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Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host.
mBio. 2018 09 25; 9(5)MBIO

Abstract

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.

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Authors+Show Affiliations

Cernak P
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
Estrela R
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
Poddar S
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. Innovative Genomics Institute, University of California, Berkeley, California, USA.
Skerker JM
Department of Bioengineering, University of California, Berkeley, California, USA. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Cheng YF
Energy Biosciences Institute, Berkeley, California, USA.
Carlson AK
Department of Bioengineering, University of California, Berkeley, California, USA.
Chen B
Department of Chemistry, University of California, Berkeley, California, USA.
Glynn VM
Department of Environmental Science, Policy, and Management, University of California, Berkeley, California, USA.
Furlan M
Department of Genetics, Evolution and Bioagents, University of Campinas, Campinas, São Paulo, Brazil.
Ryan OW
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA.
Donnelly MK
Department of Plant and Microbial Biology, University of California, Berkeley, California, USA.
Arkin AP
Department of Bioengineering, University of California, Berkeley, California, USA. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Taylor JW
Department of Plant and Microbial Biology, University of California, Berkeley, California, USA.
Cate JHD
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA jcate@lbl.gov. Department of Chemistry, University of California, Berkeley, California, USA. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

MeSH

BiotechnologyCRISPR-Associated Protein 9CRISPR-Cas SystemsGene EditingGenes, Mating Type, FungalGenetic EngineeringKluyveromycesLipid MetabolismSaccharomyces cerevisiaeSynthetic BiologyTemperatureThermotolerance

Pub Type(s)

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

Language

eng

PubMed ID

30254120

Citation

Cernak, Paul, et al. "Engineering Kluyveromyces Marxianus as a Robust Synthetic Biology Platform Host." MBio, vol. 9, no. 5, 2018.
Cernak P, Estrela R, Poddar S, et al. Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host. mBio. 2018;9(5).
Cernak, P., Estrela, R., Poddar, S., Skerker, J. M., Cheng, Y. F., Carlson, A. K., Chen, B., Glynn, V. M., Furlan, M., Ryan, O. W., Donnelly, M. K., Arkin, A. P., Taylor, J. W., & Cate, J. H. D. (2018). Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host. MBio, 9(5). https://doi.org/10.1128/mBio.01410-18
Cernak P, et al. Engineering Kluyveromyces Marxianus as a Robust Synthetic Biology Platform Host. mBio. 2018 09 25;9(5) PubMed PMID: 30254120.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Engineering Kluyveromyces marxianus as a Robust Synthetic Biology Platform Host. AU - Cernak,Paul, AU - Estrela,Raissa, AU - Poddar,Snigdha, AU - Skerker,Jeffrey M, AU - Cheng,Ya-Fang, AU - Carlson,Annika K, AU - Chen,Berling, AU - Glynn,Victoria M, AU - Furlan,Monique, AU - Ryan,Owen W, AU - Donnelly,Marie K, AU - Arkin,Adam P, AU - Taylor,John W, AU - Cate,Jamie H D, Y1 - 2018/09/25/ PY - 2018/9/27/entrez PY - 2018/9/27/pubmed PY - 2019/3/5/medline KW - CRISPR-Cas9 KW - Kluyveromyces marxianus KW - lipogenesis KW - mating KW - renewable chemicals KW - thermotolerant yeast JF - mBio JO - mBio VL - 9 IS - 5 N2 - Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications. SN - 2150-7511 UR - https://www.unboundmedicine.com/medline/citation/30254120/Engineering_Kluyveromyces_marxianus_as_a_Robust_Synthetic_Biology_Platform_Host_ DB - PRIME DP - Unbound Medicine ER -