Shinobu Takagi - Academia.edu (original) (raw)
Papers by Shinobu Takagi
Cette invention concerne des variants promoteurs sous forme d’un polynucleotide isole comprenant ... more Cette invention concerne des variants promoteurs sous forme d’un polynucleotide isole comprenant : i) une sequence nucleotidique constituee par la sequence de promoteur DAS de Pichia ou une partie fonctionnelle de celle-ci, ledit promoteur DAS etant contenu dans la sequence SEQ ID NO : 1 ; et ii) au moins un autre UAS, ledit UAS etant compris dans la sequence SEQ ID NO: 2.
Bioscience, Biotechnology, and Biochemistry, 2018
Whole-genome sequencing was conducted on two Aspergillus oryzae strains used for the manufacturin... more Whole-genome sequencing was conducted on two Aspergillus oryzae strains used for the manufacturing of food enzymes, Acrylaway® and Shearzyme®, with the aim of identifying the inserted locus of randomly integrated expression plasmid and obtaining flanking sequences for safety assessment. Illumina paired-end sequencing was employed, and the obtained reads were mapped to two references: the public genome sequence of Aspergillus oryzae RIB40 and the in-house sequence of the used expression plasmid. Introducing the concept of linking-reads, one locus for each was successfully identified as the integrated site. In the case of Acrylaway®, the obtained sequences suggested that the expression plasmid had been integrated as multiple copies in tandem form. In the case of Shearzyme®, however, information on one edge of the insert was missing, which required extra polymerase chain reaction (PCR) cloning for safety assessment. A 4-kb deletion was detected at the integrated site. There was also ev...
The invention relates to enzymes for starch processing, and specifically relates to polypeptides ... more The invention relates to enzymes for starch processing, and specifically relates to polypeptides comprising a carbohydrate-binding module amino acid sequence and an alpha-amylase amino acid sequence as well as to the application of such polypeptides.
AccessScience
A mold that is one of the most widely used fungi in industry. Especially with regard to the Japanes…
Industrial Biotechnology, 2009
Applied and Environmental Microbiology, 2008
We investigated the effect of codon optimization on the expression levels of heterologous protein... more We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae , using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3′ rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carri...
A simple intracellular proteomic study was conducted to investigate the biological activities of ... more A simple intracellular proteomic study was conducted to investigate the biological activities of <i>Aspergillus niger</i> during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identi...
Bioscience, Biotechnology, and Biochemistry, 2020
A simple intracellular proteomic study was conducted to investigate the biological activities of ... more A simple intracellular proteomic study was conducted to investigate the biological activities of Aspergillus niger during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identified conserved-prot...
FEMS Yeast Research, 2019
The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) w... more The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another import...
Applied Microbiology and Biotechnology, 2001
Journal of Bioscience and Bioengineering, 2018
Cette invention concerne des variants promoteurs sous forme d’un polynucleotide isole comprenant ... more Cette invention concerne des variants promoteurs sous forme d’un polynucleotide isole comprenant : i) une sequence nucleotidique constituee par la sequence de promoteur DAS de Pichia ou une partie fonctionnelle de celle-ci, ledit promoteur DAS etant contenu dans la sequence SEQ ID NO : 1 ; et ii) au moins un autre UAS, ledit UAS etant compris dans la sequence SEQ ID NO: 2.
Bioscience, Biotechnology, and Biochemistry, 2018
Whole-genome sequencing was conducted on two Aspergillus oryzae strains used for the manufacturin... more Whole-genome sequencing was conducted on two Aspergillus oryzae strains used for the manufacturing of food enzymes, Acrylaway® and Shearzyme®, with the aim of identifying the inserted locus of randomly integrated expression plasmid and obtaining flanking sequences for safety assessment. Illumina paired-end sequencing was employed, and the obtained reads were mapped to two references: the public genome sequence of Aspergillus oryzae RIB40 and the in-house sequence of the used expression plasmid. Introducing the concept of linking-reads, one locus for each was successfully identified as the integrated site. In the case of Acrylaway®, the obtained sequences suggested that the expression plasmid had been integrated as multiple copies in tandem form. In the case of Shearzyme®, however, information on one edge of the insert was missing, which required extra polymerase chain reaction (PCR) cloning for safety assessment. A 4-kb deletion was detected at the integrated site. There was also ev...
The invention relates to enzymes for starch processing, and specifically relates to polypeptides ... more The invention relates to enzymes for starch processing, and specifically relates to polypeptides comprising a carbohydrate-binding module amino acid sequence and an alpha-amylase amino acid sequence as well as to the application of such polypeptides.
AccessScience
A mold that is one of the most widely used fungi in industry. Especially with regard to the Japanes…
Industrial Biotechnology, 2009
Applied and Environmental Microbiology, 2008
We investigated the effect of codon optimization on the expression levels of heterologous protein... more We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae , using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3′ rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carri...
A simple intracellular proteomic study was conducted to investigate the biological activities of ... more A simple intracellular proteomic study was conducted to investigate the biological activities of <i>Aspergillus niger</i> during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identi...
Bioscience, Biotechnology, and Biochemistry, 2020
A simple intracellular proteomic study was conducted to investigate the biological activities of ... more A simple intracellular proteomic study was conducted to investigate the biological activities of Aspergillus niger during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identified conserved-prot...
FEMS Yeast Research, 2019
The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) w... more The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another import...
Applied Microbiology and Biotechnology, 2001
Journal of Bioscience and Bioengineering, 2018