad, Hercules, CA).

Trypsin digestion and LC-MSMS analysis
Five µg of total protein were digested with trypsin in-solution and analyzed by high pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously (Ozturkoglu Budak et al., 2014).Five µL of resuspended peptides spiked with trypsin digested bovine serum albumin (BSA) at a concentration of 4 µmol/µL as internal standard were injected into a 10 mm × 0.75 µm AQUASIL C18 microcapillary column coupled with a Velos LTQ-Orbitrap mass spectrometer (Thermo-Fisher, San Jose, CA).Proteome Discoverer 1.4 software using the precursor ion quantification workflow with precursor ion peak area value-based protein abundance calculation was used to identify proteins and peptides.MS/MS data was matched to the P. subrubescens FBCC1632/CBS132785 protein sequence database from the Joint Genome Institute.BSA was used as an internal standard as a mean to normalize the detected signals of the different injections.The area values of individual proteins were determined from BSA internal standard and expressed as a fold value.Functional annotations were assigned based on the IPR descriptions and the signal peptide was predicted using SignalP from JGI.

Orthologous mapping
Orthologous mapping was performed as described in Peng et al., 2018.

CAZymes are the main extracellular enzymes produced by P. subrubescens grown on biomass
Identification of extracellular proteins during the growth of P. subrubescens on different biomass offered a detailed insight into the secreted plant biomass degrading enzymes by this fungus as well as its potential degradation mechanism.The culture broths from P. subrubescens grown in wheat bran or sugar beet pulp medium for two and three days were filtered and the supernatants were collected.The samples from the first day were not included for proteomic analysis, because the protein amount was too low for a reliable analysis.In total, 321 proteins were detected in the extracellular proteome, of which 254 and 194 were detected in cultures grown in wheat bran and sugar beet pulp, respectively (Fig. 1).Functional classification of the identified proteins showed that glycoside hydrolases (GHs) were the major secreted enzymes (36% and 43%, respectively) on either substrate.Together with esterases, lipases, lyases and oxidoreductases they made up over 50% of all detected proteins.Proteases, transferases, isomerases and kinases were minor components (< 20%).The remaining proteins were other functions and proteins of unknown function.
CAZymes play a major role in plant biomass degradation, and on either substrate approximately 100 CAZymes were secreted by P. subrubescens covering 40 CAZy families, but specific sets of enzymes were produced on each biomass (Fig. 2).For simplicity, we grouped the main enzymes which can catalyze the hydrolysis of the same polysaccharide together, i.e. xylan-, pectin-, cellulose-, xyloglucan-, inulin-& starch-, and β-glucan-, mannan-& arabinogalactan-protein (AGP) degrading enzymes (Figs.2-4).The accessory enzymes that can hydrolyze multiple substrates are divided into two main groups: 1) the enzymes that can hydrolyze both pectin (side chain) and hemicelluloses (e.g.α-L-arabinofuranosidase, β-galactosidase) were referred to as the Hemicellulose + Pectin group, and 2) the enzymes that can hydrolyze different hemicelluloses but not pectin (e.g.β-glucosidase/β-xylosidase, acetylesterases) were referred to as the Hemicellulose group.P. subrubescens clearly uses different approaches to degrade the two plant biomasses (Fig. 2), indicating an ability to modify the produced enzymes to match the composition of the substrate.This is a phenomenon that has also been observed in some other fungi (Benocci et al., 2019;Berka et al., 2011;Mäkelä et al., 2017).

P. subrubescens uses different approaches and adapts enzyme production to degrade different biomasses
During degradation of wheat bran, P. subrubescens produced mainly xylanolytic enzymes (> 75% of the total CAZymes detected as determined from exoproteome data) targeting both main chain and side residues on Day 2 (Fig. 2 and Fig. 3, Xylan (main chain) and Xylan (side residue) groups).On Day 3, a higher production level of CAZymes targeting cellulose, starch and inulin (> 40% in total CAZyme signal) was observed, even though the xylanolytic enzymes still represented the major part of the proteins (almost 50%).Wheat bran contains a small amount of fructose and fructose-related polysaccharides (Hemdane et al., 2016;Onipe et al., 2015;Saunders & Walker, 1969), which could explain the induction of inulin-degrading enzymes.However, these enzymes were mainly produced on Day 3, suggesting that they may be controlled by similar regulatory systems as starch-or cellulose-degrading enzymes.
This degradation approach is also found in Aspergilli (Benoit et al., 2015), a sister genus of Penicillium.Our results provided molecular support for the previous report in which the activities of endo-β-1,4xylanase, α-L-arabinofuranosidase and β-xylosidase were shown to be very high when growing P. subrubescens on wheat bran (Mäkelä et al., 2016).In addition, when compared to published proteomes of other Penicillia, Penicillium funiculosum, Penicillium oxalicum and Penicillium decumbens (Liu et al., 2013;Ogunmolu et al., 2015;Song et al., 2016), the main cellulose-degrading enzymes detected were quite similar.In contrast, endo-xylanases from GH10 and GH30 were the main xylanases produced by P. subrubescens and P. oxalicum (Song et al., 2016), whereas P. funiculosum and P. decumbens, mainly produced GH11 xylanases (Liu et al., 2013;Ogunmolu et al., 2015).This could be explained by the closer relationship between P. subrubescens and P. oxalicum than to the other Penicillia, based on phylogenetic analysis, even  though P. subrubescens has a significantly higher number of CAZymes than P. oxalicum (Peng et al., 2017).Previously, it was shown that P. subrubescens an efficient inulin degrader (Mansouri et al., 2013).Five putative inulinases from GH32 were highly produced on wheat bran on Day 3 together with starch-and cellulose-degrading enzymes, whereas sugar beet pulp cultivation resulted in the production of low levels of starch degrading enzymes and poor production of a single inulinase (see below).In contrast, cellulolytic enzymes were highly produced on the Day 3 in sugar beet pulp.This indicates that GH32 genes may be controlled by the same regulator as amylolytic genes, but different from cellulolytic genes.Even though wheat bran also contains small amounts of fructose and fructose-containing di-and polysaccharides such as sucrose and fructans (Hemdane et al., 2016;Onipe et al., 2015;Saunders & Walker, 1969), these predicted inulinases only increased their levels on Day 3, indicating that fructose and fructose-containing saccharides may not act as the main inducers unless they are less preferred carbon sources.Furthermore, no inulinase was reported among the main proteins detected from P. funiculosum, P. oxalicum and P. decumbens; thus confirming that the inulinase production by P. subrubescens is unique to this species.
Towards degradation of sugar beet pulp P. subrubescens tended to degrade side chains of pectin before attacking the pectin main chain and cellulose (Fig. 4).During growth on sugar beet pulp, pectin-and pectin/hemicellulose-degrading enzymes (~80%, in particular debranching ones) were the major CAZymes produced by P. subrubescens on Day 2 (Fig. 4 in Pectin (side chain/residue) and Hemicellulose + Pectin groups).Enzymes acting on the pectin main chain became more pronounced on Day 3 (Fig. 4).Interestingly, the cellulolytic enzymes almost reached 50% of all produced CAZymes on Day 3, similar to those in wheat bran, suggesting that cellulose is a secondary substrate for P. subrubescens.In addition, four putative α-L-arabinofuranosidases (Protein ID 13297 from GH51 and Protein IDs 1940, 4850 and 6724 from GH54) were among the highest CAZymes produced by P. subrubescens grown in sugar beet pulp on Day 2. These predicted α-L-arabinofuranosidases were different from those predominantly detected in wheat bran, supporting the premise that P. subrubescens produced specific set of enzymes targeting different polysaccharides.These results also suggest that some of the expanded gene families in this species allow it to respond to plant biomass with diverse enzyme sets.Overall, P. subrubescens is an effective pectin degrading fungus, because it produced an almost complete set of required pectin-degrading enzymes at high level including pectin/pectate lyases, exo-polygalacturonases, endopolygalacturonase, and rhamnogalacturonan endolyases together with several pectic debranching enzymes (e.g.α-L-arabinofuranosidases, endo-arabinanases, β-galactosidases, endo-β-1,4-galactanase, α-L-fucosidase, and feruloyl esterases) (Fig. 4).Inulin-and starch-degrading enzymes were produced at low levels in sugar beet pulp.
To further explore the possible cellular machinery for plant biomass degradation of P. subrubescens, transcriptome analysis of Day 3 mycelium of P. subrubescens grown in wheat bran or sugar beet pulp was performed (Fig. 5).Transcriptomics confirmed the high expression level of genes encoding xylan-, cellulose-, starch-and inulin-degrading enzymes in wheat bran, as well as the high expression level of genes encoding pectin-degrading enzymes in sugar beet pulp.The genes encoding enzymes in Hemicellulose + Pectin and cellulose groups were different from the exoproteome data, which could indicate a delay in the detected upregulation of some of these genes between transcriptome and proteome profiles.The transcriptome also showed high expression levels of genes encoding enzymes degrading β-glucan and arabinogalactan-proteins in both wheat bran and sugar beet pulp, however their corresponding enzymes were barely detected in the proteomic analysis.
3.3.Putative regulators of plant biomass degrading enzymes from P. subrubescens Expression of genes encoding plant biomass degrading enzymes occurs via a network of transcriptional regulators.The sensing of inducers starts a signaling pathway leading to the activation of transcriptional regulators (or transcription factors), which then regulate the production of plant biomass degrading enzymes (Benocci et al., 2017;de Vries & Visser, 2001).It has been suggested that the inducers (monoand short oligosaccharides) are produced by hydrolysis of polysaccharides by small amounts of constitutively produced enzymes or scouting enzymes, which then trigger the transcriptional factors of the main hydrolytic response.Genes encoding putative transcription factors involved in plant biomass conversion were identified based on Benocci et al. (2017).Most of these were expressed at similar levels on either substrate (Table 1), which is as expected because even though transcription factors are essential for the regulation of gene expression, only small amounts are needed to modulate the regulatory network.In addition, several regulators are produced at constitutive levels and are controlled by post-transcriptional activation.We observed indications of a fine-tuning mechanism between the putative repressors (e.g.CreA, ACE1 and HCR-1 and GaaX) and the putative activators (e.g.CLR-1, CLR-2, McmA) for expression of genes encoding plant biomass degrading enzymes, possibly since the transcriptomic analysis was performed on the third day, at which point the fungus had already grown past the initial stage of plant biomass degradation.
3.4.A potential new fungal cell factory for novel CAZymes P. subrubescens contains > 700 predicted CAZyme-encoding genes in its genome, which is approximately 20-50% higher than most other genome-sequenced Penicilli (MycoCosm, https://mycocosm.jgi.doe.gov/mycocosm/home) (Grigoriev et al., 2013;Peng et al., 2017).Furthermore, the CAZyme-encoding genes related to plant biomass degradation as well as enzyme production levels, and saccharification abilities were similar to that of the industrial species A. niger (Mäkelä et al., 2016).Based on the exoproteome data, P. subrubescens produced complete sets of enzymes required to degrade complex polysaccharides, e.g.xylan and pectin, even though only few unique CAZymes, having no orthologs to other related species, were produced (Figs. 3, 4).Characterization of some of the unique CAZymes in expanded families demonstrated that their corresponding genes are functional and not pseudogenes (Coconi Linares et al., 2020).The absence of some of them in our proteomics and transcriptomics data may indicate that they are produced under specific growth conditions not covered by our study.Based on its ability to produce specific CAZymes (Mäkelä et al., 2016), the unusually high number of CAZy genes in its genome (Peng et al., 2017) and its ease of cultivation and genetic modification (Salazar-Cerezo et al., 2020), P. subrubescens has a high potential to become a novel fungal enzyme factory, particularly for the production of plant biomass degrading enzyme cocktails.

Conclusion
Exoproteomic analysis has expanded our understanding of the different approaches used by P. subrubescens to degrade two common feedstocks.To degrade xylan, P. subrubescens attacked both the main chain and side residues at the same time, whereas to degrade pectin it attacked the branches first.Our study points to the potential of P. subrubescens as a novel fungal cell factory for production of lignocellulolytic enzymes.Our future studies will address the regulatory mechanisms underlying lignocellulolytic enzyme production by P. subrubescens to improve the strain as a cell factory for extracellular enzyme production.

Table 1
Predicted regulators involved in plant biomass degradation in P. subrubescens with their expression level based on transcriptome analysis.

Fig. 1 .
Fig. 1.Distribution of extracellular proteins showing an overlap as well as differential protein secretion by P. subrubescens grown on wheat bran or sugar beet pulp.Numbers in the diagram represent amount and percentage of proteins/enzymes detected in each category.

Fig. 2 .
Fig. 2. Distribution of CAZymes produced by P. subrubescens (grouped based on activity targeting main polysaccharide components) grown on wheat bran or sugar beet pulp for two and three days as analyzed by exoproteomics.

Fig. 3 .
Fig. 3. Distribution of CAZymes in the exoproteome of P. subrubescens grown in liquid minimal medium containing 1% wheat bran for two and three days, and their transcriptome profile on Day 3. Orange, light orange, yellow, light pink, green, light green, blue, purple, pink and gray indicate xylan-(main chain), xylan-(side residue), hemicellulose, hemicellulose + pectin, pectin-(main chain), pectin-(side chain), cellulose-, xyloglucan-, inulin-& starch-, and β-glucan-, mannan-& arabinogalactan-protein (AGP) degrading enzymes.U indicates the protein/gene that has no orthologs in related species.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .
Fig. 5. Distribution of genes encoding CAZymes by P. subrubescens (grouped based on activity targeting main polysaccharide components) grown on wheat bran or sugar beet pulp for three days as analyzed by transcriptomics.
Benocci et al. (2017).(2017)b Genes among the top 10% are in bold and italics; genes among the top 20% are in bold.
Transcription factor a Accession number Candidate in P. subrubescens % identity Expression level (FPKM) b Wheat bran Sugar beet pulp Cellulose, hemicellulose and pectin regulators a