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Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3.
Genes (Basel) 2019; 10(8)G

Abstract

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.

Authors+Show Affiliations

Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China. College of Basic Science, Tianjin Agricultural University, Tianjin 300384, China.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.College of Basic Science, Tianjin Agricultural University, Tianjin 300384, China.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China. lyh@tust.edu.cn.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31409011

Citation

Li, Tao, et al. "Transcriptomic Analysis of the Influence of Methanol Assimilation On the Gene Expression in the Recombinant Pichia Pastoris Producing Hirudin Variant 3." Genes, vol. 10, no. 8, 2019.
Li T, Ma J, Xu Z, et al. Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3. Genes (Basel). 2019;10(8).
Li, T., Ma, J., Xu, Z., Wang, S., Wang, N., Shao, S., ... Liu, Y. (2019). Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3. Genes, 10(8), doi:10.3390/genes10080606.
Li T, et al. Transcriptomic Analysis of the Influence of Methanol Assimilation On the Gene Expression in the Recombinant Pichia Pastoris Producing Hirudin Variant 3. Genes (Basel). 2019 08 12;10(8) PubMed PMID: 31409011.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant Pichia pastoris Producing Hirudin Variant 3. AU - Li,Tao, AU - Ma,Jieying, AU - Xu,Zehua, AU - Wang,Shuang, AU - Wang,Nan, AU - Shao,Shulin, AU - Yang,Wei, AU - Huang,Lin, AU - Liu,Yihan, Y1 - 2019/08/12/ PY - 2019/07/27/received PY - 2019/08/07/accepted PY - 2019/8/15/entrez PY - 2019/8/15/pubmed PY - 2019/8/15/medline KW - Pichia pastoris KW - anticoagulant KW - fermentation KW - hirudin KW - methanol induction JF - Genes JO - Genes (Basel) VL - 10 IS - 8 N2 - Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3. SN - 2073-4425 UR - https://www.unboundmedicine.com/medline/citation/31409011/Transcriptomic_Analysis_of_the_Influence_of_Methanol_Assimilation_on_the_Gene_Expression_in_the_Recombinant_Pichia_pastoris_Producing_Hirudin_Variant_3 L2 - http://www.mdpi.com/resolver?pii=genes10080606 DB - PRIME DP - Unbound Medicine ER -