Deletion of either the regulatory gene ara1 or metabolic gene xki1 in Trichoderma reesei leads to increased CAZyme gene expression on crude plant biomass.

BackgroundTrichoderma reesei is one of the major producers of enzymes for the conversion of plant biomass to sustainable fuels and chemicals. Crude plant biomass can induce the production of CAZymes in T. reesei, but there is limited understanding of how the transcriptional response to crude plant biomass is regulated. In addition, it is unknown whether induction on untreated recalcitrant crude plant biomass (with a large diversity of inducers) can be sustained for longer. We investigated the transcriptomic response of T. reesei to the two industrial feedstocks, corn stover (CS) and soybean hulls (SBH), over time (4 h, 24 h and 48 h), and its regulatory basis using transcription factor deletion mutants (Δxyr1 and Δara1). We also investigated whether deletion of a xylulokinase gene (Δxki1) from the pentose catabolic pathway that converts potential inducers could lead to increased CAZyme gene expression.ResultsBy analyzing the transcriptomic responses using clustering as well as differential and cumulative expression of plant biomass degrading CAZymes, we found that corn stover induced a broader range and higher expression of CAZymes in T. reesei, while SBH induced more pectinolytic and mannanolytic transcripts. XYR1 was the major TF regulating CS utilization, likely due to the significant amount of d-xylose in this substrate. In contrast, ARA1 had a stronger effect on SBH utilization, which correlates with a higher abundance of l-arabinose in SBH that activates ARA1. Blocking pentose catabolism by deletion of xki1 led to higher expression of CAZyme encoding genes on both substrates at later time points. Surprisingly, this was also observed for Δara1 at later time points. Many of these genes were XYR1 regulated, suggesting that inducers for this regulator accumulated over time on both substrates.ConclusionOur data demonstrates the complexity of the regulatory system related to plant biomass degradation in T. reesei and the effect the feedstock composition has on this. Furthermore, this dataset provides leads to improve the efficiency of a T. reesei enzyme cocktail, such as by the choice of substrate or by deleting xki1 to obtain higher production of plant biomass degrading CAZymes

Dissecting semi-specialist and specialist plant biomass utilization strategies using the fungi Trichoderma reesei and Podospora anserina

Fungi are the most important and efficient plant biomass degrading microorganisms and use different strategies to degrade plant biomass, reflecting their ecological niches. Only a deep understanding of these strategies, especially during growth on crude plant biomass and not only on simple mono- or polysaccharides, will lead to further improvements of biorefinery processes that use them as a substrate. The aim of this PhD thesis is to dissect the plant biomass degrading strategies of specialist (Podospora anserina), semi-specialist (Trichoderma reesei) and generalist (Aspergillus niger) fungi. Chapter 2 described the characterization of the missing regulator responding to L-arabinose and D-galactose (two of the main monosaccharides in plant biomass) in T. reesei: ARA1, which is a functional but not sequence ortholog of the arabinanolytic regulator AraR from the generalist Aspergillus niger, demonstrating a clear case of parallel evolution. Chapter 3 described a simple method to overcome senescence in P. anserina, which was used in Chapter 4 for a time-course transcriptome during growth on two industrial feedstocks, soybean hulls (SBH) (dicot, richer in pectin) and corn stover (CS) (monocot, richer in hemicellulose). Overall, SBH resulted in a larger diversity of expressed genes, confirming previous proteomics studies. Our results provide an in depth view of the transcriptomic adaptation of P. anserina to substrate composition, but also pointed out strategies to improve saccharification of plant biomass at the industrial level, such as novel enzymes or harvesting time for enzyme production. The same feedstocks (CS and SBH) were used in Chapter 5 to investigate the time course transcriptomic response of T. reesei. Two regulatory (Δxyr1 and Δara1) and one catabolic (Δxki1) mutant were used together with the wild type to deeply investigate its degrading strategy. CS induced a broader range and higher expression of CAZyme encoding genes in T. reesei, while SBH induced more pectinolytic and mannanolytic genes. XYR1 was the major TF regulating CS utilization, while ARA1 had a stronger effect on SBH utilization, matching with the substrate composition. Blocking pentose catabolism by deletion of xki1 led to higher expression of CAZyme encoding genes on both substrates at later time points. Surprisingly, this was also observed for Δara1 at later time points. Many of these genes were XYR1 regulated, suggesting that inducer(s) for this regulator accumulated over time on both substrates. This dataset provides leads to improve the efficiency of a T. reesei enzyme cocktail, such as by the choice of substrate or by deleting xki1 to obtain higher production of plant biomass degrading CAZymes. Finally in Chapter 6 I discussed the initial question that leads this thesis: what makes a fungus a generalist, a semi-specialist or a specialist? This answer is only at its initial stage and more studies are required, but using the data from Chapters 2, 4 and 5 and previously studies in A. niger, it appears that CAZyme gene content in fungal genomes can only partially indicate the fungal strategy, while their regulation may be the key factor, supporting our initial hypothesis (see Chapter 1)

Dissecting semi-specialist and specialist plant biomass utilization strategies using the fungi Trichoderma reesei and Podospora anserina

Fungi are the most important and efficient plant biomass degrading microorganisms and use different strategies to degrade plant biomass, reflecting their ecological niches. Only a deep understanding of these strategies, especially during growth on crude plant biomass and not only on simple mono- or polysaccharides, will lead to further improvements of biorefinery processes that use them as a substrate. The aim of this PhD thesis is to dissect the plant biomass degrading strategies of specialist (Podospora anserina), semi-specialist (Trichoderma reesei) and generalist (Aspergillus niger) fungi. Chapter 2 described the characterization of the missing regulator responding to L-arabinose and D-galactose (two of the main monosaccharides in plant biomass) in T. reesei: ARA1, which is a functional but not sequence ortholog of the arabinanolytic regulator AraR from the generalist Aspergillus niger, demonstrating a clear case of parallel evolution. Chapter 3 described a simple method to overcome senescence in P. anserina, which was used in Chapter 4 for a time-course transcriptome during growth on two industrial feedstocks, soybean hulls (SBH) (dicot, richer in pectin) and corn stover (CS) (monocot, richer in hemicellulose). Overall, SBH resulted in a larger diversity of expressed genes, confirming previous proteomics studies. Our results provide an in depth view of the transcriptomic adaptation of P. anserina to substrate composition, but also pointed out strategies to improve saccharification of plant biomass at the industrial level, such as novel enzymes or harvesting time for enzyme production. The same feedstocks (CS and SBH) were used in Chapter 5 to investigate the time course transcriptomic response of T. reesei. Two regulatory (Δxyr1 and Δara1) and one catabolic (Δxki1) mutant were used together with the wild type to deeply investigate its degrading strategy. CS induced a broader range and higher expression of CAZyme encoding genes in T. reesei, while SBH induced more pectinolytic and mannanolytic genes. XYR1 was the major TF regulating CS utilization, while ARA1 had a stronger effect on SBH utilization, matching with the substrate composition. Blocking pentose catabolism by deletion of xki1 led to higher expression of CAZyme encoding genes on both substrates at later time points. Surprisingly, this was also observed for Δara1 at later time points. Many of these genes were XYR1 regulated, suggesting that inducer(s) for this regulator accumulated over time on both substrates. This dataset provides leads to improve the efficiency of a T. reesei enzyme cocktail, such as by the choice of substrate or by deleting xki1 to obtain higher production of plant biomass degrading CAZymes. Finally in Chapter 6 I discussed the initial question that leads this thesis: what makes a fungus a generalist, a semi-specialist or a specialist? This answer is only at its initial stage and more studies are required, but using the data from Chapters 2, 4 and 5 and previously studies in A. niger, it appears that CAZyme gene content in fungal genomes can only partially indicate the fungal strategy, while their regulation may be the key factor, supporting our initial hypothesis (see Chapter 1)

Silenziamento del gene putativo ModA in Fusarium oxysporum per lo studio della compatibilita vegetativa

L'incompatibilità vegetativa nei funghi è studiata da molto tempo, ma ancora oggi alcuni aspetti rimangono poco chiari. Questa ricerca si è proposta di studiare in F. oxysporum f. sp. lycopersici (Fol) un gene omologo al gene ModA di P. anserina, implicato nella morte cellulare per incompatibilità vegetativa. Tramite ricerche in banca dati e allineamento blast è stato individuato il gene Foxg-03819 in Fusarium oxysporum, codificante per una proteina ipotetica di 699 aminoacidi, come omologo di ModA di P. anserina. Tramite blast tra la proteina codificata dal gene ModA di P. anserina e i putativi geni omologhi in alcune specie appartenenti al genere Fusarium, è emerso che in Fox il gene codifica, molto probabilmente, per una famiglia proteica composta da 7 proteine simili, così come in F. verticilloides (4 proteine) e in F. graminearum (3 proteine). Da ricerche effettuate anche su altri organismi fungini, il genere Fusarium sembrerebbe, quindi, l'unico fungo in cui si ipotizza la presenza di geni codificanti per una famiglia proteica. Al fine di studiare questo gene nell’isolato 4287 di Fol, è stato effettuato un knock out, sostituendo il putativo gene ModA con quello per la resistenza all’Igromicina. Sulla base dei risultati dei saggi di patogenicità è stato possibile dedurre che il gene ModA non gioca un ruolo nella patogenicità del fungo, così come dimostrato dai saggi condotti su mela, frutto e pianta di pomodoro (infezione ed adesione alle radici) dove il trasformante ∆ModA ha mantenuto un comportamento perfettamente confrontabile con il ceppo wild type. Dai risultati ottenuti dai test fisiologici, si è potuto dedurre che, in generale, il gene ModA ha un limitato ruolo nella fisiologia di Fol. Sebbene il trasformante ∆ModA abbia mantenuto la capacità di penetrare il cellophane, formare aggregati miceliali, sporulare e crescere su diversi substrati, il gene sembrerebbe, tuttavia, regolare il chemiotropismo nei confronti di un essudato radicale e di un ferormone, come dimostrato in studi condotti in parallelo alla presente tesi. Ulteriori indagini sono necessarie per chiarire questo aspetto. Quando utilizzato nei test di complementazione mediante l’impiego di mutanti nit-, a livello qualitativo il trasformante non ha mostrato alcun difetto nei meccanismi di fusione, dando luogo ad una perfetta compatibilità con tutti i mutanti nit- complementari del ceppo 4287. Essendo noto che in Fol l’efficienza di fusione è inferiore al 100%, sono stati condotti saggi quantitativi di complementazione al fine di verificare il ruolo di ModA nell’efficienza di fusione e sopravvivenza dell’eterocarion. A causa dell’elevato numero di reverenti non è stato possibile ottenere le informazioni attese. Per verificare, infine, se il silenziamento del locus ModA fosse in grado di sopprimere o ridurre la morte cellulare per incompatibilità vegetativa anche in Fox, sarebbe stato opportuno quantificare la frequenza di fusione incompatibile con appositi saggi. Purtroppo ciò non è stato possibile poiché gli unici ceppi disponibili sono risultati essere anche autoincompatibili (Fod75-GFP e Fom18M-Co5). Ulteriori studi, soprattutto a livello quantitativo con ceppi incompatibili (non autoincompatibili), saranno necessari per chiarire il ruolo di ModA in Fol. Molto interessante sarebbe ottenere anche altri trasformanti knock-out sia per il gene ModA che per altri geni con funzioni analoghe per valutarne il ruolo nella compatibilità vegetativa

Sugar catabolism in Aspergillus and other fungi related to the utilization of plant biomass

Fungi are found in all natural and artificial biotopes and can use highly diverse carbon sources. They play a major role in the global carbon cycle by decomposing plant biomass and this biomass is the main carbon source for many fungi. Plant biomass is composed of cell wall polysaccharides (cellulose, hemicellulose, pectin) and lignin. To degrade cell wall polysaccharides to different monosaccharides, fungi produce a broad range of enzymes with a large variety in activities. Through a series of enzymatic reactions, sugar-specific and central metabolic pathways convert these monosaccharides into energy or metabolic precursors needed for the biosynthesis of biomolecules. This chapter describes the carbon catabolic pathways that are required to efficiently use plant biomass as a carbon source. It will give an overview of the known metabolic pathways in fungi, their interconnections, and the differences between fungal species