[4S02a] 03. Fermentation Physiology/ Technology - Part 2

3:20 PM - 3:40 PM JST(6:20 AM - 6:40 AM UTC)

[4Ferm-08] Linking Hypoxia-Response Analysis to Improved Productivity in Industrially Relevant Stirred-Tank Fermentation of Aspergillus oryzae

○Soma Araki¹, Shunya Susukida¹, Jikian Tokashiki¹, Kiyoaki Muto¹, Ken Miyazawa¹, Akira Yoshimi², Keietsu Abe¹ (1. Tohoku Univ. (Japan), 2. Kyoto Univ. (Japan))

Keywords:

Hypoxia,Aspergillus oryzae,ROS,Stirred-tank fermentation,Productivity

Filamentous fungi, such as Aspergillus oryzae, are widely used for production of diverse bioactive and industrially relevant organic compounds. In industrial applications, stirred-tank bioreactor (STBR) fermentation is common as it provides efficient oxygen transfer to meet the high oxygen demand of filamentous fungi. However, during cultivation, oxygen transfer becomes limiting for various reasons (e.g., biomass increase, unique morphology), leading to hypoxic environments despite continuous aeration. Such oxygen limitation is highly relevant to biomass growth and productivity in industrial fermentation; therefore, it is important to understand the responses to such environments. It is conceivable that the largest cellular responses occur in the short term; however, they remain poorly understood. To address this gap, we conducted short-term (≦6 hours) multi-omics profiling of a hyphal-dispersion strain of A. oryzae grown in a 4 L STBR in which the dissolved oxygen (DO) concentration was reduced to ≦1% (hypoxia). Additionally, we performed a genetic modification based on our findings and tested the productivity of a target enzyme.Transcriptome analysis revealed that several genes related to antioxidant enzymes were significantly upregulated in hypoxia, most prominently within 1 hour. Genes involved in some known antioxidant systems (e.g., glutathione system) were also upregulated. Based on these data, we conducted a reactive oxygen species (ROS) assay. Interestingly, a transient rise in intracellular ROS levels was observed after 1 hour of hypoxia, followed by rapid normalization. Our metabolome data showed a decrease in the reduced/oxidized glutathione ratio in hypoxia, suggesting enhanced ROS detoxification. These results provide new insights into how early hypoxia triggers a transient increase in intracellular ROS levels, and several responses, represented by the upregulation of antioxidant enzymes and related systems, buffer the oxidative stress in A. oryzae during STBR fermentation.On the basis of these findings, we further investigated whether the insights obtained from short-term multi-omics profiling could be applied to strain improvement. An overexpression strain carrying an antioxidant-related gene was therefore constructed, and the productivity of an endogenous target enzyme driven by a high-expression promoter was evaluated. Fermentation tests in both 250 mL and 5 L scale bioreactors confirmed that enzyme productivity was increased in the engineered strain. Collectively, these results demonstrate the utility of our approach not only for dissecting the early cellular responses to hypoxia, but also for identifying biological and genetic targets for improvement in productivity in the STBR fermentation.