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A regulated synthetic operon facilitates stable overexpression of multigene terpenoid pathway in Bacillus subtilis

  • Metabolic Engineering and Synthetic Biology - Short Communication
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C30 carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C30 carotenoid production.

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Acknowledgements

We thank I. Maeda for providing the pHYcrtMN plasmid. Funding for this work was obtained through EuroCoRes SYNBIO (SYNMET), NWO-ALW 855.01.161, EU FP-7 grant 289540 (PROMYSE). IIA is a recipient of Erasmus Mundus Action 2, Strand 1, Fatima Al Fihri project ALFI1200161 scholarship and is on study leave from Faculty of Pharmacy, Alexandria University. HP is a recipient of Bernoulli scholarship from University of Groningen.

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  1. Ingy I. Abdallah, Dan Xue and Hegar Pramastya have contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands

    Ingy I. Abdallah, Dan Xue, Hegar Pramastya, Ronald van Merkerk, Rita Setroikromo & Wim J. Quax

  2. Pharmaceutical Biology Research Group, School of Pharmacy, Institut Teknologi Bandung, Bandung, 40132, Indonesia

    Hegar Pramastya

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  1. Ingy I. Abdallah
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Abdallah, I.I., Xue, D., Pramastya, H. et al. A regulated synthetic operon facilitates stable overexpression of multigene terpenoid pathway in Bacillus subtilis. J Ind Microbiol Biotechnol 47, 243–249 (2020). https://doi.org/10.1007/s10295-019-02257-4

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