Publications by authors named "Bart Oud"

7 Publications

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Multiple downy mildew effectors target the stress-related NAC transcription factor LsNAC069 in lettuce.

Plant J 2019 09 4;99(6):1098-1115. Epub 2019 Jul 4.

Plant-Microbe Interactions, Department of Biology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, the Netherlands.

To cause disease in lettuce, the biotrophic oomycete Bremia lactucae secretes potential RxLR effector proteins. Here we report the discovery of an effector-target hub consisting of four B. lactucae effectors and one lettuce protein target by a yeast-two-hybrid (Y2H) screening. Interaction of the lettuce tail-anchored NAC transcription factor, LsNAC069, with B. lactucae effectors does not require the N-terminal NAC domain but depends on the C-terminal region including the transmembrane domain. Furthermore, in Y2H experiments, B. lactucae effectors interact with Arabidopsis and potato tail-anchored NACs, suggesting that they are conserved effector targets. Transient expression of RxLR effector proteins BLR05 and BLR09 and their target LsNAC069 in planta revealed a predominant localization to the endoplasmic reticulum. Phytophthora capsici culture filtrate and polyethylene glycol treatment induced relocalization to the nucleus of a stabilized LsNAC069 protein, lacking the NAC-domain (LsNAC069 ). Relocalization was significantly reduced in the presence of the Ser/Cys-protease inhibitor TPCK indicating proteolytic cleavage of LsNAC069 allows for relocalization. Co-expression of effectors with LsNAC069 reduced its nuclear accumulation. Surprisingly, LsNAC069 silenced lettuce lines had decreased LsNAC069 transcript levels but did not show significantly altered susceptibility to B. lactucae. In contrast, LsNAC069 silencing increased resistance to Pseudomonas cichorii bacteria and reduced wilting effects under moderate drought stress, indicating a broad role of LsNAC069 in abiotic and biotic stress responses.
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http://dx.doi.org/10.1111/tpj.14383DOI Listing
September 2019

Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae.

Proc Natl Acad Sci U S A 2013 Nov 21;110(45):E4223-31. Epub 2013 Oct 21.

Department of Biotechnology, Delft University of Technology, 2628 BC, Delft, The Netherlands.

Laboratory evolution of the yeast Saccharomyces cerevisiae in bioreactor batch cultures yielded variants that grow as multicellular, fast-sedimenting clusters. Knowledge of the molecular basis of this phenomenon may contribute to the understanding of natural evolution of multicellularity and to manipulating cell sedimentation in laboratory and industrial applications of S. cerevisiae. Multicellular, fast-sedimenting lineages obtained from a haploid S. cerevisiae strain in two independent evolution experiments were analyzed by whole genome resequencing. The two evolved cell lines showed different frameshift mutations in a stretch of eight adenosines in ACE2, which encodes a transcriptional regulator involved in cell cycle control and mother-daughter cell separation. Introduction of the two ace2 mutant alleles into the haploid parental strain led to slow-sedimenting cell clusters that consisted of just a few cells, thus representing only a partial reconstruction of the evolved phenotype. In addition to single-nucleotide mutations, a whole-genome duplication event had occurred in both evolved multicellular strains. Construction of a diploid reference strain with two mutant ace2 alleles led to complete reconstruction of the multicellular-fast sedimenting phenotype. This study shows that whole-genome duplication and a frameshift mutation in ACE2 are sufficient to generate a fast-sedimenting, multicellular phenotype in S. cerevisiae. The nature of the ace2 mutations and their occurrence in two independent evolution experiments encompassing fewer than 500 generations of selective growth suggest that switching between unicellular and multicellular phenotypes may be relevant for competitiveness of S. cerevisiae in natural environments.
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http://dx.doi.org/10.1073/pnas.1305949110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831460PMC
November 2013

Evolutionary engineering of a glycerol-3-phosphate dehydrogenase-negative, acetate-reducing Saccharomyces cerevisiae strain enables anaerobic growth at high glucose concentrations.

Microb Biotechnol 2014 Jan 4;7(1):44-53. Epub 2013 Sep 4.

Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC, The Netherlands; Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, The Netherlands.

Glycerol production by Saccharomyces cerevisiae, which is required for redox-cofactor balancing in anaerobic cultures, causes yield reduction in industrial bioethanol production. Recently, glycerol formation in anaerobic S. cerevisiae cultures was eliminated by expressing Escherichia coli (acetylating) acetaldehyde dehydrogenase (encoded by mhpF) and simultaneously deleting the GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase, thus coupling NADH reoxidation to reduction of acetate to ethanol. Gpd⁻ strains are, however, sensitive to high sugar concentrations, which complicates industrial implementation of this metabolic engineering concept. In this study, laboratory evolution was used to improve osmotolerance of a Gpd⁻ mhpF-expressing S. cerevisiae strain. Serial batch cultivation at increasing osmotic pressure enabled isolation of an evolved strain that grew anaerobically at 1 M glucose, at a specific growth rate of 0.12 h⁻¹. The evolved strain produced glycerol at low concentrations (0.64 ± 0.33 g l⁻¹). However, these glycerol concentrations were below 10% of those observed with a Gpd⁺ reference strain. Consequently, the ethanol yield on sugar increased from 79% of the theoretical maximum in the reference strain to 92% for the evolved strains. Genetic analysis indicated that osmotolerance under aerobic conditions required a single dominant chromosomal mutation, and one further mutation in the plasmid-borne mhpF gene for anaerobic growth.
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http://dx.doi.org/10.1111/1751-7915.12080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896938PMC
January 2014

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae.

Microb Cell Fact 2012 Sep 15;11:131. Epub 2012 Sep 15.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Background: Pyruvate-decarboxylase negative (Pdc⁻) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc⁻S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc⁻ strains cannot grow on high glucose concentrations and require C₂-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry.

Results: Genetic analysis of a Pdc⁻ strain previously evolved to overcome these deficiencies revealed a 225 p in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc⁻ strain enabled growth on 20 g l⁻¹ glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h⁻¹) similar to that of the evolved Pdc⁻ strain (0.23 h⁻¹). Furthermore, the reverse engineered Pdc⁻ strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h⁻¹) than the evolved strain (0.20 h⁻¹). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc⁻S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc⁻ strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C₂-compound auxotrophy.

Conclusions: In this study we have discovered and characterised a mutation in MTH1 enabling Pdc⁻ strains to grow on glucose as the sole carbon source. This successful example of reverse engineering not only increases the understanding of the glucose tolerance of evolved Pdc⁻ S. cerevisiae, but also allows introduction of this portable genetic element into various industrial yeast strains, thereby simplifying metabolic engineering strategies.
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http://dx.doi.org/10.1186/1475-2859-11-131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503853PMC
September 2012

Laboratory evolution of new lactate transporter genes in a jen1Δ mutant of Saccharomyces cerevisiae and their identification as ADY2 alleles by whole-genome resequencing and transcriptome analysis.

FEMS Yeast Res 2012 05 18;12(3):359-374. Epub 2012 Jan 18.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands; Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.

Laboratory evolution is a powerful approach in applied and fundamental yeast research, but complete elucidation of the molecular basis of evolved phenotypes remains a challenge. In this study, DNA microarray-based transcriptome analysis and whole-genome resequencing were used to investigate evolution of novel lactate transporters in Saccharomyces cerevisiae that can replace Jen1p, the only documented S. cerevisiae lactate transporter. To this end, a jen1Δ mutant was evolved for growth on lactate in serial batch cultures. Two independent evolution experiments yielded growth on lactate as sole carbon source (0.14 and 0.18 h(-1) , respectively). Transcriptome analysis did not provide leads, but whole-genome resequencing showed different single-nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Introduction of these ADY2 alleles in a jen1Δ ady2Δ strain enabled growth on lactate (0.14 h(-1) for Ady2p(Leu219Val) and 0.12 h(-1) for Ady2p(Ala252Gly) ), demonstrating that these alleles of ADY2 encode efficient lactate transporters. Depth of coverage of DNA sequencing, combined with karyotyping, gene deletions and diagnostic PCR, showed that an isochromosome III (c. 475 kb) with two additional copies of ADY2(C755G) had been formed via crossover between retrotransposons YCLWΔ15 and YCRCΔ6. The isochromosome formation shows how even short periods of selective pressure can cause substantial karyotype changes.
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http://dx.doi.org/10.1111/j.1567-1364.2012.00787.xDOI Listing
May 2012

Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast.

FEMS Yeast Res 2012 Mar 10;12(2):183-96. Epub 2012 Jan 10.

Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.

Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages.
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http://dx.doi.org/10.1111/j.1567-1364.2011.00776.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615171PMC
March 2012

Metabolome, transcriptome and metabolic flux analysis of arabinose fermentation by engineered Saccharomyces cerevisiae.

Metab Eng 2010 Nov 8;12(6):537-51. Epub 2010 Sep 8.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

One of the challenges in strain improvement by evolutionary engineering is to subsequently determine the molecular basis of the improved properties that were enriched from the natural genetic variation during the selective conditions. This study focuses on Saccharomyces cerevisiae IMS0002 which, after metabolic and evolutionary engineering, ferments the pentose sugar arabinose. Glucose- and arabinose-limited anaerobic chemostat cultures of IMS0002 and its non-evolved ancestor were subjected to transcriptome analysis, intracellular metabolite measurements and metabolic flux analysis. Increased expression of the GAL-regulon and deletion of GAL2 in IMS0002 confirmed that the galactose transporter is essential for growth on arabinose. Elevated intracellular concentrations of pentose-phosphate-pathway intermediates and upregulation of TKL2 and YGR043c (encoding transketolase and transaldolase isoenzymes) suggested an involvement of these genes in flux-controlling reactions in arabinose fermentation. Indeed, deletion of these genes in IMS0002 caused a 21% reduction of the maximum specific growth rate on arabinose.
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http://dx.doi.org/10.1016/j.ymben.2010.08.003DOI Listing
November 2010