Transcriptome-based Mining of the Constitutive Promoters for Tuning Gene Expression in Aspergillus oryzae

Laoteng Kobkul,Anantayanon Jutamas,Chutrakul Chanikul,Panchanawaporn Sarocha,Jeennor Sukanya 한국미생물학회 2023 The journal of microbiology Vol.61 No.2

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.

Dissecting Metabolic Regulation in Mycelial Growth and Fruiting Body Developmental Stages of Cordyceps militaris through Integrative Transcriptome Analysis

Roypim Thananusak,Kobkul Laoteng,Nachon Raethong,Mattheos Koffas,Wanwipa Vongsangnak 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.3

Dissecting the cellular metabolism of Cordyceps militaris is important for the efficient production of bioactive compounds of a target with medicinal and industrial applications. However, the metabolic functions during developmental stages in this fungus at a system level are still unexplored. In this study, we aimed to reveal the metabolic functions and regulation of C. militaris TBRC6039 relevant to its developmental stages, including mycelial growth (MY) and fruiting body (FB) stages through integrative transcriptome analysis. The transcriptome analysis showed that 9,256 genes of C. militaris were expressed in both stages. Of them, 1,877 genes, residing primarily in the cell division cycle and amino acid, carbohydrate, and lipid metabolisms, exhibited significant differences in transcript levels between the MY and FB stages. Through integration with genome-scale networks analysis, the unique reporter metabolites (e.g., α-D-glucose, β-D-glucose, D-galactose, triacylglycerol, and diacylglycerol) and key regulators (e.g., AtfA, Atf2, and Yap1 transcription factors) were identified in C. militaris when grown at FB stage, linking to up-regulation of the metabolic genes involved in galactose and polysaccharide metabolisms, as well as glycerolipid and glycerophospholipid biosynthesis. Moreover, the high cordycepin content is related to the upregulated genes in lipid metabolism during the FB stage. Our findings suggest that the transcriptional regulation of these metabolic pathways played a crucial role in specific developmental stages of C. militaris. This study serves for cultivation process improvement for overproduction of valuable metabolites in C. militaris through an emerging systems and synthetic biology approach.

Microbial Production of γ-Linolenic Acid: Submerged versus Solid-state Fermentations

Milan Čertík,Zuzana Adamechová,Kobkul Laoteng 한국식품과학회 2012 Food Science and Biotechnology Vol.21 No.4

One of the major interests of lipid biotechnology is targeted on natural manufacturing of healthy oils containing polyunsaturated fatty acids. Of them, γ-linolenic acid (C18:3 n-6; GLA) as the key intermediate in the n-6 fatty acid family is involved to maintain the proper mammalian cell functions. Insufficient supply of GLA from agricultural and animal sources resulted in ‘hunting’ for appropriate microorganisms suitable to produce this essential fatty acid in high yield. Extensive studies on oleaginous lower filamentous fungi have led to development of two basic fermentation techniques for GLA production: submerged and solid- state fermentations. Each of the processes provide specific advantages in various applications depending on the GLA product form (GLA-rich oil, whole cells, and fermented mass) and might bring new prospects to fill marketing claims in food, feed, pharmaceutical, and veterinary fields.

A survey of pre-service teachers’ acceptance of technology in Thailand

Timothy Teo,Jintavee Khlaisang,Thapanee Thammetar,Kobkul Sunphakitjumnong,Anirut Satiman,Nammon Ruangrit 서울대학교 교육연구소 2014 Asia Pacific Education Review Vol.15 No.4

In the recent decade, Thailand has launched many initiatives to ensure that technology is integrated into the school curriculum. Despite the investment and efforts of the government, few studies have been conducted to examine users’ acceptance of technology. Given that educators are the change agents in many educational initiatives, it is important to gain insights into the factors that influence their uses of technology for teaching and learning. The aim of this study is to survey the level of technology acceptance among pre-service teachers in Thailand. In so doing, the level of technology acceptance among a national sample of 969 pre-service teachers was measured and whether their acceptance was significantly differentiated by gender, age, perceived technology competence, and computer experience was assessed. Using the Technology Acceptance Measure for Pre-service Teachers (Teo in Educ Psychol Meas 70(6):990–1006, 2010a; Multicult Educ Technol J 4(3):163–172, 2010b), the results provided preliminary evidence which suggests that pre-service teachers possess an above-average level of technology acceptance and that computer experience acted as a mediator of technology acceptance. To some degree, this study has contributed to the ensuing debates on technology acceptance in education and offered additional insights into the advancement of technology integration in Asia.

Promoter exchange of the cryptic nonribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae

Chutrakul Chanikul,Panchanawaporn Sarocha,Jeennor Sukanya,Anantayanon Jutamas,Laoteng Kobkul 한국미생물학회 2022 The journal of microbiology Vol.60 No.1

Oligopeptides with functional activities are of current interest in the nutraceutical and medical sectors. The development of the biosynthetic process of oligopeptides through a nonribosomal peptide synthetase (NRPS) system has become more challenging. To develop a production platform for nonribosomal peptides (NRPs), reprogramming of transcriptional regulation of the acv gene encoded ACV synthetase (ACVS) was implemented in Aspergillus oryzae using the CRISPRCas9 system. Awakening silent acv expression was successfully achieved by promoter substitution. Among the three exchanged promoters, AoPgpdA, AoPtef1, and PtPtoxA, the replacement of the native promoter with AoPgpdA led to the highest ACV production in A. oryzae. However, the ACV production of the AoPGpdA strain was also dependent on the medium composition, in which urea was the best nitrogen source, and a C:N ratio of 20:1 was optimal for tripeptide production. In addition to cell growth, magnesium ions are an essential element for ACV production and might participate in ACVS activity. It was also found that ACV was the growthassociated product of the engineered strain that might be a result of constitutive transcriptional control by the AoPgpdA promoter. This study offers a potential strategy for nonribosomal ACV production using the fungal system, which is applicable for redesigning bioactive oligopeptides with industrial relevance.