The biobased-economy aims to create a circular biotechnology ecosystem to transition from a fossil fuel-based to a sustainable industry based on biomass. For this, new microbial cell-factories are essential. We present the draft genome of the CEN.PK-derived Saccharomyces cerevisiae SpyCas9 expressing strain (IMX2600), that serve as chassis of new cell-factories.

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The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

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Over the past century, the world population has increased fourfold. This increase in human population is accompanied by an increased demand in food, water, energy and consumer goods. The resulting intensification of agriculture, deforestation, emission of green-house gasses and high utilization of natural compounds such as coal, minerals, metals and fossil-fuels have resulted in global warming and a depletion of Earth’s natural reserves. The application of biotechnology can aid in the transition to a more sustainable, circular and bio-based economy. For example, by offering novel production processes for a range of different compounds, such as therapeutics, beverages, food, chemicals and fuels from renewable sources. For instance, Baker’s yeast is known for its natural ability to produce CO2 and ethanol from sugars, characteristics that were historically exploited for the production of alcoholic beverages and bread. Today, bio-ethanol as transportation fuel made by yeasts also provides a more sustainable alternative to gasoline. Additionally, due to the enormous increase in knowledge and the establishment of genome editing tools and sequencing possibilities, biotechnology can now apply genetically engineered microbes to produce an ever-increasing range of products, both native or heterologous, to the microorganism. For example, micro-organisms have been engineered to produce complex molecules such as human insulin, the flavoring compound vanillin and the antimalarial drug artemisinin. Insulin, which is essential for treatment of diabetes, was conventionally produced by extraction from pig pancreas, while artemisinin and vanillin were extracted from plants, or in case of vanillin, also synthesized chemically. However, microbial production of such industrially valuable compounds, from simple substrates such as glucose or second-generation feedstocks, offers a more reliable and sustainable production method compared to these classical methods. In this thesis special emphasis is given to the Baker’s yeast Saccharomyces cerevisiae and its application in the production of aromatic compounds. There is an increasing interest in the microbial production of aromatic molecules, such as in the flavor and fragrance industry. The economic potential of this field is partly due to European legislation, that allows the production and sale of microbially produced molecules, as long as the final product is devoid of genetically modified organisms (GMOs). S. cerevisiae is able to natively synthesize several aromatic compounds, although their production is limited by tight regulation of the involved pathways. Many other industrially attractive aromatic compounds find their origin in plants. In order to establish yeast-based production of these aromatic molecules, it is necessary to both introduce plant genes, and modify, the metabolism of S. cerevisiae to obtain fast and efficient production...@en

Engineered strains of the yeast Saccharomyces cerevisiae are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use L-phenylalanine and/or L-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds. Activity of the Ehrlich pathway requires any of four S. cerevisiae 2-oxo-acid decarboxylases (2-OADCs): Aro10 or the pyruvate-decarboxylase isoenzymes Pdc1, Pdc5, and Pdc6. Elimination of pyruvate-decarboxylase activity from S. cerevisiae is not straightforward as it plays a key role in cytosolic acetyl-CoA biosynthesis during growth on glucose. In a search for pyruvate decarboxylases that do not decarboxylate aromatic 2-oxo acids, eleven yeast and bacterial 2-OADC-encoding genes were investigated. Homologs from Kluyveromyces lactis (KlPDC1), Kluyveromyces marxianus (KmPDC1), Yarrowia lipolytica (YlPDC1), Zymomonas mobilis (Zmpdc1) and Gluconacetobacter diazotrophicus (Gdpdc1.2 and Gdpdc1.3) complemented a Pdc⁻ strain of S. cerevisiae for growth on glucose. Enzyme-activity assays in cell extracts showed that these genes encoded active pyruvate decarboxylases with different substrate specificities. In these in vitro assays, ZmPdc1, GdPdc1.2 or GdPdc1.3 had no substrate specificity towards phenylpyruvate. Replacing Aro10 and Pdc1,5,6 by these bacterial decarboxylases completely eliminated aromatic fusel-alcohol production in glucose-grown batch cultures of an engineered coumaric acid-producing S. cerevisiae strain. These results outline a strategy to prevent formation of an important class of by-products in ‘chassis’ yeast strains for production of non-native aromatic compounds.

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The organic compound 2-phenylethanol (2PE) has a pleasant floral scent and is intensively used in the cosmetic and food industries. Microbial production of 2PE by phenylalanine bioconversion or de novo biosynthesis from sugar offer sustainable, reliable and natural production processes compared to chemical synthesis. Despite the ability of Saccharomyces cerevisiae to naturally synthesize 2PE, de novo synthesis in high concentration and yield remains a metabolic engineering challenge. Here, we demonstrate that improving phosphoenolpyruvate supply by expressing pyruvate kinase variants and eliminating the formation of p-hydroxy-phenylethanol without creating tyrosine auxotrophy significantly contributed to improve 2PE production in S. cerevisiae. In combination with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, these mutations enabled better connection between glycolysis and pentose phosphate pathway optimizing carbon flux towards 2PE. However, attempts to further connect these two parts of central carbon metabolism by redirecting fructose-6P towards erythrose-4P by expressing a phosphoketolase-phosphotransacetylase pathway did not result in improved performance. The best performing strains were capable of producing 13mM of 2PE at a yield of 0.113 mol mol^-1, which represents the highest yield for de novo produced 2PE in S. cerevisiae and other yeast species.

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CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH). In anaerobic, glucose-grown batch cultures this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h^-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol g biomass^-1 h^-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h^-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.

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