Transcriptomic Analysis of the Influence of Methanol Assimilation on the Gene Expression in the Recombinant <i>Pichia pastoris</i> Producing Hirudin Variant 3

• Main Authors: Tao Li, Jieying Ma, Zehua Xu, Shuang Wang, Nan Wang, Shulin Shao, Wei Yang, Lin Huang, Yihan Liu
• Format: Article
• Language: English
• Published: MDPI AG 2019-08-01
• Series: Genes
• Subjects: anticoagulant; methanol induction; fermentation; hirudin; <i>Pichia pastoris</i>
• Online Access: https://www.mdpi.com/2073-4425/10/8/606

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 <i>Pichia pastoris</i> GS115/pPIC9K-<i>hv3</i> under the regulation of the promoter of <i>AOX1</i> 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.