Unraveling the regulation of sugar beet pulp utilization in the industrially relevant fungus Aspergillus niger

Garrigues, Sandra; Kun, Roland S.; Peng, Mao; Bauer, Diane; Keymanesh, Keykhosrow; Lipzen, Anna; Ng, Vivian; Grigoriev, Igor V.; Vries, Ronald P. de

doi:10.1016/j.isci.2022.104065

Cited by 7 publications

(6 citation statements)

References 46 publications

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“…First, the gaaR and araR genes were simultaneously knocked out. The double deletion strain Δ gaaR Δ araR was used as background for the deletion of rhaR and galX , resulting in the quadruple deletion strain Δ gaaR Δ araR Δ rhaR Δ galX , which was used for the generation of the Δ gaaR Δ araR Δ rhaR Δ galX Δ xlnR Δ clrB sextuple deletion strain ( 56 ). Finally, the octuple Δ gaaR Δ araR Δ rhaR Δ galX Δ xlnR Δ clrB Δ amyR Δ inuR deletion strain was generated by the deletion of amyR and inuR in the sextuple deletion strain, while the decuple deletion strain Δ gaaR Δ araR Δ rhaR Δ galX Δ xlnR Δ clrB Δ amyR Δ inuR Δ clrA Δ creA was generated by the double deletion of clrA and creA in the octuple deletion strain (R. S. Kun, S. Garriegues, R. P. de Vries, unpublished data).…”

Section: Methodsmentioning

confidence: 99%

Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9

Garrigues

Peng

Kun

et al. 2023

mBio

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CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain Δ kusA . Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci—known as off-target mutations—could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development.

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Section: Methodsmentioning

confidence: 99%

Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9

Garrigues

Peng

Kun

et al. 2023

mBio

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“…The fungi Aspergillus niger, A. oryzae, and Trichoderma reesei were shown to possess a variety of enzymes that could degrade the complex structure of lignocellulosic substrates [48]. Fungi, such as A. niger, were reported to have a flexible regulatory network adapted to the utilisation of hemicellulose and cellulose when pectin degradation was impaired [49]. In the literature, it was reported that the cultivation pH and temperature are significant factors affecting fungal hydrolytic activity [19,21].…”

Section: One-factor Experimentsmentioning

confidence: 99%

“…The optimisation of fermentation conditions for the production of enzymes pectinase and cellulase by A. niger NCIM 548 was performed on different substrates, such as wheat bran, corn bran, and kinnow peel [19]. In the study of Garrigues et al, A. niger was involved in the SBP degradation to assess the role of pectinolytic and hemicelulolytic enzyme regulators [49]. In the work of Berlowska and co-authors, the efficiency of lactic acid production using simultaneous enzymatic hydrolysis and the fermentation of SPB was analysed [10].…”

Section: One-factor Experimentsmentioning

confidence: 99%

Optimised Degradation of Lignocelluloses by Edible Filamentous Fungi for the Efficient Biorefinery of Sugar Beet Pulp

Gaizauskaite,

Zvirdauskiene,

Svazas

et al. 2024

Polymers

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The degradation of the complex structure of lignocellulosic biomass is important for its further biorefinery to value-added bioproducts. The use of effective fungal species for the optimised degradation of biomass can promote the effectiveness of the biorefinery of such raw material. In this study, the optimisation of processing parameters (temperature, time, and s/w ratio) for cellulase activity and reducing sugar (RS) production through the hydrolysis of sugar beet pulp (SBP) by edible filamentous fungi of Aspergillus, Fusarium, Botrytis, Penicillium, Rhizopus, and Verticillium spp. was performed. The production of RS was analysed at various solid/water (s/w) ratios (1:10–1:20), different incubation temperatures (20–35 °C), and processing times (60–168 h). The Aspergillus niger CCF 3264 and Penicillium oxalicum CCF 3438 strains showed the most effective carboxymethyl cellulose (CMC) degrading activity and also sugar recovery (15.9–44.8%) from SBP biomass in the one-factor experiments. Mathematical data evaluation indicated that the highest RS concentration (39.15 g/100 g d.w.) and cellulolytic activity (6.67 U/g d.w.) could be achieved using A. niger CCF 3264 for the degradation of SBP at 26 °C temperature with 136 h of processing time and a 1:15 solid/water ratio. This study demonstrates the potential of fungal degradation to be used for SBP biorefining.

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“…The transcriptomes of the control and deletion mutant strains cultivated for 2 and 8 h in liquid cultures and cultivated for 40 h on solid cultures were analyzed using RNA-seq. RNA isolation, purification, and quantitative and qualitative evaluation were performed as previously described [32].…”

Section: Transcriptomics Analysismentioning

confidence: 99%

“…For liquid culture samples, purification of mRNA, synthesis of cDNA library and sequencing were performed at the Joint Genome Institute (JGI, US). RNA sequencing, processing of raw fastq reads and evaluation of raw gene counts were performed as previously reported [32]. Three biological replicates were generated and sequenced for each condition.…”

Section: Transcriptomics Analysismentioning

confidence: 99%

The Amylolytic Regulator AmyR of Aspergillus niger Is Involved in Sucrose and Inulin Utilization in a Culture-Condition-Dependent Manner

Kun

Salazar-Cerezo

Peng

et al. 2023

JoF

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Filamentous fungi degrade complex plant material to its monomeric building blocks, which have many biotechnological applications. Transcription factors play a key role in plant biomass degradation, but little is known about their interactions in the regulation of polysaccharide degradation. Here, we deepened the knowledge about the storage polysaccharide regulators AmyR and InuR in Aspergillus niger. AmyR controls starch degradation, while InuR is involved in sucrose and inulin utilization. In our study, the phenotypes of A. niger parental, ΔamyR, ΔinuR and ΔamyRΔinuR strains were assessed in both solid and liquid media containing sucrose or inulin as carbon source to evaluate the roles of AmyR and InuR and the effect of culture conditions on their functions. In correlation with previous studies, our data showed that AmyR has a minor contribution to sucrose and inulin utilization when InuR is active. In contrast, growth profiles and transcriptomic data showed that the deletion of amyR in the ΔinuR background strain resulted in more pronounced growth reduction on both substrates, mainly evidenced by data originating from solid cultures. Overall, our results show that submerged cultures do not always reflect the role of transcription factors in the natural growth condition, which is better represented on solid substrates. Importance: The type of growth has critical implications in enzyme production by filamentous fungi, a process that is controlled by transcription factors. Submerged cultures are the preferred setups in laboratory and industry and are often used for studying the physiology of fungi. In this study, we showed that the genetic response of A. niger to starch and inulin was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment did not fully match the behavior of the fungus in a solid environment. These results have direct implications in enzyme production and would help industry choose the best approaches to produce specific CAZymes for industrial purposes.

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Unraveling the regulation of sugar beet pulp utilization in the industrially relevant fungus Aspergillus niger

Cited by 7 publications

References 46 publications

Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9

Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9

Optimised Degradation of Lignocelluloses by Edible Filamentous Fungi for the Efficient Biorefinery of Sugar Beet Pulp

The Amylolytic Regulator AmyR of Aspergillus niger Is Involved in Sucrose and Inulin Utilization in a Culture-Condition-Dependent Manner

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