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Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor

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Abstract

Background

Reprogramming in transcriptional regulation provides an effective tool for adjusting cellular metabolic activities. The strong methanol-inducible alcohol oxidase-1 promoter (pAOX1) is commonly used for heterologous gene expression in the yeast Pichia pastoris. Here, we present a novel Pichia pastoris strain engineered to co-express methanol-induced transcription factor 1 (Mit1) and the target protein. Mit1 upregulates pAOX1 in response to methanol.

Methods and Results

Two model proteins (VEGF and eGFP) have been used as the target proteins under the control of pAOX1. The sequence of Mit1 had obtained from the yeast genome and likewise cloned under the control of pAOX1. The results indicated a 1.9 and 2.2 fold increase in the detected VEGF and eGFP, respectively, when co-expressed with Mit1. Furthermore, the double-recombinant cells, containing Mit-1 and eGFP, produced 1.3 fold more eGFP when the methanol feeding concentration was doubled. The real-time PCR indicated a slight increase in the Mit1 expression, probably due to the negative regulatory feedback loop that exists for the intrinsic yeast Mit1. Overexpression of Mit1 also led to duplication of AOX1 enzyme activity, which may enhance the yeast cells’ capacity for methanol detoxification.

Conclusion

Overexpression of Mit1 could be considered a promising strategy for upregulation of target recombinant proteins in Pichia pastoris.

Graphical Abstract

Intracellular overexpression of Mit1 upregulates the heterologous target gene (eGFP) production, which is expressed under the control of pAOX1.

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The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

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Authors and Affiliations

  1. Protein Research Center, Shahid Beheshti University, P.O. Box: 19839, 69411, Tehran, Iran

    Samin Haghighi Poodeh, Seyed Omid Ranaei Siadat, Sareh Arjmand & Maryam Khalifeh Soltani

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  1. Samin Haghighi Poodeh
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  2. Seyed Omid Ranaei Siadat
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  3. Sareh Arjmand
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  4. Maryam Khalifeh Soltani
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Contributions

S.H.P. carried out experiments and contributed to the writing, S.O.R.S. supervised the study, S.A. planned, coordinated, and supervised the project and wrote the manuscript, M.K.S. assisted in conducting experiments.

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Correspondence to Sareh Arjmand.

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Haghighi Poodeh, S., Ranaei Siadat, S.O., Arjmand, S. et al. Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor. Mol Biol Rep 49, 9379–9386 (2022). https://doi.org/10.1007/s11033-022-07790-7

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  • DOI: https://doi.org/10.1007/s11033-022-07790-7

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