Improving industrial yeast strains: exploiting natural and artificial diversity

Abstract

Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as 'global transcription machinery engineering' (gTME), to induce genetic variation, providing a new source of yeast genetic diversity.

Keywords: GMO; Saccharomyces cerevisiae; evolutionary engineering; genetic engineering; metabolic engineering; non-Saccharomyces.

Figure 1

Overview of strategies to obtain superior industrial yeast strains. In order to select novel yeast strains for industrial applications, several strategies can be applied. First, the existing natural diversity can be explored by genotyping and phenotyping isolated feral strains or strains from yeast collections to select the most interesting variants. Apart from investigating naturally occurring yeasts, diversity can also be generated artificially. There are multiple strategies to induce genetic diversity in a single strain or shuffle the genomes of multiple strains. Strains resulting from these strategies are all considered non-genetically modified yeasts, implying that they can be freely used in industrial fermentations. These strategies will be further discussed in the section on ‘Generation of artificial diversity’. Lastly, strategies based on genetic engineering, where a recombinant piece of DNA is transformed in a target strain to confer a specific, industrially relevant phenotype to this strain, can be very efficient. However, this technique genetically modifies yeasts, currently limiting their use in food or beverage fermentations because of consumer concerns.

Figure 2

Origins of genetic variation in yeast. Genetic variation can be caused by several different mechanisms. For sake of simplicity, only one chromosome per yeast cell is displayed (green or purple). Different color shades represent homologous chromosomes. In (e), a second chromosome is represented in red. (a) Sexual reproduction: after sporulation and concomitant meiotic cross-over events in the parental strains (2n), genomes of two haploid (n) segregants can hybridize, a process called mating. (b) Point mutations: changes in single nucleotides. These mutations can be synonymous or nonsynonymous: synonymous mutations do not change the amino acid sequence, while nonsynonymous mutations do. Nonsynonymous mutations are therefore more likely to alter the phenotype. (c) InDels: insertion and deletion events of relatively short pieces of DNA. (d) Transposons: insertion of transposable elements in the genome. (e) Changes in ploidy level: the whole genome, or large parts, is duplicated or lost, which can result in poly- or aneuploidies. (f) Horizontal gene transfer: transfer of genes by means other than regular sexual reproduction. (g) Genetic recombination: reorganization of parts of the genome. It can act on both homologous (cross-over and gene conversion) and nonhomologous loci (ectopic recombination). Homologous recombination such as gene conversion (nonreciprocal transfer of genetic material between highly homologous genes) occurs relatively frequently and can sometimes give rise to novel or modified traits. Ectopic recombination events such as TY-promoted chromosomal translocations are more rare, but can drastically rearrange the genome, and even generate novel genes.

Figure 3

Life cycle of S. cerevisiae. Yeast cells can exist in both a haploid and diploid state. (a) Diploid cells are heterozygous for the mating type locus (mating type a/α), which makes diploids incapable of mating. Haploid cells have either mating type a or mating type α, making them capable of mating with a cell of the opposite mating type. In nutrient-rich conditions, both haploid and diploid cells can proliferate asexually by budding. When exposed to some nutrient-poor conditions, diploids can undergo sporulation (meiosis followed by spore formation), resulting in the conversion of a diploid cell into four haploid spores, two possessing mating type a and two having mating type α, which can germinate into haploid cells when conditions improve. In homothallic strains, the haploid derivatives can undergo a mating type switch (together with the mother cell), mediated by an endonuclease encoded by the HO gene. In this way, a mating type-switched cell can mate with neighboring sister cells of the opposite mating type, resulting in a homozygous (except for the MAT locus, which determines the mating type) diploid. In heterothallic strains, the HO gene is typically inactive and therefore haploid derivatives cannot switch mating type. (b) Mechanism of the mating type switch of homothallic strains. On chromosome III, the MAT locus is flanked by Hidden MAT Left and Right (HML and HMR, respectively), carrying a silenced copy of MATα and MATa, respectively. Homothallic strains contain the HO gene, a gene coding for an endonuclease that cleaves DNA specifically at the MAT locus. After breakdown of the MAT locus by exonucleases, a gene conversion event occurs, where HML or HMR is used as a template to repair the DNA strand. Because cells prefer to change their mating type, that is, a MATα cell will rather use HMR as a template and vice versa, mating-type switch occurs frequently.

Figure 4

Overview of different strain improvement techniques using hybridization. Sexual and asexual hybridization is a powerful technique to generate artificial diversity in yeast. Due to the sometimes complex genetics (ploidy, sporulation, …) of yeast, different techniques have been developed. Most techniques start from two parental (P) strains, selected for the target phenotype. The color scheme indicates the strength of the phenotype, for example red = strong ethanol tolerance, yellow = weak ethanol tolerance. In these examples, the parental strains are selected for the same phenotype, but combining different phenotypes of both parents is also possible. (a) In direct mating, two haploid cells or spores of opposite mating types are crossed. When the parental strains are both heterothallic, these haploids can be prescreened and selected, and cell-to-cell mating can be applied. When both or one of the parental strains is homothallic, spore-to-spore or spore-to-cell mating, respectively, can be used. In these latter cases, the selection step (indicated with *) cannot be applied. (b) In rare mating, strains are crossed without a sporulation step. This is possible because diploid yeasts occasionally (but rarely) undergo a homothallic mating-type switch, yielding an a/a or α/α diploid cell. These cells can subsequently hybridize with a haploid cell of the opposite mating type. It is important to note that rare mating is not limited to the development of triploid yeasts. For example, tetraploid hybrids can be obtained if P2 would be an a/a type yeast. (c) In mass mating, multiple parental strains, or a heterogeneous population of the same parental strain, can be used. After mass sporulation and mixing of the resulting spores, mass mating will occur. These rounds of mass sporulation and mass mating can be repeated multiple times, a process which is one way to perform so-called genome shuffling. In genome shuffling, the mass sporulation and mass mating steps can also be replaced by protoplast fusion. (d) Cytoduction can be used to transfer cytoplasmically inherited traits. First, the KAR1 gene of the parental strain containing the targeted cytoplasmic trait is deleted. Next, both parental strains are crossed (or fused by protoplast fusion), but because karyogamy is blocked, the heterokaryon segregates into cells containing a nucleus of only one parent but the cytoplasmic components of both parents (=heteroplasmons). With proper selection, this technique can also yield so-called disomic strains that contain the full chromosome complement of one parent plus one chromosome from the other parent. (e) In protoplast fusion, cells are asexually merged after cell wall removal in osmotically supportive medium. After cell wall regeneration, the formed transient heterokaryons may undergo karyogamy and form hybrids.

Figure 5

Novel techniques for genetic modification of industrial strains. Schematic overview of selected techniques to generate phenotypic diversity in (industrial) S. cerevisiae strains. The wild-type situation is always shown on the left, and the altered situation on the right. The thickness of the arrows indicates the transcription level of the genes. Global techniques (a–c) include global transcription machinery engineering (a), which exploits genome-wide transcriptional re-wiring generated by a mutated general transcription factor; artificial zinc finger transcription factors (b), creating altered transcription profiles resulting from transcription factors with a wide variety of specificity, for instance by coupling randomized zinc fingers to a repression domain; and transposon mutagenesis (c), which knocks out genes in a random fashion. Targeted techniques comprise in vitro generation of genetic variability using random mutagenesis (d) or DNA shuffling (e), TaGTEAM (f), which can drastically elevate the local mutation rate by targeting a mutator protein (=DNA glycosylase + DNA-binding protein) to a specific genomic region, and the use of oligonucleotide-based approaches like synthetic oligonucleotides (g) to introduce specific gene deletions or mutations. See text for more details.