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Synthetic Biology-Guided Metabolic Engineering

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Front. Bioeng. Biotechnol. | doi: 10.3389/fbioe.2019.00097

Biological parts for Kluyveromyces marxianus synthetic biology

  • 1School of Microbiology, College of Science, Engineering and Food Science, University College Cork, Ireland
  • 2Environmental Research Institute, College of Science, Engineering and Food Science, University College Cork, Ireland
  • 3APC Microbiome Institute, College of Medicine and Health, University College Cork, Ireland
  • 4Department of Biotechnology, Delft University of Technology, Netherlands
  • 5Microbiology Department, University College Cork, Ireland

Kluyveromyces marxianus is a non-conventional yeast whose physiology and metabolism lends itself to diverse biotechnological applications. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. In this paper, we bring together and characterise a set of native K. marxianus parts for the expression of multiple genes for metabolic engineering and synthetic biology. All parts are cloned and stored according to the MoClo/Yeast Tool Kit standard for quick sharing and rapid construction. Using available genomic and transcriptomic data, we have selected promoters and terminators to fine-tune constitutive and inducible gene expression. The collection includes a number of known centromeres and autonomously replication sequences (ARS). We also provide a number of chromosomal integration sites selected for efficiency or visible phenotypes for rapid screening. Finally, we provide a single-plasmid CRISPR/Cas9 platform for genome engineering and facilitated gene targeting, and rationally create auxotrophic strains to expand the common range of selection markers available to K. marxianus. The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. Plasmids containing all parts are available at Addgene for public distribution.

Keywords: Kluyveromyces, Synthetic Biology, Metabolic Engineering, genome engineering, yeast, Golden gate, in vitro assembly

Received: 25 Nov 2018; Accepted: 16 Apr 2019.

Edited by:

Rodrigo Ledesma-Amaro, Imperial College London, United Kingdom

Reviewed by:

Dae-Hee Lee, Korea Research Institute of Bioscience and Biotechnology (KRIBB), South Korea
Hyun Ah Kang, Chung-Ang University, South Korea  

Copyright: © 2019 Rajkumar, Varela, Juergens, Daran and Morrissey. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. John P. Morrissey, University College Cork, Microbiology Department, Cork, Ireland, j.morrissey@ucc.ie