Presentation Information
[P02-296]Deletion of transcription factor CreA eliminates carbon catabolite repression and improves cell growth in Aspergillus oryzae by activation of the pentose phosphate pathway
○Fami Baihaqqi1, Tomohiro Suzuki1, Satoshi Wakai1,2, Chiaki Ogino1 (1. Kobe University (Japan), 2. JAMSTEC (Japan))
Keywords:
Aspergillus oryzae,CreA,Carbon catabolite repression,Pentose phosphate pathway,EMP pathway
[Purpose]
Aspergillus oryzae has a high capacity to secrete enzymes, making this fungus an ideal host for overexpression. Recently, in our endeavor to optimize IpeA enzyme production through fermentation engineering, we observed a carbon catabolite repression (CCR) pattern in our engineered strain when using >6% glucose as a substrate. Despite employing a strong promoter set of P-sodM/T-glaB for expression, we speculate that significant CCR control may still influence protein synthesis. In Aspergillus spp., CreA and its orthologs are known as major CCR repressor proteins in the presence of glucose or xylose in the media. Although CreA primarily binds in the upstream regions of amylolytic genes, its relationship to protein overexpression has not yet been explored.
[Method]
In this study, a CreA deletion plasmid, pUPG-ΔcreA was used to construct the ΔcreA-strain A. oryzae. Constitutive expression of CreA with different promoters and truncated CreA114 was also introduced into this ΔcreA-strain. We then used the ΔcreA-strain and its derivatives for comparative study through metabolic profiling using LCMS analysis, transcriptomic analysis (qPCR), and protein overexpression.
[Results]
When cultivated in 3% GPY medium, the ΔcreA strain produced higher levels of innate α-amylase compared to the reference strain RIB40, indicating a derepression effect from creA deletion. Additionally, the ΔcreA strain produced higher dry mycelial weight. Interestingly, we observed slower glucose assimilation in the ΔcreA strain compared to RIB40. When CreAwt and truncated CreA114 were constitutively expressed in the deletion strain, glucose assimilation was restored. This suggests that CreA plays a significant role in the glucose metabolism pathway in A. oryzae. Further analysis of metabolic profile and transcriptomic reveals that in the absence of CreA in A. oryzae, carbon metabolism was redirected through the pentose phosphate (PP) pathway instead of EMP pathway. This carbon metabolism redirection via PP pathway suggest increase provisional supply of NADPH and cell building blocks, which resulted in higher specific biomass per substrate in ΔcreA strain.
[Consideration]
Our study reveal broader influence of CreA as CCR regulator in A. oryzae where is diverting carbon metabolism and improvement of biomass via activation of PP pathway and less dominant in EMP pathway, which could reduce by product metabolism such as ethanol. We are currently investigating an functional practicality of CreA deletion for protein overexpression.
[Conclusion]
This result may provide valuable insight into construction of CCR free and more efficient host Aspergillus oryzae for protein production.
Aspergillus oryzae has a high capacity to secrete enzymes, making this fungus an ideal host for overexpression. Recently, in our endeavor to optimize IpeA enzyme production through fermentation engineering, we observed a carbon catabolite repression (CCR) pattern in our engineered strain when using >6% glucose as a substrate. Despite employing a strong promoter set of P-sodM/T-glaB for expression, we speculate that significant CCR control may still influence protein synthesis. In Aspergillus spp., CreA and its orthologs are known as major CCR repressor proteins in the presence of glucose or xylose in the media. Although CreA primarily binds in the upstream regions of amylolytic genes, its relationship to protein overexpression has not yet been explored.
[Method]
In this study, a CreA deletion plasmid, pUPG-ΔcreA was used to construct the ΔcreA-strain A. oryzae. Constitutive expression of CreA with different promoters and truncated CreA114 was also introduced into this ΔcreA-strain. We then used the ΔcreA-strain and its derivatives for comparative study through metabolic profiling using LCMS analysis, transcriptomic analysis (qPCR), and protein overexpression.
[Results]
When cultivated in 3% GPY medium, the ΔcreA strain produced higher levels of innate α-amylase compared to the reference strain RIB40, indicating a derepression effect from creA deletion. Additionally, the ΔcreA strain produced higher dry mycelial weight. Interestingly, we observed slower glucose assimilation in the ΔcreA strain compared to RIB40. When CreAwt and truncated CreA114 were constitutively expressed in the deletion strain, glucose assimilation was restored. This suggests that CreA plays a significant role in the glucose metabolism pathway in A. oryzae. Further analysis of metabolic profile and transcriptomic reveals that in the absence of CreA in A. oryzae, carbon metabolism was redirected through the pentose phosphate (PP) pathway instead of EMP pathway. This carbon metabolism redirection via PP pathway suggest increase provisional supply of NADPH and cell building blocks, which resulted in higher specific biomass per substrate in ΔcreA strain.
[Consideration]
Our study reveal broader influence of CreA as CCR regulator in A. oryzae where is diverting carbon metabolism and improvement of biomass via activation of PP pathway and less dominant in EMP pathway, which could reduce by product metabolism such as ethanol. We are currently investigating an functional practicality of CreA deletion for protein overexpression.
[Conclusion]
This result may provide valuable insight into construction of CCR free and more efficient host Aspergillus oryzae for protein production.
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