Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the ...past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.
Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. ...Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro‐fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short‐term dynamics of the transcriptome revealed a remarkably fast decrease in the average half‐life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose‐induced regulation of gene expression.
Synopsis
In natural environment, nutrient availability may vary ‘from feast to famine’ and vice versa very suddenly and frequently causing excessive nutritional stresses. The robustness of an organism pictures its ability to cope with these transitions. Among the essential nutrient for growth, the more investigated of all is still the carbon source with a preference for glucose. The sudden increase in glucose concentration causes fast changes in the concentration of metabolites acting as secondary messengers, for example, cAMP. However, with the exception of adenosine nucleotides (Theobald et al, 1997) and central carbon metabolism intermediates (Visser et al, 2004), little is known about the fast changes at metabolome level occurring within the first minutes following the addition of excess glucose. By integrating metabolome and transcriptome data collected within the first minutes following the sudden relief of glucose limitation, this work provides the first comprehensive study, clearly picturing how yeast cells adapt to new environmental conditions.
For the quantitative systems analysis of the dynamic response to glucose availability, it is essential that experimental conditions are tightly controlled. To achieve such analysis, the yeast Saccharomyces cerevisiae was grown under glucose limitation at a growth rate of 0.05/h in chemostat culture. At steady state, the glucose concentration was instantaneously increased from 0.15 to 5 mM. The culture was sampled after 30, 60, 90, 120, 210, 300 and 330 s for metabolite and transcript analysis.
As the metabolites varied within seconds after the addition of glucose to the steady‐state culture, the transcriptome response only changed between 120 and 210 s. Despite this time shift, a very high correlation in the nature of these responses was observed. Among the fast metabolite fluctuations, an important drop in AXP pool was measured. This drop was accompanied by a time‐delayed upregulation of the genes encoding the complete purine biosynthetic pathway. Interestingly, the transcript data revealed that besides the purine biosynthesis genes, purine salvage, one carbon (C1), sulfur assimilation pathways were all coordinately upregulated, strengthening the metabolome data pointing out a requirement of purines (Figures 3 and 4). The coordinated elicitation of purine synthetic and salvage, one carbon (C1) and sulfur assimilation pathways suggests that methylation reactions mediated via S‐adenosylmethionine might play a crucial role in the growth acceleration process. Further analysis of the transcriptome response by incorporating overrepresentation of functional categories and location analysis data from more than 100 transcription factors allowed mapping of the regulatory circuit taking place in this metabolic transition. It pictured that the cells were gearing up to accelerate growth as shown by the reprogramming of the transcription and translation machinery and were trying to recover from severe redox stress.
Very early on (after 120 s), a large part of the genes involved in translation of ribosomal DNA (subunits of the RNA pol I) and ribosome processing were significantly upregulated. In conjunction, fine‐tuning of the translation seems to set up, as many elements of the translation initiation machinery were upregulated as well.
In addition to the ‘energetic stress’, as shown by a loss of the cellular energy charge, the cells faced redox stress. Based on the data presented here, we hypothesized that the imbalance in intermediates of the top (high) and the bottom (low) of the glycolysis was a direct consequence of the inhibition of glyceraldehyde‐3‐phosphate dehydrogenase by the increased NADH/NAD ratio. To restore redox homeostasis, the cell used both metabolic and transcriptional regulations. Increase of the intracellular trehalose‐6‐phosphate concentration was in line with its inhibitory role of the hexokinases preventing the cell to die from the so‐called ‘glucose accelerated death’. The modulation of the expression of the genes encoding tricarboxylic acid enzymes was also essential in the restoration of the redox cellular status.
More surprising was the rapidity of the transcript turnover. Measurement of mRNA half‐lives undoubtedly showed that a much faster decay of transcript was occurring upon the relief of glucose limitation. The average half‐life of mRNA displaying significant downregulation was nine‐fold shorter than reported earlier (Wang et al, 2001), suggesting that mRNA degradation participates actively in the regulation of translation. Consequently, one could consider that this accelerated mRNA decay represents a widespread regulatory level of glucose‐triggered catabolite repression.
Conversely to that one could think, the nutritional transition from limitation to excess glucose is not only limited to growth acceleration but it represents a more balanced process where the cell has to overcome glucose‐induced stress while the cellular processes required for gearing up growth are building up.
Experiments that, in addition to transcriptome and metabolome data, include information at other relevant information levels (e.g. proteome, phosphoproteome and fluxome, references) will be essential to meet the longstanding challenge of cellular physiology/systems biology: to come to an integral understanding of the responses of living cells to their physical and chemical environment.
Quantitative interpretation of dynamic experiments requires a tightly controlled experimental set‐up.
The kinetic of the metabolome is at least one order of magnitude faster than the kinetic of the transcriptome.
Integration of metabolome and transcriptome approaches strengthen the biological data interpretation: the nature of the coordinated transcriptome and metabolome responses are tightly correlated upon relief of glucose limitation condition.
A fast mRNA decay participates actively to the adaptation of cell facing an increased glucose concentration.
Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. ...Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.
Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of
strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of ...biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate μ ≈ 0.01 h
). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h
) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of
, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of
were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of
in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h
). Mutations in the membrane transporter genes
and/or
were found in several of the evolved strains. Deletion of
and
in a
-overexpressing strain increased its specific growth rate to 0.25 h
The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that
strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.
Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.
In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and ...differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growth-limiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms.
While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple ...yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.
A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of
Saccharomyces cerevisiae. Introduction of a ...tyrosine-insensitive allele of
ARO4 (encoding 3-deoxy-
d-
arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of
ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive
ARO4 allele in combination with the Tyr-insensitive
ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.
Genome-wide analysis of transcriptional regulation is generally studied by determining sets of "signature transcripts" that are up- or down-regulated relative to a reference situation when a single ...culture parameter or genetic modification is changed. This approach is especially relevant for defining small subsets of transcripts for use in high throughput, cost-effective diagnostic analyses. However, this approach may overlook the simultaneous control of transcription by more than one environmental parameter. This study represents the first quantitative assessment of the impact of transcriptional cross-regulation by different environmental parameters. As a model, we compared the response of aerobic as well as anaerobic chemostat cultures of the yeast Saccharomyces cerevisiae to growth limitation by four different macronutrients (carbon, nitrogen, phosphorus, and sulfur). The identity of the growth-limiting nutrient was shown to have a strong impact on the sets of transcripts that responded to oxygen availability and vice versa. We concluded that identification of reliable signature transcripts for specific environmental parameters can be obtained only by combining transcriptome data sets obtained under several sets of reference conditions. Furthermore, the two-dimensional approach to transcriptome analysis is a valuable new tool to study the interaction of different transcriptional regulation systems.
Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-arabinose pathway and the Penicillium ...chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h-1 on l-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on l-arabinose in the presence of d-glucose and d-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher Km for d-glucose transport than wild-type Gal2. Introduction of the Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on l-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.
Industrial penicillin production with the filamentous fungus Penicillium chrysogenum is based on an unprecedented effort in microbial strain improvement. To gain more insight into penicillin ...synthesis, we sequenced the 32.19 Mb genome of P. chrysogenum Wisconsin54-1255 and identified numerous genes responsible for key steps in penicillin production. DNA microarrays were used to compare the transcriptomes of the sequenced strain and a penicillinG high-producing strain, grown in the presence and absence of the side-chain precursor phenylacetic acid. Transcription of genes involved in biosynthesis of valine, cysteine and alpha-aminoadipic acid-precursors for penicillin biosynthesis-as well as of genes encoding microbody proteins, was increased in the high-producing strain. Some gene products were shown to be directly controlling beta-lactam output. Many key cellular transport processes involving penicillins and intermediates remain to be characterized at the molecular level. Genes predicted to encode transporters were strongly overrepresented among the genes transcriptionally upregulated under conditions that stimulate penicillinG production, illustrating potential for future genomics-driven metabolic engineering.
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