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 M.Sc. and Ph.D. 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.

中文翻译：

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演示EasyClone和CRISPR / Cas9在酿酒酵母和解脂耶氏酵母代谢工程中的应用的教学方案。

我们提出了一个教学协议，该案例适合于证明使用EasyClone和CRISPR / Cas9进行工业相关酵母酿酒酵母和解脂耶氏酵母的代谢工程，并以β-胡萝卜素的生产为例。该协议详细介绍了生成DNA部分，转化酵母和基因型酵母并进行表型筛选以确定β-胡萝卜素生成所需的所有步骤。该协议旨在用作针对M.Sc.的为期两周的实践课程的指导手册。和博士学位 学生们。该协议详细介绍了学生工程化酵母以生产β-胡萝卜素的所有必要步骤，并作为代谢工程原理的实用介绍，包括增加天然前体供应和减轻限速步骤的概念。它还强调了两种与工业相关的酵母物种在代谢和异源生产能力方面的关键差异。该协议分为两周的每日实验，并提供每个步骤的详细说明，这意味着该协议可以“按原样”用于教学课程，也可以作为案例研究来研究如何设计酵母以产生附加值分子。