The hybrid yeast Saccharomyces pastorianus is the microbial ‘workhorse’ responsible for production of lager beer. With an annual production of 1.9 billion hectolitres, this type of beer is the most popular alcoholic beverage worldwide. S. pastorianus ferments sugars present in wort to ethanol and a variety of flavour molecules that contribute to the taste and perception of the beverage. This yeast has emerged from a natural hybridization event in a human-caused environment, which probably occurred in late medieval times, between the mesophilic ale yeast Saccharomyces cerevisiae and the cryotolerant yeast Saccharomyces eubayanus. As a consequence, S. pastorianus strains harbour chromosomes from both these parental species. In addition, prolonged domestication in lager brewing processes has led to many additional changes in S. pastorianus: individual chromosomes underwent extensive recombination and occur in different copy numbers that, moreover, vary between strains. The advent of whole genome sequencing technologies has contributed to insights into the complex genomic ‘blueprints’ of S. pastorianus strains.

The allo-aneupolyploid genomes of currently used brewing yeast strains were shaped in centuries of evolution, with industrial brewing conditions providing selective pressure. Today, targeted genetic engineering enables researchers to alter or add traits beyond what is achievable by solely applying selective pressure. The rapid development of CRISPR-Cas-based tools for genome editing have, particularly in baker’s yeast S. cerevisiae and to a lesser extent in non-conventional yeast species, greatly accelerated strain development. However, applying these tools in heterozygous alloaneupolyploid contexts such as those in S. pastorianus poses substantial technological challenges.

The aim of the research described in this thesis was to develop novel genome editing strategies for yeasts, improving the genetic accessibility of hybrid yeasts and subsequently applying these methodologies to generate novel lager brewing strains with enhanced brewing characteristics...

Building synthetic cells is extremely interesting from a fundamental perspective, as the ability to rationally build viable, dividing, and self-maintaining cells provides knowledge on the minimal requirements to sustain life. This should enhance our understanding about which biological parts are minimally required for a cell to live, and how different cellular functions work and interact with each other. Besides a fundamental understanding of life, synthetic cells can also be applied as synthetic biology tools, for example for synthesis and delivery of therapeutics, or the production of compounds that cannot be produced with currently available organisms used as cell factories. The overarching goal of the research described in this thesis was to devise a strategy for building genomes for synthetic cell, using baker’s yeast Saccharomyces cerevisiae. Two methods were explored, the first being building a genome de novo using yeast in vivo assembly, and secondly, it was investigated whether the preexisting minimal genome of S. cerevisiae mitochondria could be expanded. To this end, yeast mitochondrial DNA and RNA was characterized using novel methods and various strategies for engineering the yeast mitochondrial genome were tested.

The production of ethanol by the yeast Saccharomyces cerevisiae remains the process in industrial biotechnology with the largest product volume (ca. 100 million litres annually in 2022). Carbon losses occur due to the formation of biomass and glycerol, which can account for at least 8% of the product. Under anaerobic conditions, formation of yeast biomass and glycerol are coupled via redox-cofactor balances, as a net generation of NADH during biomass formation needs to be compensated by NADH-dependent formation of glycerol from sugar. This thesis discusses redox-engineering strategies for maximizing ethanol yields on substrate.

One of the most conserved and central parts of metabolism is the pathway of glycolysis, that breaks down hexose sugars. While multiple variants of glycolytic pathway exist, the Emden-Meyerhof-Parnas pathway which converts glucose to two molecules of pyruvate with the net gain of two ATP and two reduced NADH moieties is the one predominantly found in eukaryotes. This well-studied metabolic pathway is important for industrial biotechnology, because sugars are often used as substrate for microbial production of fuels and chemicals, as well as human health, because of the involvement of glycolytic dysfunction and deregulation in diseases such as cancer. The analysis of glycolytic enzyme function is complicated by the presence in many organisms of a large range of secondary ‘moonlighting’ functions besides the main glycolytic function. Because of its robustness, genetic accessibility, ease of culture and highly expressed glycolytic pathway, baker’s yeast, Saccharomyces cerevisiae has been key to research on glycolysis. Besides its role in food and beverage production and as industrial host for e.g. bioethanol and insulin production, this yeast has been intensively used as a model organism to study fundamental processes and pathways...

The yeast Saccharomyces cerevisiae has been used by humans for many centuries in microbial fermentation processes for the production of, for example, bread and alcoholic beverages. This long history of use and, in addition, its fast growth, its ability to rapidly convert sugars into ethanol and the ease with which it can be genetically modified, have contributed to this yeast becoming a very popular model organism. S. cerevisiae is currently used in large-scale industrial processes for the production of biofuels and a broad range of other chemicals. The ability of S. cerevisiae to grow in the absence of oxygen is quite unique among yeasts, and not only important for the production of beer and wine, but also for industrial production of bulk chemicals. The high product yields that are required in these types of processes can in theory only be achieved when sugars are completely converted into product, instead of being partially or completely oxidized to CO2 via aerobic respiration. Fast anaerobic growth of S. cerevisiae does require that standard synthetic media, that are used to grow this yeast in the laboratory, are supplemented with a number of additional components. These additional nutritional requirements originate from the fact that oxygen is required for biosynthesis of some important components of the yeast cell. While most yeast species are actually able to ferment, they usually cannot grow in the complete absence of oxygen at all, not even when such cell components or their precursors are added to anaerobic growth media. As a consequence, some yeast species that have industrially relevant traits that are absent or less pronounced in S. cerevisiae, such as resistance to higher temperatures, cannot at the moment be used in anaerobic industrial processes. This PhD thesis describes research on oxygen requirements related to membrane synthesis in yeast, using S. cerevisiae as the main model organism, with the goal to understand these requirements and eliminate them by genetic modification. Inspiration is obtained from evolutionary adaptations of eukaryotic microorganisms that naturally occur in anaerobic or oxygen-poor environments. Metabolic engineering strategies developed in this way may then possibly be applied to other industrially relevant yeast species and thus aid the elucidation of additional, as yet unknown oxygen requirements, or even enable anaerobic growth of those yeasts as well.

Saccharomyces cerevisiae is a natural producer of ethanol and industrial strains can produce ethanol at high volumetric rates and near-theoretical yields. In addition to its fast fermentative metabolism, its GRAS (generally recognized as safe) status, ease of genetic engineering, tolerance to low pH and high ethanol concentrations contribute to the popularity of S. cerevisiae as an industrial platform organism. Ethanolic fermentation is, however, not unique to S. cerevisiae. Other facultatively fermentative yeast species share many performance characteristics with S. cerevisiae and may even hold additional advantages for industrial application. However, they typically lack one key distinctive phenotype of S. cerevisiae: its capability to grow fast in the absence of oxygen on simple media with minimal addition of vitamins and anaerobic growth factors. This characteristic is essential for industrial application, as aeration of large bioreactors is expensive and near-theoretical yields of fermentation products can only be achieved in the absence of respiratory dissimilation of sugars.

The increase in world population together with an unsustainable and fossil fuel-based economy are at the root of the significant increase in the average global temperature since the pre-industrial era. Switching to a more sustainable and circular bio-based economy is one of the main pillars included in the Paris agreement aimed at reducing green-house gasses emissions in the next decades. Biotechnology promises to play a crucial role in this context by providing novel, more sustainable processes for the production of food, drugs, fuels and chemicals.

Already for millennia, microbial fermentation is used for the production of dairy products, alcoholic beverages and bread. In the last decades, the field of biotechnology has tremendously expanded and nowadays, a wide range of compounds ranging from biofuels to chemicals and pharmaceuticals is produced using microbial cell factories. The development of genetic engineering tools has greatly contributed to this rapid development. Catalysing the conversion of renewable carbohydrate feedstocks into fuels and chemicals, microbial cell factories offer a sustainable alternative to fossil resources-based production, and thereby contribute to reduce greenhouse gas emissions. The yeast Saccharomyces cerevisiae plays an important role in industrial biotechnology. Its popularity for applied research and industrial production can be attributed to several factors as its fast fermentative metabolism, its tolerance to low pH, high sugar and alcohol concentrations and its genetic tractability. S. cerevisiae possesses one of the best furbished molecular toolboxes, which makes it possible to assemble complex heterologous pathways, as was recently illustrated by the successful biosynthesis of opioids in yeast. Despite this great progress, extensive genetic remodelling of native pathways remains challenging. This can largely be explained by the high genetic redundancy present in the yeast genome, in which multiple genes encode proteins with redundant functions, and by the fact that the genes belonging to a pathway are scattered over the entire genome. The goal of this thesis was to design, set up and validate a strategy aiming at facilitating the remodelling of (essential) pathways, based on simplifying and reorganizing the yeast genome. The starting point of this research is the central carbon metabolism and in particular, as proof of concept, the glycolytic pathway.

The first production of alcoholic beverages by fermentation of plant-derived materials occurred many thousands of years ago. In the 15th century, production of lager beer with pale barley malt, soft water and Saaz noble hops, first resulted in pilsner, a clear golden, crispy tasting beer. Today, this type of lager beer is the most produced alcoholic beverage in the world. The yeast responsible for conversion of wort sugars into ethanol, CO2 and aromatic compounds is S. pastorianus, a hybrid of S. cerevisiae and the recently discovered cryotolerant species S. eubayanus. Properties from both parents make S. pastorianus a superior fermenter of brewer’s wort at temperatures below 15 °C. Since S. cerevisiae is not a cryotolerant yeast, the low-temperature performance of S. pastorianus hybrids has been attributed to the S. eubayanus sub-genome. However, the geographical origin of the S. eubayanus sub-genome of S. pastorianus hybrids, as well as its contribution to metabolism of key oligosaccharides in brewer’s wort, is not fully understood. The research described in this thesis therefore focused on the genetics of the metabolism of maltose and maltotriose, two key sugars in brewer’s wort, in S. eubayanus.

Contemporary society heavily depends on fossil sources. The energy and materials derived from fossil reserves were major contributors to the acceleration and intensification of agriculture and industry over the past 100 years. Such reserves are finite, hence, after expanding geographically, our economy is now consuming natural reserves that should not just support our generation but also those of the future. This unsustainable scenario becomes even more concerning when environmental impacts are taken into account. Even in the most ― and probably unrealistic ― optimistic climate scenarios, which assume no further increase in CO2 emissions in the next decades, the global temperature would still raise by 2 °C at the end of this century with respect to pre-industrial era, which could already have a negative impact on, for instance, food security.

Microbial production of fuels and chemicals provides opportunities for replacing conventional production processes, which are based on chemical synthesis from non-renewable raw materials or on labour- and capital-intensive extraction from animal or plant tissues. Aeons of evolution, in which astronomical numbers of microorganisms competed for scarce resources, have optimized and streamlined the thousands of biochemical conversions in their cells for growth in specific natural environments. The resulting metabolic diversity, represented by many millions of microbial species, offers a great potential for developing novel microbial conversions of renewable substrates to products. Major advances in (recombinant) DNA technology have enabled the engineering of several microorganisms into efficient production platforms, which can be further modified for the production of a wide range of fuels and chemicals. For high-volume products based on microbial fermentation, such as transport fuels and commodity chemicals, the use of substrate can comprise up to 70% of the total product costs. These high substrate costs make a high product yield on the substrate essential for economic viability.

CRISPR/Cas9 is a novel Swiss Army Knife in the field of synthetic biology. The thesis of Harmen van Rossum (Biotechnology/TNW) shows that this tool enables rapid genetic modification of baker’s yeast; six genetic changes could be introduced within one week – previously this could easily take a few months.
The goal of the PhD project was to optimise yeast as a cell factory for the production of a wide variety of compounds, like jet fuels and diesel replacements from renewable sources. An important precursor for such products is acetyl-CoA, a universal building block that also participates in many metabolic and physiological processes. However, its synthesis in yeast is suboptimal, requiring more sugar than necessary. Competition with fossil-based production processes is challenged by such inefficiencies. The thesis showed that this can be addressed by a combination of synthetic biology, laboratory evolution and genetic engineering. Besides replacing the native acetyl-CoA production pathway in baker’s yeast by more efficient routes from other organisms, it was also demonstrated that the direction of an important shuttle system (the carnitine shuttle) could be reversed. Such changes are key to improve acetyl-CoA production from sugars. Harmen’s PhD project was part of the BE-Basic program and in close collaboration with DSM (Delft) and Amyris (Emeryville, CA, USA). Currently, Harmen van Rossum works as a scientist at the biotech company Zymergen (Emeryville, USA).