Periodic Reporting for period 1 - NURoFE (Nuclear Regulators of Fungal Enzymes)
Okres sprawozdawczy: 2021-05-26 do 2023-05-25
1. Explanation of the work carried out by the beneficiaries and overview of the proposal
This proposal aims to uncover new nuclear regulators of fungal enzymes regulating important enzymes for renewable biofuels and secondary metabolite production under Carbon Catabolite Repression (CCR) that respond to glucose-mediated signaling using Aspergillus nidulans as a model. The degradation of biomass and usage for renewable biofuels have a bottleneck in the limited amount of enzymes produced and the high cost, this proposal aims to find out new nuclear regulators allowing to improve the secretion of hydrolytic enzymes such as xylanases and cellulases production turning feasible the conversion of biomass cellulose-based into green biofuel, reducing the usage of biomass waste for the production of electricity generated by combustion, impairing the CO2 emissions and supporting the reduction of global warming. Furthermore, glucose repression also plays a role in secondary metabolite production that holds importance in pharmaceutical, medical, and agricultural applications such as food poisoning by the toxin aflatoxin, the targets identified can be used for agricultural applications that aim the reduction of food waste, another major worldwide concern once the food could become limited in the future.
This report shows the data collected from the initial experiments executed that correspond to the period from May 26th to October 1st, 2021.
1.2 Objectives
The overall aim of this project is to identify uncharacterized proteins regulating CCR and how these proteins are controlling xylanase, cellulase, and secondary metabolite production. To achieve this, the proposal was break-down into work packages (Fig. 1) described below, showing how the project should be developed during the execution of the Marie-Curie action.
1.2.1 Work Package 1
The wild-type Aspergillus strain will be grown in minimal media supplemented with 1% xylan (w/v) as a carbon source for 24 h and then 2% (w/v) glucose will be added to this media for 1 h to activate CCR. Nuclear fractions from both xylan and glucose plus xylan grown media will be prepared as described previously1,2. The aim is to identify proteins with a role in CCR that translocate to the nucleus in the presence of glucose, compared with xylan nuclear profile using mass spectrometry.
This proposal aims to uncover new nuclear regulators of fungal enzymes regulating important enzymes for renewable biofuels and secondary metabolite production under Carbon Catabolite Repression (CCR) that respond to glucose-mediated signaling using Aspergillus nidulans as a model. The degradation of biomass and usage for renewable biofuels have a bottleneck in the limited amount of enzymes produced and the high cost, this proposal aims to find out new nuclear regulators allowing to improve the secretion of hydrolytic enzymes such as xylanases and cellulases production turning feasible the conversion of biomass cellulose-based into green biofuel, reducing the usage of biomass waste for the production of electricity generated by combustion, impairing the CO2 emissions and supporting the reduction of global warming. Furthermore, glucose repression also plays a role in secondary metabolite production that holds importance in pharmaceutical, medical, and agricultural applications such as food poisoning by the toxin aflatoxin, the targets identified can be used for agricultural applications that aim the reduction of food waste, another major worldwide concern once the food could become limited in the future.
This report shows the data collected from the initial experiments executed that correspond to the period from May 26th to October 1st, 2021.
1.2 Objectives
The overall aim of this project is to identify uncharacterized proteins regulating CCR and how these proteins are controlling xylanase, cellulase, and secondary metabolite production. To achieve this, the proposal was break-down into work packages (Fig. 1) described below, showing how the project should be developed during the execution of the Marie-Curie action.
1.2.1 Work Package 1
The wild-type Aspergillus strain will be grown in minimal media supplemented with 1% xylan (w/v) as a carbon source for 24 h and then 2% (w/v) glucose will be added to this media for 1 h to activate CCR. Nuclear fractions from both xylan and glucose plus xylan grown media will be prepared as described previously1,2. The aim is to identify proteins with a role in CCR that translocate to the nucleus in the presence of glucose, compared with xylan nuclear profile using mass spectrometry.
2 Work Performed
The proposal describes in the WP1, preparation of the nuclear fraction protocol isolating nuclear fractions from the samples grown in xylan and glucose plus xylan, finally, mass spectrometry using nuclear fractions should be applied and samples analyzed. The enrichment of the nuclear fraction was observed by Western blot using anti-histone H3 antibody (Abcam ab1791) rabbit polyclonal and as a positive control was used calf thymus histone purified (Roche #10223565001). The samples were divided into control (Histone H3 purified), XT (Xylan total cell lysate), XC (Xylan cytoplasm fraction), and XN (Xylan nuclear fraction), GT (Glucose total cell lysate), GC (Glucose cytoplasm fraction), and GN (Glucose nuclear fraction), as observed in Fig. 2 (attached), the fraction XN and GN showed higher concentration of histone H3 while the fractions XC didn’t show histone detection confirming that nuclei enrichment was achieved. As expected, XT and GT holding the total cell lysate fractions showed a weaker band detecting histone H3 due to the presence of both cytoplasm and nuclear fractions (Fig. 2). Additionally, the amount of ß-actin was measured by Western blot, as expected, total cell lysate and cytoplasm fractions showed the highest amount of ß-actin than the nuclear fraction in both conditions, ß-actin abundance is higher in the cytoplasm than nucleus5 confirming again the absence in the nuclear preparation.
Proteins from the cytoplasm and nuclear fractions from strains grown in xylan or xylan supplemented with glucose as a carbon source were prepared for mass spectrometry as described previously1,2. The data analysis was done using the software Proteome Discoverer 2.0 Daemon (Thermo Fisher) using Aspergillus nidulans database for peptide identification. The results were submitted to a second analysis using filtering from Microsoft Office Excel checking communal proteins identified in each replicate for consistency of the data. The results from cells grown in xylan (cytoplasm and nuclear fractions) and xylan supplemented with glucose (cytoplasm and nuclear fractions) were submitted to a third filtering process selecting communal and unique proteins for each condition. Indeed, after the addition of glucose, it was identified unique proteins in glucose treatment that moved to the nucleus (Fig. 3), among them was found the transcription factor CreA (AN6195) already described as a nuclear regulator of CCR in the presence of glucose. CreA also can be used as an experimental control showing that CCR is in place. Regarding the presence of the nuclear proteins due to posttranslational modification and movement to the nucleus or increased expression in the presence of glucose, the analysis of RNAseq using A. nidulans AGB551 in the same conditions showed lower fold-increase variation (<1) for all proteins of interest (POI) identified (Fig. 3). Moreover, the POIs have submitted to functional classification grouping the target proteins and showing the nuclear regulators highlighted in red (Fig. 4).
The results are promising and the delivery of the WP1 shown in the Gantt Chart was done, it was predicted to be executed into 6 months since the project was started, showing that the project was 2 months forward in the schedule, however, due to premature termination of the grant, the step for dissemination and exploitation could not be executed as planned. The researcher was able to acquire skills in nuclear fraction preparation and mass spectrometer device learning and analysis including mass spectrometry analysis and functional classification for the POI.
The proposal describes in the WP1, preparation of the nuclear fraction protocol isolating nuclear fractions from the samples grown in xylan and glucose plus xylan, finally, mass spectrometry using nuclear fractions should be applied and samples analyzed. The enrichment of the nuclear fraction was observed by Western blot using anti-histone H3 antibody (Abcam ab1791) rabbit polyclonal and as a positive control was used calf thymus histone purified (Roche #10223565001). The samples were divided into control (Histone H3 purified), XT (Xylan total cell lysate), XC (Xylan cytoplasm fraction), and XN (Xylan nuclear fraction), GT (Glucose total cell lysate), GC (Glucose cytoplasm fraction), and GN (Glucose nuclear fraction), as observed in Fig. 2 (attached), the fraction XN and GN showed higher concentration of histone H3 while the fractions XC didn’t show histone detection confirming that nuclei enrichment was achieved. As expected, XT and GT holding the total cell lysate fractions showed a weaker band detecting histone H3 due to the presence of both cytoplasm and nuclear fractions (Fig. 2). Additionally, the amount of ß-actin was measured by Western blot, as expected, total cell lysate and cytoplasm fractions showed the highest amount of ß-actin than the nuclear fraction in both conditions, ß-actin abundance is higher in the cytoplasm than nucleus5 confirming again the absence in the nuclear preparation.
Proteins from the cytoplasm and nuclear fractions from strains grown in xylan or xylan supplemented with glucose as a carbon source were prepared for mass spectrometry as described previously1,2. The data analysis was done using the software Proteome Discoverer 2.0 Daemon (Thermo Fisher) using Aspergillus nidulans database for peptide identification. The results were submitted to a second analysis using filtering from Microsoft Office Excel checking communal proteins identified in each replicate for consistency of the data. The results from cells grown in xylan (cytoplasm and nuclear fractions) and xylan supplemented with glucose (cytoplasm and nuclear fractions) were submitted to a third filtering process selecting communal and unique proteins for each condition. Indeed, after the addition of glucose, it was identified unique proteins in glucose treatment that moved to the nucleus (Fig. 3), among them was found the transcription factor CreA (AN6195) already described as a nuclear regulator of CCR in the presence of glucose. CreA also can be used as an experimental control showing that CCR is in place. Regarding the presence of the nuclear proteins due to posttranslational modification and movement to the nucleus or increased expression in the presence of glucose, the analysis of RNAseq using A. nidulans AGB551 in the same conditions showed lower fold-increase variation (<1) for all proteins of interest (POI) identified (Fig. 3). Moreover, the POIs have submitted to functional classification grouping the target proteins and showing the nuclear regulators highlighted in red (Fig. 4).
The results are promising and the delivery of the WP1 shown in the Gantt Chart was done, it was predicted to be executed into 6 months since the project was started, showing that the project was 2 months forward in the schedule, however, due to premature termination of the grant, the step for dissemination and exploitation could not be executed as planned. The researcher was able to acquire skills in nuclear fraction preparation and mass spectrometer device learning and analysis including mass spectrometry analysis and functional classification for the POI.
3. Progress beyond the state of the art
The original proposal was predicting the identification of nuclear regulators in the presence of glucose, as an additional experiment, a previous unpublished RNAseq was analyzed and used to verify the fold-increase in the expression of the POIs identified as a nuclear regulator. Indeed, the variation of these proteins was <1 validating those proteins identified in the enriched nuclear fraction moved into the nucleus due to posttranslational modifications instead of a fold-increase in the expression after glucose addition. As expected, the POIs include DNA-binding proteins responsible for DNA regulation and protein expression (Fig. 4), highlighting the presence of proteins related to the Saga complex responsible for transcription of a subset of RNA polymerase II regulating genes regarding environmental response to stresses6.
Unfortunately, the premature termination of the project compromised the real impact of the proposal, although the POIs are very precious once lacks the description and characterization of unique glucose nuclear regulators for xylanase and cellulase production and secondary metabolite repression. The full characterization of the targets could provide possible ways for bioengineering using filamentous fungus aiming to improve green biofuel production via increased hydrolytic enzymes secretion and food preservation by avoiding the production of secondary metabolites such as toxins.
The original proposal was predicting the identification of nuclear regulators in the presence of glucose, as an additional experiment, a previous unpublished RNAseq was analyzed and used to verify the fold-increase in the expression of the POIs identified as a nuclear regulator. Indeed, the variation of these proteins was <1 validating those proteins identified in the enriched nuclear fraction moved into the nucleus due to posttranslational modifications instead of a fold-increase in the expression after glucose addition. As expected, the POIs include DNA-binding proteins responsible for DNA regulation and protein expression (Fig. 4), highlighting the presence of proteins related to the Saga complex responsible for transcription of a subset of RNA polymerase II regulating genes regarding environmental response to stresses6.
Unfortunately, the premature termination of the project compromised the real impact of the proposal, although the POIs are very precious once lacks the description and characterization of unique glucose nuclear regulators for xylanase and cellulase production and secondary metabolite repression. The full characterization of the targets could provide possible ways for bioengineering using filamentous fungus aiming to improve green biofuel production via increased hydrolytic enzymes secretion and food preservation by avoiding the production of secondary metabolites such as toxins.