Molecular Biology and Genetic Engineering of Yeasts

Henri Heslot

• Publisher: CRC Press
• Year: 2018
• Print ISBN:
  9781315895635
• eBook ISBN:
  9781351091633
• Topic: Technology & Engineering
• Subtopic: Food Science
• Index: Technology & Engineering

Molecular Biology and Genetic Engineering of Yeasts presents a comprehensive examination of how yeasts are used in genetic engineering. The book discusses baker's yeast, in addition to a number of unconventional yeasts being used in an increasing number of studies. 175 figures help illustrate the information presented. Topics discussed include yeast transformation, yeast plasmids, protein localization and processing in yeast, protein secretion, various aspects of Saccharomyces cerevisiae, and heterologous expression and secretion.

Chapter 1
Yeast Transformation

I. Yeast Transformation

Early efforts leading to yeast transformation^1,2 have led to the development of a sophisticated array of vectors and systems for manipulating the genome of Saccharomyces cerevisiae.³ Most of these techniques and approaches now seem to be readily extendable to other yeast species: transformation of, e.g., Schizosaccharomyces pombe⁴ or Kluyveromyces lactis⁵ was reported quite early. Other nonconventional yeasts received attention more recently (for review, see Reference 6). The same general principles seem to apply to the ten or more species so far tested; for example, integrative vectors do generally integrate by homologous recombination, and chromosomal sequences (ARS) can confer extrachromosomal replication. This makes yeasts quite different from other eukaryotic cells including fungi, where nonhomologous recombination events are frequent or predominant and where ARS sequences could not be convincingly demonstrated up to now.

A. Procedures for Yeast Transformation

Early work on yeast transformation used spheroplasts stabilized in an isotonic medium.^1,4,5 In the presence of calcium ions and polyethylene glycol (PEG), DNA is taken up by a poorly understood process.⁷ The efficiency of transformation (transformed cells per surviving protoplast) depends on the genetic background of the recipient⁸ and on the type of selection applied: under nonselective conditions, several percent of transformed cells can be obtained.⁹ Fusion of yeast spheroplasts with DNA-containing liposomes¹⁰ or with Escherichia coli minicells harboring yeast E. coli shuttle plasmids¹¹ can be considered as variations of the above procedure and result in a higher transformation efficiency (up to 10% with minicells). Many strains, however, do not exhibit such a high transformation efficiency, and selective markers have to be used in order to select transformants.

The spheroplast method, although still widely used, is time consuming, and its efficiency can be severely limited by the regeneration step where protoplasts are allowed to regenerate into whole cells. Several methods using whole cells have been developed which are, in some cases, as efficient as the spheroplast method. Cells of Saccharomyces cerevisiae treated with alkali cations (lithium, for example)¹² or with 2-mercaptoethanol¹³ were transformed at high efficiency in the presence of PEG and after a heat shock. The specificity for cation requirement (i.e., monovalent vs. divalent) was found to be strain specific, some strains being transformable only in the presence Ca²⁺, others only with Li⁺.¹⁴ Although PEG is generally needed to obtain a significant transformation frequency, probably by triggering irreversible adsorption of plasmid DNA,¹⁴ some strains can be transformed without PEG.¹⁵ Whole cell transformation using the LiCl method have been adapted to other yeast species such as Schizosaccharomyces pombe,¹⁶ K. lactis,¹⁷ or Yarrowia lipolytica.¹⁸

Although the mechanisms involved in spheroplast and whole cell transformation are equally poorly understood, the two procedures probably rely on quite different mechanisms. Cotransformation seems to be the rule when spheroplasts are used, indicating that many plasmids enter the cells. Initial experiments with two integrative plasmids carrying different marker genes gave 25 to 33% cotransformation in Saccharomyces cerevisiae,¹⁹ On the contrary, few plasmids seem to enter cells transformed by the LiCl method.²⁰ On the other hand, cells made competent by the LiCl method seem to take up linear DNA more readily than circular DNA, whereas no such difference is observed when the spheroplasting method is used: linearized ARS plasmids transform spheroplasts of S. cerevisiae at the same frequency as circular plasmids, but they transform 80-fold more efficiently than uncut plasmids of LiCl-treated cells.²¹ A similar increase of transformation efficiency of linearized ARS pla...

Table of contents

• Cover
• Title
• Copyright
• THE AUTHORS
• Contents
• Chapter 1 Yeast Transformation
• Chapter 2A Yeast Plasmids: The 2-μm Circle Plasmid of Saccharomyces cerevisiae
• Chapter 2B Yeast Plasmids: Linear and Circular Plasmids from Kluyveromyces
• Chapter 3 Transposable Elements in Yeast
• Chapter 4 Transcriptional Control of Gene Expression
• Chapter 5 Regulation of the Galactose Genes of Saccharomyces cerevisiae
• Chapter 6A Protein Localization and Processing in Yeast: Nuclear Targeting
• Chapter 6B Protein Localization and Processing in Yeast: Vacuolar Protein Targeting
• Chapter 6C Protein Localization and Processing in Yeast: Targeting Proteins into Mitochondria
• Chapter 6D Protein Localization and Processing in Yeast: Peroxisomes
• Chapter 6E Protein Localization and Processing in Yeast: Targeting of Integral Membrane Proteins
• Chapter 7 Protein Secretion
• Chapter 8A Saccharomyces cerevisiae Mating Pheromones
• Chapter 8B The Killer Toxin of Saccharomyces cerevisiae
• Chapter 8C Saccharomyces cerevisiae Invertase
• Chapter 8D Saccharomyces cerevisiae Acid Phosphatase
• Chapter 9 The Ubiquitin System and Its Role in the Genetic Control of Protein Half-Life
• Chapter 10 Heterologous Expression and Secretion
• Index