Title Transcriptome‑based Mining of the Constitutive Promoters for Tuning Gene Expression in Aspergillus oryzae
Author Kobkul Laoteng, Jutamas Anantayanon, Chanikul Chutrakul, Sarocha Panchanawaporn, and Sukanya Jeennor*
Address Industrial Bioprocess Technology Research Team, Functional Ingredients and Food Innovation Research Group (IFIG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand
Bibliography Journal of Microbiology, 61(2),199-210, 2023,
DOI 10.1007/s12275-023-00020-0
Key Words Aspergillus oryzae · Carbon source · Constitutive promoter · Glyceraldehyde triphosphate dehydrogenase · Transcriptional regulation · Ubiquitin
Abstract Transcriptional regulation has been adopted for developing metabolic engineering tools. The regulatory promoter is a crucial genetic element for strain optimization. In this study, a gene set of Aspergillus oryzae with highly constitutive expression across different growth stages was identified through transcriptome data analysis. The candidate promoters were functionally characterized in A. oryzae by transcriptional control of β-glucuronidase (GUS) as a reporter. The results showed that the glyceraldehyde triphosphate dehydrogenase promoter (PgpdA1) of A. oryzae with a unique structure displayed the most robust strength in constitutively controlling the expression compared to the PgpdA2 and other putative promoters tested. In addition, the ubiquitin promoter (Pubi) of A. oryzae exhibited a moderate expression strength. The deletion analysis revealed that the 5' untranslated regions of gpdA1 and ubi with the length of 1028 and 811 nucleotides, counted from the putative translation start site (ATG), respectively, could efficiently drive the GUS expression. Interestingly, both promoters could function on various carbon sources for cell growth. Glucose was the best fermentable carbon source for allocating high constitutive expressions during cell growth, and the high concentrations (6–8% glucose, w/v) did not repress their functions. It was also demonstrated that the secondary metabolite gene coding for indigoidine could express under the control of PgpdA1 or Pubi promoter. These strong and moderate promoters of A. oryzae provided beneficial options in tuning the transcriptional expression for leveraging the metabolic control towards the targeted products.