Presentation Information
[P02-294]Evaluation of promoters suitable for culturing Aspergillus oryzae using Sorghum juice
○Tomohiro Suzuki1, Satoshi Wakai1,2, Yutaro Mori1, Takashi Sazuka3, Chiaki Ogino1 (1. Grad. Sch. Eng., Kobe Univ. (Japan), 2. X-star., JAMSTEC. (Japan), 3. Grad. Sch. Bioagri. Sci., Nagoya Univ. (Japan))
Keywords:
Aspergillus oryzae,Sorghum,Promotor,Protein production
[Purpose]
Aspergillus oryzae has a high protein secretion capacity. In research on enzyme production using A. oryzae, refined sugars such as glucose and maltose have traditionally been used as substrates; however, there is a growing need for new culture substrates to replace these in order to realize a circular economy. Sorghum juice, extracted from sorghum stalks, is rich in sugars such as sucrose, glucose, and fructose. Furthermore, it has been found to contain amino acids and minerals. Therefore, sorghum juice is a candidate for a novel growth medium that can serve not only as a carbon source but also as a substitute for peptone and yeast extract, which are typically used as nitrogen sources. Furthermore, from the perspective of suitable cultivation areas, sorghum is expected to see an expansion of its cultivation area by utilizing fallow land within Japan, making local production for local consumption feasible.In this study, we searched for a promoter capable of achieving high protein production in A. oryzae cultivation using sorghum juice.
[Method]
Using the A. oryzae NS4ΔligD strain, we cultured the cells in 3% sorghum PY medium adjusted to have the same sugar concentration as the 3% glucose PY medium commonly used as a liquid medium and extracted RNA from the cells after 48 hours of culture to perform transcriptome analysis. We selected the top 10 genes with the highest read counts in the 3% sorghum medium and analyzed their gene expression levels via RT-qPCR using RNA extracted from the cells after 24, 48, 72, and 96 hours of cultivation. Based on these analyses, we used the promoter regions of promising genes to express cellulase (CBHI) and evaluated the promoters through production tests.
[Result]
The heatmap analysis of gene expression on each chromosome based on transcriptomic analysis revealed differences in gene expression depending on the culture medium composition. Furthermore, we were able to identify candidate promoters suitable for cultivation in sorghum juice, including those with high expression levels in the late stages of cultivation and those with consistently high expression levels throughout the entire cultivation process. In particular, the hlyA (AO090010000018) promoter was confirmed to possess high enzymatic activity.
[Consideration]
Unlike the high-expression promoter held by our laboratory, where enzyme production declines in the late stages of cultivation, enzyme production from the hlyA promoter increases over time. This finding demonstrates the need for a promoter suitable for biomanufacturing using sorghum juice.
[Conclusion]
These results confirm the feasibility of using sorghum juice in an enzyme production system using A. oryzae, and suggest that the system could be extended to the production of various other substances based on gene expression profiles.
Aspergillus oryzae has a high protein secretion capacity. In research on enzyme production using A. oryzae, refined sugars such as glucose and maltose have traditionally been used as substrates; however, there is a growing need for new culture substrates to replace these in order to realize a circular economy. Sorghum juice, extracted from sorghum stalks, is rich in sugars such as sucrose, glucose, and fructose. Furthermore, it has been found to contain amino acids and minerals. Therefore, sorghum juice is a candidate for a novel growth medium that can serve not only as a carbon source but also as a substitute for peptone and yeast extract, which are typically used as nitrogen sources. Furthermore, from the perspective of suitable cultivation areas, sorghum is expected to see an expansion of its cultivation area by utilizing fallow land within Japan, making local production for local consumption feasible.In this study, we searched for a promoter capable of achieving high protein production in A. oryzae cultivation using sorghum juice.
[Method]
Using the A. oryzae NS4ΔligD strain, we cultured the cells in 3% sorghum PY medium adjusted to have the same sugar concentration as the 3% glucose PY medium commonly used as a liquid medium and extracted RNA from the cells after 48 hours of culture to perform transcriptome analysis. We selected the top 10 genes with the highest read counts in the 3% sorghum medium and analyzed their gene expression levels via RT-qPCR using RNA extracted from the cells after 24, 48, 72, and 96 hours of cultivation. Based on these analyses, we used the promoter regions of promising genes to express cellulase (CBHI) and evaluated the promoters through production tests.
[Result]
The heatmap analysis of gene expression on each chromosome based on transcriptomic analysis revealed differences in gene expression depending on the culture medium composition. Furthermore, we were able to identify candidate promoters suitable for cultivation in sorghum juice, including those with high expression levels in the late stages of cultivation and those with consistently high expression levels throughout the entire cultivation process. In particular, the hlyA (AO090010000018) promoter was confirmed to possess high enzymatic activity.
[Consideration]
Unlike the high-expression promoter held by our laboratory, where enzyme production declines in the late stages of cultivation, enzyme production from the hlyA promoter increases over time. This finding demonstrates the need for a promoter suitable for biomanufacturing using sorghum juice.
[Conclusion]
These results confirm the feasibility of using sorghum juice in an enzyme production system using A. oryzae, and suggest that the system could be extended to the production of various other substances based on gene expression profiles.
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