A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica

Nicholas Milne, Larissa Ribeiro Ramos Tramontin, Irina Borodina*

*Corresponding author for this work

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Abstract

We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.
Original languageEnglish
JournalFEMS Yeast Research
ISSN1567-1356
DOIs
Publication statusAccepted/In press - 2020

Cite this

@article{20c619bcea7d4a0fb1b25a98a6af4b06,
title = "A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica",
abstract = "We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.",
author = "Nicholas Milne and {Ribeiro Ramos Tramontin}, Larissa and Irina Borodina",
year = "2020",
doi = "10.1093/femsyr/foz062",
language = "English",
journal = "F E M S Yeast Research",
issn = "1567-1356",
publisher = "Oxford University Press",

}

TY - JOUR

T1 - A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica

AU - Milne, Nicholas

AU - Ribeiro Ramos Tramontin, Larissa

AU - Borodina, Irina

PY - 2020

Y1 - 2020

N2 - We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.

AB - We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.

U2 - 10.1093/femsyr/foz062

DO - 10.1093/femsyr/foz062

M3 - Journal article

C2 - 31556952

JO - F E M S Yeast Research

JF - F E M S Yeast Research

SN - 1567-1356

ER -