School of Medicine, Department of Microbiology

Job title

Assistant Professor

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Research Areas 【 display / non-display

  • Life sciences   Virology  

  • Life sciences   Bacteriology  

  • Life sciences   Applied microbiology  

Affiliation 【 display / non-display

  • Sapporo Medical University   Department of Microbiology  


Research Interests 【 display / non-display

  • lipase

  • denitrification

  • biofilm

  • wastewater treatment

  • phage

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Papers 【 display / non-display

  • Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity

    Yoshiaki Yasutake, Kenji Konishi, Shuji Muramatsu, Keitaro Yoshida, Sachiyo Aburatani, Shin-ichi Sakasegawa, Tomohiro Tamura

    International Journal of Biological Macromolecules ( Elsevier BV )  167   578 - 586  2021.01  [Refereed]  [International journal]

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    Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

    DOI PubMed

  • Peculiarities of biofilm formation by Paracoccus denitrificans.

    Kana Morinaga, Keitaro Yoshida, Kohei Takahashi, Nobuhiko Nomura, Masanori Toyofuku

    Applied microbiology and biotechnology    2020.01  [Refereed]  [International journal]

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    Most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. Biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. Paracoccus denitrificans is a non-motile, Gram-negative bacterium that forms a very thin, unique biofilm. A key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein A (BapA), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-GMP or c-di-GMP). Cyclic-di-GMP is a major second messenger involved in biofilm formation in many bacteria. Though cyclic-di-GMP is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in P. denitrificans. Furthermore, quorum sensing (QS) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. The QS signal used in P. denitrificans is hydrophobic and is delivered through membrane vesicles. Studies on QS show that P. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. In this review, we discuss the peculiarities of biofilm formation by P. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions.

    DOI PubMed

  • Production of recombinant extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis

    Keitaro Yoshida, Kenji Konishi, Arturo Magana-Mora, Adrien Rougny, Yoshiaki Yasutake, Shuji Muramatsu, Satomi Murata, Toshitaka Kumagai, Sachiyo Aburatani, Shin-ichi Sakasegawa, Tomohiro Tamura

    Bioscience, Biotechnology, and Biochemistry ( Oxford University Press (OUP) )  83 ( 10 ) 1974 - 1984  2019.10  [Refereed]  [International journal]

    Authorship:   Lead author

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    ABSTRACT Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL−1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.

    DOI PubMed

  • Genome Sequence of Rhodococcus erythropolis Type Strain JCM 3201

    Keitaro Yoshida, Wataru Kitagawa, Koji Ishiya, Yasuo Mitani, Nobutaka Nakashima, Sachiyo Aburatani, Tomohiro Tamura

    Microbiology Resource Announcements ( American Society for Microbiology )  8 ( 14 )  2019.04  [Refereed]  [International journal]

    Authorship:   Lead author

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    Rhodococcus erythropolis JCM 3201 can express several recombinant proteins that are difficult to express in Escherichia coli . It is used as one of the hosts for protein expression and bioconversion.

    DOI PubMed

  • Complete Genome Sequence of an Efficient Vitamin D3-Hydroxylating Bacterium, Pseudonocardia autotrophica NBRC 12743

    Keitaro Yoshida, Yoshiaki Yasutake, Tomohiro Tamura

    Microbiology Resource Announcements ( American Society for Microbiology )  7 ( 12 )  2018.09  [Refereed]

    Authorship:   Lead author

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    Pseudonocardia autotrophica NBRC 12743 contains a cytochrome P450 vitamin D 3 hydroxylase, and it is used as a biocatalyst for the commercial production of hydroxyvitamin D 3 , a valuable compound for medication. Here, we report the complete genome sequence of P. autotrophica NBRC 12743, which could be useful for improving the productivity of hydroxyvitamin D 3 .

    DOI PubMed

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Misc 【 display / non-display

Presentations 【 display / non-display

  • Recombinant production of extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis

    Keitaro Yoshida

    2019 International Conference on Biotechnology and Bioengineering 

    Presentation date: 2019.09