Marcel van den Broek’s research while affiliated with Delft University of Technology and other places

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Publications (16)


Chromosome level genome assembly and annotation of Hanseniaspora mollemarum CBS 18055 strain
  • Article

January 2025

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4 Reads

Microbiology Resource Announcements

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Harshitha Indudhar Hanji

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Marcel van den Broek

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Jean-Marc Daran

Hanseniaspora species gained attention due to the ability of these species to ferment simple sugars and to actively contribute to the development of bouquet aromas in wine and cider fermentations. We present a chromosome-level assembly of an isolate of Hanseniaspora mollemarum that would enhance its potential applications.


Discovery and Synthetic Applications of a NAD(P)H-Dependent Reductive Aminase from Rhodococcus erythropolis

December 2024

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22 Reads

ACS Catalysis

Ewald P. J. Jongkind

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Jack Domenech

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Arthur Govers

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[...]

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Caroline E. Paul

Reductive amination is one of the most synthetically direct routes to access chiral amines. Several Imine Reductases (IREDs) have been discovered to catalyze reductive amination (Reductive Aminases or RedAms), yet they are dependent on the expensive phosphorylated nicotinamide adenine dinucleotide cofactor NADPH and usually more active at basic pH. Here, we describe the discovery and synthetic potential of an IRED from Rhodococcus erythropolis (RytRedAm) that catalyzes reductive amination between a series of medium to large carbonyl and amine compounds with conversions of up to >99% and 99% enantiomeric excess at neutral pH. RytRedAm catalyzes the formation of a substituted γ-lactam and N-methyl-1-phenylethanamine with stereochemistry opposite to that of fungal RedAms, giving the (S)-enantiomer. This enzyme remarkably uses both NADPH and NADH cofactors with KM values of 15 and 247 μM and turnover numbers kcat of 3.6 and 9.0 s–1, respectively, for the reductive amination of hexanal with allylamine. The crystal structure obtained provides insights into the flexibility to also accept NADH, with residues R35 and I69 diverging from that of other IREDs/RedAms in the otherwise conserved Rossmann fold. RytRedAm thus represents a subfamily of enzymes that enable synthetic applications using NADH-dependent reductive amination to access complementary chiral amine products.


Effect of growth-limiting carbon source on the physiology of C. jadinii and proteome composition. S. cerevisiae CEN.PK113-7D and C. jadinii CBS 621 were grown in steady-state, aerobic ethanol-limited and glucose-limited chemostats at a dilution rate of 0.10 h⁻¹. A, B Biomass yields on substrate and oxygen, respectively. C Protein content as a percentage of biomass. D Protein yields on substrate. E Enzymes involved in conversion of ethanol to acetyl-CoA in yeasts. Adh alcohol dehydrogenase, Ald aldehyde dehydrogenase, Acs acetyl-CoA synthetase. F Top 10 most abundant proteins (with at least 3 identified peptides) in the proteome of ethanol-grown C. jadinii versus the levels of the same proteins in glucose-limited chemostats in the same species. Cj stands for Cyberlindnera jadinii. G Protein amino acid residue composition (as a percentage of the total amino acid residue pool). H stands for highest, and L for lowest amino acids in abundance for all analysed species and conditions. #p < 0.05, ##p < 0.01, ###p < 0.001, S. cerevisiae vs C. jadinii, same substrate. *p < 0.05, **p < 0.01, ***p < 0.001, same yeast species, ethanol- (EtOH) vs glucose-grown (Gluc), n = 2
Growth rates and physiological characterization of C. jadinii in batch and ethanol-limited chemostat cultures at varying dilution rates.A Optical density at 660 nm (OD660) as a function of time and B maximum growth rates (µmax) of glucose- and ethanol-grown batch cultures. C Biomass yield on ethanol, D biomass-specific ethanol uptake rate (qethanol) and E biomass-specific oxygen uptake rates (qO2) in aerobic, steady-state, ethanol-limited chemostat cultures of C. jadinii CBS 621. F Maintenance requirements for ethanol (methanol) and oxygen (mO2) calculated as the y-intercept of the fitted curves shown in D and E, respectively. Maintenance ATP requirements (mATP) were estimated from mO2, assuming an in vivo P/O ratio of 1.8 [16]. G, H Quantification of the main components of the biomass in % of dry weight: proteins (Prot), RNA, carbohydrates (Carb) and lipids. I Protein allocation in ethanol-limited chemostat cultures grown at different dilution rates. Categories were manually curated and represent (in ascending order) ethanol catabolism (first three steps shown in Fig. 1F), carbohydrate metabolism (Carb.), oxidative phosphorylation (OXPHOS), TCA cycle (including the glyoxylate shunt reactions), amino acid metabolism, translation and others. The last category encompasses proteins from pathways with a lower proteome allocation or for which no pathway was assigned. J Total alcohol dehydrogenase protein (CjAdh, % of proteome) as a function of dilution rate. K Estimated vmax for CjAdh, assuming a kcat of 150 min⁻¹ for all isoforms (Brenda database [76], EC 1.1.1.1, average reported value for yeasts) and a proteome comprising 50% of the dry weight (w/w), plotted together with the ethanol consumption rates (qethanol) from bioreactor experiments. 0.39 h⁻¹ represents the intersection between both lines. Open symbols show carbon recovery of around 107%, while closed symbols represent carbon recoveries of 100 ± 5%. **p < 0.001, n = 2
Growth of C. jadinii CBS 621 on single-vitamin dropout and vitamin-free media. A Growth curves of C. jadinii CBS 621 on single-vitamin dropout media in shake flasks grown on synthetic medium with ethanol as a sole carbon source. ∆ “vitamin name” indicates absence of only the indicated vitamin from the standard vitamin mix. Complete indicates presence of all vitamins and ∆vitamins indicates absence of all vitamins. B OD660 after 24 h of cultivation in shake flasks. C OD660 after further 24 h of cultivation in shake flasks for samples from indicated groups (x-axis) that were reinoculated at OD 0.5 in the presence or absence of thiamine, n = 2. D Overview of tBLASTn search to identify genes coding for enzymes in the biosynthesis of thiamine in CBS 621. The reference GenBank IDs used as queries were retrieved from UniProt, based on searches containing the respective protein name and species (C. jadinii CBS 1600). The tBLASTn algorithm was run locally using default settings
Characterization of C. jadinii strains in microtiter plates, shake flasks and bioreactors concerning vitamin dependency. A Representative growth curves of serially diluted CBS 621 in microtiter plates, ranging from 1 to 32 × dilution, initial OD660 0.2 to 0.00625, respectively. B Calculation of specific growth rates via serial dilution method. The time (h) required for a specific dilution to reach a green value of 20 was plotted against the natural logarithm of the respective dilution (1–32). C Specific growth rates for 13 C. jadinii strains grown on ethanol in the presence or absence of vitamins, calculated with the serial dilution method, n.d non-detectable. D Specific growth rates of 4 C. jadinii strains grown on ethanol in shake flasks in the presence or absence of vitamins. E Correlation of specific growth rates obtained in microtiter plates (x-axis) with those measured in shake flasks (y-axis) for strains grown in the presence of vitamins. F–H Biomass yields on ethanol, oxygen and protein content for selected strains grown in ethanol-limited chemostats (dilution rate 0.10 h⁻¹). Where indicated by ∆vit, no vitamins were added to the cultivation, n = 2–3
Growth of C. jadinii CBS 5947 on vitamin-free medium in aerobic, ethanol-limited fed-batch cultures. Aerobic fed-batch fermentation was conducted at 30 °C, at pH 5.0 and at 1 bar overpressure, with a feed of pure ethanol. A 25% (w/v) NH4OH solution was used for simultaneous pH control and nitrogen supplementation. A Scheme of reactor and critical parameters related to the process (fed-batch phase). W0: initial weight of the culture broth (kg), cx,0: initial biomass concentration (kgbiomass (kgbroth)⁻¹), µ0: initial specific growth rate in fed-batch phase (h⁻¹), DW = biomass dry weight concentration (gbiomass (kgbroth)⁻¹). Figure created in BioRender. Vieira-Lara, M. (2024) BioRender.com/z76n618. B Profile of feed rate during the fed-batch phase. C–E Consumed ethanol, biomass dry weight, and growth rate (µ) during the fed-batch phase. Symbols represent data and grey lines refer to simulations. F–G Protein percentage in the biomass and total protein content during the fed-batch phase. H Total biomass and protein yield on ethanol based on the whole fed-batch process. Data are shown for 2 independent reactors (mean ± SD)
Quantitative physiology and biomass composition of Cyberlindnera jadinii in ethanol-grown cultures
  • Article
  • Full-text available

December 2024

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53 Reads

Biotechnology for Biofuels and Bioproducts

Background Elimination of greenhouse gas emissions in industrial biotechnology requires replacement of carbohydrates by alternative carbon substrates, produced from CO2 and waste streams. Ethanol is already industrially produced from agricultural residues and waste gas and is miscible with water, self-sterilizing and energy-dense. The yeast C. jadinii can grow on ethanol and has a history in the production of single-cell protein (SCP) for feed and food applications. To address a knowledge gap in quantitative physiology of C. jadinii during growth on ethanol, this study investigates growth kinetics, growth energetics, nutritional requirements, and biomass composition of C. jadinii strains in batch, chemostat and fed-batch cultures. Results In aerobic, ethanol-limited chemostat cultures, C. jadinii CBS 621 exhibited a maximum biomass yield on ethanol (YX/SmaxY_{X/S}^{\max}) of 0.83 gbiomass (gethanol)⁻¹ and an estimated maintenance requirement for ATP (mATP) of 2.7 mmolATP (gbiomass)⁻¹ h⁻¹. Even at specific growth rates below 0.05 h⁻¹, a stable protein content of approximately 0.54 gprotein (gbiomass)⁻¹ was observed. At low specific growth rates, up to 17% of the proteome consisted of alcohol dehydrogenase proteins, followed by aldehyde dehydrogenases and acetyl-CoA synthetase. Of 13 C. jadinii strains evaluated, 11 displayed fast growth on ethanol (μmax > 0.4 h⁻¹) in mineral medium without vitamins, and CBS 621 was found to be a thiamine auxotroph. The prototrophic strain C. jadinii CBS 5947 was grown on an inorganic salts medium in fed-batch cultures (10-L scale) fed with pure ethanol. Biomass concentrations in these cultures increased up to 100 gbiomass (kgbroth)⁻¹, with a biomass yield of 0.65 gbiomass (gethanol)⁻¹. Model-based simulation, based on quantitative parameters determined in chemostat cultures, adequately predicted biomass production. A different protein content of chemostat- and fed-batch-grown biomass (54 and 42%, respectively) may reflect the more dynamic conditions in fed-batch cultures. Conclusions Analysis of ethanol-grown batch, chemostat and fed-batch cultures provided a quantitative physiology baseline for fundamental and applied research on C. jadinii. Its high maximum growth rate, high energetic efficiency of ethanol dissimilation, simple nutritional requirements and high protein content, make C. jadinii a highly interesting platform for production of SCP and other products from ethanol.

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Quantitative physiology and biomass composition of Cyberlindnera jadinii in ethanol-grown cultures

August 2024

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35 Reads

Background Elimination of greenhouse gas emissions in industrial biotechnology requires replacement of carbohydrates by alternative carbon substrates, produced from CO 2 and waste streams. Ethanol is already industrially produced from agricultural residues and waste gas and is miscible with water, self-sterilising and energy-dense. The yeast C. jadinii can grow on ethanol and has a history in the production of single-cell protein (SCP) for feed and food applications. To address a knowledge gap in quantitative physiology of C. jadinii during growth on ethanol, this study investigates growth kinetics, growth energetics, nutritional requirements, and biomass composition of C. jadinii strains in batch, chemostat and fed-batch cultures. Results In aerobic, ethanol-limited chemostat cultures, the thiamine-auxotrophic strain C. jadinii CBS 621 exhibited a maximum biomass yield on ethanol (Y x/smax ) of 0.83 g biomass ·(g ethanol ) ⁻¹ and an estimated maintenance requirement for ATP (m ATP ) of 0.28 mmol·(g biomass ) ⁻¹ ·h ⁻¹ . Even at specific growth rates below 0.05 h ⁻¹ , a stable protein content of approximately 0.54 g protein ·(g biomass ) ⁻¹ was observed. At low specific growth rates, up to 17% of the proteome consisted of alcohol dehydrogenase proteins, followed by aldehyde dehydrogenases and acetyl-CoA synthetase. Of 13 C. jadinii strains evaluated, 11 displayed fast growth on ethanol (μ max > 0.4 h ⁻¹ ) in mineral medium without vitamins. The prototrophic strain C. jadinii CBS 5947 was grown on an inorganic salts medium in fed-batch cultures fed with pure ethanol. Biomass concentrations in these cultures increased up to 100 g·L -1 , with a biomass yield of 0.65 g biomass ·(g ethanol ) ⁻¹ . Model-based simulation, based on quantitative parameters determined in chemostat cultures, adequately predicted biomass production. A different protein content of chemostat-and fed-batch-grown biomass (54% and 42%, respectively) may reflect the more dynamic conditions in fed-batch cultures. Conclusions Analysis of ethanol-grown batch, chemostat and fed-batch cultures provided a quantitative physiology baseline for fundamental and applied research on C. jadinii . Its high maximum growth rate, high energetic efficiency of ethanol dissimilation, simple nutritional requirements and high protein content, make C. jadinii a highly interesting platform for production of SCP and other products from ethanol.



Draft genome sequence of the Saccharomyces cerevisiae SpyCas9 expressing strain IMX2600, a laboratory and platform strain from the CEN.PK lineage for cell-factory research

December 2023

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35 Reads

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2 Citations

Microbiology Resource Announcements

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.


Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae reveals condition-dependent intron abundance

August 2023

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89 Reads

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5 Citations

Yeast

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for the enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification of spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as the sole carbon source, revealing the impact of growth conditions on mitochondrial RNA expression and splicing. This study uncovered a remarkable difference in the turnover of Group II introns between yeast grown in either mostly fermentative or fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to monitor mitochondrial transcriptome responses in a broad range of relevant contexts, including oxidative stress, apoptosis and mitochondrial diseases.


Figure 1. The CRI-SPA gene-transfer system. ( A ) Left: The CRI-SPA Donor (CD) strain and the recipient library strains (here YKO). Right: Graphics of the individual genetic components are shown in the legend box. The YKO strains contain a specific deletion marked by the antibiotic marker 1 (AB1) and a target site for the Cas9-sgRNA CRISPR nuclease. The CD strains contain: Kl URA3-GAL1p cassettes at the centr omeres of all chr omosomes , a cas9 gene , a selectable CRI-SPA plasmid maintained by antibiotic marker (AB2) and encoding a sgRNA targeting the insertion site of the genetic feature (blocked in the CD), a genetic feature of interest coupled to an antibiotic marker, AB3 (optional), and a Kl URA3 gene between the genetic feature of interest and the telomere. ( B ) The individual steps of the CRI-SPA procedure (see main text for details). Step 1 , a CD strain is pinned onto all strains of the library plates and incubated for mating. Step 2 , diploids are exhausted for glucose via growth on raffinose and selected for the marker in the library strains (AB1) and the marker on the CRI-SPA plasmid (AB2). In the diploid stage, the target site in the recipient strain is cleaved by Cas9-sgRNA. Repair of the resulting DNA DSB by gene conversion using the corresponding donor site in the CD strain as template transfers the genetic feature to the recipient locus. Note that the target site of the Cas9-sgRNA in the recipient chromosome is destroyed by insertion of the genetic feature of interest. Step 3 , galactose induced SPA eliminates the donor chromosomes. Step 4, haploid cells containing only recipient chromosomes are selected on 5-FOA. Step 5, the modified library strains are obtained by selecting for AB1, which marks the YKO deletion, and optionally, by selecting for the genetic feature (AB3).
Figure 3. Betaxanthin production in YKO strains. ( A ) Left: images of BY-Ref and BY-Btx colonies in visible light (top) and corresponding filtered images (bottom). Right: quantification of yellowness of BY-Ref and BY-Btx colonies by image analysis, see main text and Supplementary Figure S2 for details. ( B ) Left: image of BY-Ref and the BY-Btx strains in liquid SC medium. Right: quantification of fluorescence produced by BY-Ref and the BY-Btx strains in liquid SC medium. ( C ) Final plate of a CRI-SPA Btx-cassette transfer experiment. At the top, a scheme illustrating the Btx-cassette and how it is transferred from the CD strain to a library recipient strain. To the left, diagrams with color codes indicating selection applied at the different CRI-SPA steps. G418 selects for the kanMX marker in the gene-deletion cassettes of the YKO library, HYG selects for the CRI-SPA plasmid, NTC selects for the Btx-cassette, and 5-FOA counter-select donor strain chromosomes, which all contain Kl URA3 markers. ( D ) In the middle, yellow intensity ranking of all YKO mutant strains after CRI-SPA mediated Btx-cassette transfer. To the left, mosaic images of the four individual replicates obtained with selected YKO mutants displaying higher , similar , or lower yellowness as compared to BY-Btx. Mutants in red frames were purified and restreaked on solid YPD medium, see images to the right.
Figure 4. Reproducibility of betaxanthin CRI-SPA experiments in the presence and absence of pathway transfer selection. ( A ) The mean yellow color intensities obtained for each mutant in repeat 1 and repeat 2, which both were performed in the presence of natMX to select for the Btx-cassette genes. ( B ) The mean yellow color intensities obtained for each mutant in trial 1 and trial 3. Trial 3 was performed in the absence of natMX . The top and bottom 192 genes and their overlap across screens are colored and displayed in flanking Venn diagrams. Insets show the correlation for colony fitness across screens.
Figure 5 Betaxanthin yield and fitness r elationship r e v eals mechanistic patterns in betaxanthin production. Colony size (i.e. fitness) vs Yellow Color intensity (i.e. Betaxanthin Yield). The results of Gene Ontology Enrichment Analyses on se v eral subgroups of genes are shown as graphs for the bottom hits (white, 1.2 std below the screen mean) and top hits (yellow, 1.2 std above the screen mean). As indicated by the bottom right scale, node size indicates the significance of GO terms enrichment, edge transparency indicates the number of shared genes between GO terms. The cyan group did not show any term enrichment. The position of the BY-Btx strain is marked with a black asterisk.
Figure 6. CRI-SPA hits identified on solid medium produce enhanced amounts of betaxanthin in liquid medium. ( A ) selected Btx-hit str ains y ellow and fitness scores within the screen. ( B ) Correlation between CRI-SPA yellowness score and fluorescence of re v ersed engineered hits after 7 days cultivation in liquid SC medium. Error bars show standard deviations for 16 and 3 replicates for CRI-SPA and liquid Fluor escence, r especti v ely. Mar kers coloring scheme, below inset, is defined by the agreement between CRI-SPA scor e and fluor escence in liquid medium, and significance differ ence from WT r eported by a one-sided Welch's test.
CRI-SPA: a high-throughput method for systematic genetic editing of yeast libraries

August 2023

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162 Reads

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5 Citations

Nucleic Acids Research

Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.


Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts

April 2023

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30 Reads

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3 Citations

New Biotechnology

Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR- induced double strand break is unpredictable and preferentially uses the homoeologous chromosome as template, preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for (i) absence of loss of heterozygosity upon CRISPR-editing, (ii) efficiency of the gRNA, and (iii) absence of effect on strain physiology. Successful examples of highly efficient single and double gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development. DATA AVAILABILITY: Data underlying graphs and figures found in this manuscript are deposited at the 4TU research dat center (https://data.4tu.nl/info/en/) and available through the doi: 10.4121/21648329.


Figure 1. Annotated mitochondrial genome of CEN.PK113-7D. Primary polycistronic transcripts (light blue boxes); mRNA: messenger RNA, ncRNA: noncoding RNA, rRNA: ribosomal RNA, tRNA: transfer RNA. Exons that encode the main proteins are indicated in pink. Coding intron sequences are encoded in orange, non-doing intron sequences are indicated in white.
Figure 2. Sequencing pipeline and quality control. A) schematic overview of the sample preparation and sequencing pipeline. B,C) Waffle plots showing distribution of read origin and type of the passed reads (Q >=7) of the three replicates of ethanol-grown cultures (B) and glucose-grown cultures (C). The distribution of the origin of sequencing reads between mitochondrial, cytosolic and control RNA is shown on the left, on the right the distribution of different types of mitochondrial RNA is shown. One square represents 1/100 of the total number of reads.
Figure 4. Sequencing coverage of the mitochondrial transcriptome of cultures grown on ethanol and glucose. A) per-base coverage of the mitochondrial transcriptome of cultures grown on ethanol (top) compared to the per-base coverage of the mitochondrial transcriptome of cultures grown on glucose (bottom), normalized per million bases as Counts-Per-Million (CPM) of the heavy (forward) strand of the mitochondrial DNA. Coverage depth is represented in grey, the standard deviation between the sequencing depth of triplicate experiments is shown in orange. B) schematic overview, sequencing depth and splicing of the COX1 Open reading frame (ORF). The middle shows the COX1 ORF, where the exon sequences are shaded in pink, colorless areas are spliced out of the final COX1 mRNA. The different splicing variants of the COX1 open reading frame are shown below the COX1 ORF. Capitalized names are used to indicate protein-encoding introns, non-coding introns are shown in lowercase. (i) shows per-base coverage of the COX1 ORF, normalized per million bases as Counts-Per-Million (CPM). Coverage depth is represented in grey, the standard deviation between the sequencing depth of triplicate experiments is shown in red. (ii) shows visualization of sequencing reads mapped to the COX1 ORF visualized using Tablet, a red line indicates alignment of a sequencing read to the location on the ORF, a grey line indicates a gap in the read. The analysis was done for mitochondria of ethanolgrown cultures (top) and glucose-grown cultures (bottom). The location of the COX1 exon is shaded in pink for visualization purposes. C) schematic overview, sequencing depth and splicing of the COB ORF. D) Transcript levels of the COX1 introns on glucose (light pink) and ethanol (purple), normalized to library size in CPM and normalized to transcript levels of the COX1 exon mRNA. E) Transcript levels of the COB introns on glucose (light pink) and ethanol (purple), normalized to library size in CPM and normalized to transcript levels of the COB exon mRNA.
S. cerevisiae strains used in this study.
Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae reveals condition-dependent intron turnover

January 2023

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144 Reads

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or post-transcriptional processing and splicing. Due to inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions. Therefore, a method for enrichment of mitochondrial RNA and sequencing using Nanopore technology was developed, enabling the resolution of splicing of polycistronic genes and the quantification the spliced RNA. This method successfully captured the full mitochondrial transcriptome and resolved RNA splicing patterns with single-base resolution, and was applied to explore the transcriptome of S. cerevisiae grown with glucose or ethanol as sole carbon source, revealing the impact of growth conditions on mitochondrial RNA-expression and splicing. This study uncovered a remarkable difference in turn-over of group II introns between yeast grown in mostly fermentative and fully respiratory conditions. Whether this accumulation of introns in glucose medium has an impact on mitochondrial functions remains to be explored. Combined with the high tractability of the model yeast S. cerevisiae, the developed method enables to explore mitochondrial transcriptome regulation and processing in a broad range of conditions relevant in human context, including aging, apoptosis and mitochondrial diseases.


Citations (6)


... To verify earlier reports that C. jadinii shows higher biomass yields on ethanol than S. cerevisiae [16,18], aerobic ethanol-limited chemostat cultures of the model strains C. jadinii CBS 621 [18,57,58] and S. cerevisiae CEN. PK113-7D [39,[59][60][61] were grown at a dilution rate of 0.10 h −1 . Consistent with earlier reports [18], steadystate cultures of S. cerevisiae CEN.PK113-7D showed a biomass yield of 0.59 ± 0.01 g biomass (g ethanol ) −1 (Fig. 1A). ...

Reference:

Quantitative physiology and biomass composition of Cyberlindnera jadinii in ethanol-grown cultures
Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth

Metabolic Engineering

... FACS data was analyzed using the Flowing Software version 2.5.1 (Turku Centre for Biotechnology, Finland). Broek et al. 2023 ) to which four extra contigs containing the ErCas12a and Xdcrt integration cassettes were added (supplementary sequences). The alignments were further processed with SAMtools (Danecek et al. 2021 ) and visualized using the Integr ativ e Genomics Vie wer (IGV) (Robinson et al. 2011 ). ...

Draft genome sequence of the Saccharomyces cerevisiae SpyCas9 expressing strain IMX2600, a laboratory and platform strain from the CEN.PK lineage for cell-factory research

Microbiology Resource Announcements

... (Meta)transcriptome allows for functional profiling of microbial communities. It is suitable for RNA viruses (Fritch et al., 2023;Kim et al., 2023), eukaryotic microbes (Wongsurawat et al., 2022;Albacar et al., 2023;Krawczyk et al., 2023;Koster et al., 2024), and only a few studies have been conducted on prokaryotic microbes (Grü nberger et al., 2020(Grü nberger et al., , 2022Forquet et al., 2022;Pust et al., 2022;Poursalavati et al., 2023). Pitt et al. employed nanopore sequencing to identify the resistant RNA of Klebsiella pneumoniae, successfully detecting up to three resistance gene operons (Pitt et al., 2020). ...

Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae reveals condition-dependent intron abundance

Yeast

... T he limited studies to date seem to indicate that an inter-c hr omosomal r ecombination is a diploid-r elated e v ent, but it r emains to be determined whether it is more prevalent in hybrids where the higher sequence divergence between sister c hr omosomes ma y ha ve an impact. In fact, data from a recent study in S. pastorianus supports this idea as those authors observed less recombination in cases where divergent c hr omosomes wer e not pr esent (Bennis et al. 2023 ). Although not central to this study, it is w ell kno wn that the process of domestication often gives rise to polyploidization, aneuploidy and loss of heterozygosity, and it is interesting to speculate that similar forces may be at play here. ...

Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts
  • Citing Article
  • April 2023

New Biotechnology

... Postma et al. [103] assembled linear and circular chromosomes containing 41 genes in Saccharomyces cerevisiae. The resulting strain, IMF48, produced a titer of 0.049 ± 0.007 µM of pelargonidin-3-O-glucoside, establishing the first microbial cell factory for the de novo production of pelargonidin-3-O-glucoside (a type of anthocyanin). ...

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism
  • Citing Article
  • January 2022

Metabolic Engineering

... Strains expressing the yeast-optimized fluorescent protein ymTur-quoise2 (ymTq2; Botman et al., 2019) in the mitochondria (IMC251, IMC252 and IMC253; Table 1) were transformed with plasmid pUDC329 (pTEF1-preSU9-ymTq2-tENO2, URA3, CEN/ARS, bla and ColE1). Plasmid pUDC329 was cloned using Golden Gate assembly performed according to Lee et al. (2015), from part plasmids pUD538 (GFP-dropout backbone, URA3, CEN/ARS, bla and ori), pYTK013 (part 2, pTEF1), pGGkp320 (part 3a, preSU9), pGGkp308 (part 3b, ymTurquoise2) and pYTK055 (part 4, tENO2) (Supporting Information: Table S2) (Boonekamp et al., 2022;Bouwknegt et al., 2021). The part plasmids were either obtained from the Yeast Toolkit collection (Lee et al., 2015) or made according to the author's instructions using primers with Golden Gate flanking regions corresponding to the respective part type. ...

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

Fungal Biology and Biotechnology