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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Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Responses of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.

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The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.

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Background: The lager brewing yeast, S. pastorianus, is a hybrid between S. cerevisiae and S. eubayanus with extensive chromosome aneuploidy. S. pastorianus is subdivided into Group 1 and Group 2 strains, where Group 2 strains have higher copy number and a larger degree of heterozygosity for S. cerevisiae chromosomes. As a result, Group 2 strains were hypothesized to have emerged from a hybridization event distinct from Group 1 strains. Current genome assemblies of S. pastorianus strains are incomplete and highly fragmented, limiting our ability to investigate their evolutionary history. Results: To fill this gap, we generated a chromosome-level genome assembly of the S. pastorianus strain CBS 1483 from Oxford Nanopore MinION DNA sequencing data and analysed the newly assembled subtelomeric regions and chromosome heterozygosity. To analyse the evolutionary history of S. pastorianus strains, we developed Alpaca: a method to compute sequence similarity between genomes without assuming linear evolution. Alpaca revealed high similarities between the S. cerevisiae subgenomes of Group 1 and 2 strains, and marked differences from sequenced S. cerevisiae strains. Conclusions: Our findings suggest that Group 1 and Group 2 strains originated from a single hybridization involving a heterozygous S. cerevisiae strain, followed by different evolutionary trajectories. The clear differences between both groups may originate from a severe population bottleneck caused by the isolation of the first pure cultures. Alpaca provides a computationally inexpensive method to analyse evolutionary relationships while considering non-linear evolution such as horizontal gene transfer and sexual reproduction, providing a complementary viewpoint beyond traditional phylogenetic approaches.

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Many organisms have several similar transporters with different affinities for the same substrate. Typically, high-affinity transporters are expressed when substrate is scarce and low-affinity ones when it is abundant. The benefit of using low instead of high-affinity transporters remains unclear, especially when additional nutrient sensors are present. Here, we investigate two hypotheses. It was previously hypothesized that there is a trade-off between the affinity and the catalytic efficiency of transporters, and we find some but no definitive support for it. Additionally, we propose that for uptake by facilitated diffusion, at saturating substrate concentrations, lowering the affinity enhances the net uptake rate by reducing substrate efflux. As a consequence, there exists an optimal, external-substrate-concentration dependent transporter affinity. A computational model of Saccharomyces cerevisiae glycolysis shows that using the low affinity HXT3 transporter instead of the high affinity HXT6 enhances the steady-state flux by 36%. We tried to test this hypothesis with yeast strains expressing a single glucose transporter modified to have either a high or a low affinity. However, due to the intimate link between glucose perception and metabolism, direct experimental proof for this hypothesis remained inconclusive. Still, our theoretical results provide a novel reason for the presence of low-affinity transport systems.

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The haploid Saccharomyces cerevisiae strain CEN.PK113–7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias. In this study, we obtained a near-complete genome assembly of CEN.PK113–7D using only Oxford Nanopore Technology's MinION sequencing platform. Fifteen of the 16 chromosomes, the mitochondrial genome and the 2-μm plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere repeat. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113–7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII that caused misidentification of a MAL locus in the previous CEN.PK113–7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113–7D and places a caveat on assumed genome stability of microorganisms.@en

Stationary-phase (SP) batch cultures of Saccharomyces cerevisiae, in which growth has been arrested by carbon-source depletion, are widely applied to study chronological lifespan, quiescence and SP-associated robustness. Based on this type of experiments, typically performed under aerobic conditions, several roles of oxygen in aging have been proposed. However, SP in anaerobic yeast cultures has not been investigated in detail. Here, we use the unique capability of S. cerevisiae to grow in the complete absence of oxygen to directly compare SP in aerobic and anaerobic bioreactor cultures. This comparison revealed strong positive effects of oxygen availability on adenylate energy charge, longevity and thermotolerance during SP. A low thermotolerance of anaerobic batch cultures was already evident during the exponential growth phase and, in contrast to the situation in aerobic cultures, was not substantially increased during transition into SP. A combination of physiological and transcriptome analysis showed that the slow post-diauxic growth phase on ethanol, which precedes SP in aerobic, but not in anaerobic cultures, endowed cells with the time and resources needed for inducing longevity and thermotolerance. When combined with literature data on acquisition of longevity and thermotolerance in retentostat cultures, the present study indicates that the fast transition from glucose excess to SP in anaerobic cultures precludes acquisition of longevity and thermotolerance. Moreover, this study demonstrates the importance of a preceding, calorie-restricted conditioning phase in the acquisition of longevity and stress tolerance in SP yeast cultures, irrespective of oxygen availability.@en