Publications by authors named "John van der Oost"

239 Publications

Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas.

Nucleic Acids Res 2021 Oct 6. Epub 2021 Oct 6.

Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.
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http://dx.doi.org/10.1093/nar/gkab893DOI Listing
October 2021

SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation.

Nat Commun 2021 08 19;12(1):5033. Epub 2021 Aug 19.

Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands.

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.
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http://dx.doi.org/10.1038/s41467-021-25337-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376896PMC
August 2021

Towards a synthetic cell cycle.

Nat Commun 2021 07 26;12(1):4531. Epub 2021 Jul 26.

Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.

Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.
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http://dx.doi.org/10.1038/s41467-021-24772-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313558PMC
July 2021

(R)evolution-on-a-chip.

Trends Biotechnol 2021 May 25. Epub 2021 May 25.

Laboratory of Microbiology, Wageningen University and Research, 6708, WE, Wageningen, The Netherlands. Electronic address:

Billions of years of Darwinian evolution has led to the emergence of highly sophisticated and diverse life forms on Earth. Inspired by natural evolution, similar principles have been adopted in laboratory evolution for the fast optimization of genes and proteins for specific applications. In this review, we highlight state-of-the-art laboratory evolution strategies for protein engineering, with a special emphasis on in vitro strategies. We further describe how recent progress in microfluidic technology has allowed the generation and manipulation of artificial compartments for high-throughput laboratory evolution experiments. Expectations for the future are high: we foresee a revolution on-a-chip.
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http://dx.doi.org/10.1016/j.tibtech.2021.04.009DOI Listing
May 2021

High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica.

Metab Eng 2021 07 7;66:239-258. Epub 2021 May 7.

Wageningen University, Bioprocess Engineering, PO Box 16, 6700 AA, Wageningen, Netherlands.

The microalga Nannochloropsis oceanica is considered a promising platform for the sustainable production of high-value lipids and biofuel feedstocks. However, current lipid yields of N. oceanica are too low for economic feasibility. Gaining fundamental insights into the lipid metabolism of N. oceanica could open up various possibilities for the optimization of this species through genetic engineering. Therefore, the aim of this study was to discover novel genes associated with an elevated neutral lipid content. We constructed an insertional mutagenesis library of N. oceanica, selected high lipid mutants by five rounds of fluorescence-activated cell sorting, and identified disrupted genes using a novel implementation of a rapid genotyping procedure. One particularly promising mutant (HLM23) was disrupted in a putative APETALA2-like transcription factor gene. HLM23 showed a 40%-increased neutral lipid content, increased photosynthetic performance, and no growth impairment. Furthermore, transcriptome analysis revealed an upregulation of genes related to plastidial fatty acid biosynthesis, glycolysis and the Calvin-Benson-Bassham cycle in HLM23. Insights gained in this work can be used in future genetic engineering strategies for increased lipid productivity of Nannochloropsis.
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http://dx.doi.org/10.1016/j.ymben.2021.04.012DOI Listing
July 2021

Gut bacteriophage dynamics during fecal microbial transplantation in subjects with metabolic syndrome.

Gut Microbes 2021 Jan-Dec;13(1):1-15

Department of Plant Sciences & Plant Pathology, Montana State University, Bozeman, MT, USA.

Metabolic Syndrome (MetS) is a growing public health concern worldwide. Individuals with MetS have an increased risk for cardiovascular (CV) disease and type 2 diabetes (T2D). These diseases - in part preventable with the treatment of MetS - increase the chances of premature death and pose a great economic burden to health systems. A healthy gut microbiota is associated with a reduction in MetS, T2D, and CV disease. Treatment of MetS with fecal microbiota transplantation (FMT) can be effective, however, its success rate is intermediate and difficult to predict. Because bacteriophages significantly affect the microbiota membership and function, the aim of this pilot study was to explore the dynamics of the gut bacteriophage community after FMT in MetS subjects. We performed a longitudinal study of stool bacteriophages from healthy donors and MetS subjects before and after FMT treatment. Subjects were assigned to either a control group (self-stool transplant, n = 3) or a treatment group (healthy-donor-stool transplant; n-recipients = 6, n-donors = 5). Stool samples were collected over an 18-week period and bacteriophage-like particles were purified and sequenced. We found that FMT from healthy donors significantly alters the gut bacteriophage community. Subjects with better clinical outcome clustered closer to the heathy donor group, suggesting that throughout the treatment, their bacteriophage community was more similar to healthy donors. Finally, we identified bacteriophage groups that could explain these differences and we examined their prevalence in individuals from a larger cohort of MetS FMT trial.Trial information- http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2705; NTR 2705.
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http://dx.doi.org/10.1080/19490976.2021.1897217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023239PMC
April 2021

Synthetic Biology Approaches To Enhance Microalgal Productivity.

Trends Biotechnol 2021 10 1;39(10):1019-1036. Epub 2021 Feb 1.

Bioprocess Engineering, AlgaePARC, Wageningen University, Wageningen, The Netherlands. Electronic address:

The major bottleneck in commercializing biofuels and other commodities produced by microalgae is the high cost associated with phototrophic cultivation. Improving microalgal productivities could be a solution to this problem. Synthetic biology methods have recently been used to engineer the downstream production pathways in several microalgal strains. However, engineering upstream photosynthetic and carbon fixation metabolism to enhance growth, productivity, and yield has barely been explored in microalgae. We describe strategies to improve the generation of reducing power from light, as well as to improve the assimilation of CO by either the native Calvin cycle or synthetic alternatives. Overall, we are optimistic that recent technological advances will prompt long-awaited breakthroughs in microalgal research.
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http://dx.doi.org/10.1016/j.tibtech.2020.12.010DOI Listing
October 2021

Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles.

CRISPR J 2021 02 4;4(1):82-91. Epub 2021 Feb 4.

Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands.

The ability of CRISPR-Cas12a nucleases to function reliably in a wide range of species has been key to their rapid adoption as genome engineering tools. However, so far, Cas12a nucleases have been limited for use in organisms with growth temperatures up to 37 °C. Here, we biochemically characterize three Cas12a orthologs for their temperature stability and activity. We demonstrate that Cas12a (FnCas12a) has great biochemical potential for applications that require enhanced stability, including use at temperatures >37°C. Furthermore, by employing the moderate thermophilic bacterium as our experimental platform, we demonstrate that FnCas12a is active at temperatures up to 43°C. Subsequently, we develop a single-plasmid FnCas12a-based genome editing tool for , combining the FnCas12a targeting system with plasmid-borne homologous recombination (HR) templates that carry the desired modifications. Culturing of cells at 45°C allows for the uninhibited realization of the HR-based editing step, while a subsequent culturing step at reduced temperatures induces the efficient counterselection of the non-edited cells by FnCas12a. The developed gene-editing tool yields gene-knockout mutants within 3 days, and does not require tightly controllable expression of FnCas12a to achieve high editing efficiencies, indicating its potential for other (thermophilic) bacteria and archaea, including those with minimal genetic toolboxes. Altogether, our findings provide new biochemical insights into three widely used Cas12a nucleases, and establish the first Cas12a-based bacterial genome editing tools for moderate thermophilic microorganisms.
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http://dx.doi.org/10.1089/crispr.2020.0086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898400PMC
February 2021

Eat1-Like Alcohol Acyl Transferases From Yeasts Have High Alcoholysis and Thiolysis Activity.

Front Microbiol 2020 29;11:579844. Epub 2020 Oct 29.

Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University and Research, Wageningen, Netherlands.

Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment , accepting a wide range of alcohols (C2-C10) but only a small range of acyl donors (C2-C4). We show that alcoholysis occurs in several Crabtree negative yeast species but also in engineered strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters by overexpressing Eat1 in . Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.
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http://dx.doi.org/10.3389/fmicb.2020.579844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658179PMC
October 2020

(Hyper)Thermophilic Enzymes: Production and Purification.

Methods Mol Biol 2021 ;2178:469-478

Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where "normal" proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.
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http://dx.doi.org/10.1007/978-1-0716-0775-6_29DOI Listing
March 2021

Transcriptomic and Phenotypic Analysis of a Mutant in .

Front Microbiol 2020 15;11:556064. Epub 2020 Sep 15.

Wageningen Food and Biobased Research, Wageningen, Netherlands.

SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ , during sporulation. A Δ mutant of NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the Δ mutant are elongated with the presence of multiple septa. This observation suggests that in , SpoIIE is necessary for the completion of the sporulation process, as seen in and . Moreover, when grown in reactors, the mutant produced higher levels of solvents than the wild type strain. The impact of the inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ , σ , σ , σ ) were strongly down-regulated in the mutant. Inactivation of also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in .
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http://dx.doi.org/10.3389/fmicb.2020.556064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522474PMC
September 2020

The Ongoing Quest to Crack the Genetic Code for Protein Production.

Mol Cell 2020 10 2;80(2):193-209. Epub 2020 Oct 2.

Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, the Netherlands. Electronic address:

Understanding the genetic design principles that determine protein production remains a major challenge. Although the key principles of gene expression were discovered 50 years ago, additional factors are still being uncovered. Both protein-coding and non-coding sequences harbor elements that collectively influence the efficiency of protein production by modulating transcription, mRNA decay, and translation. The influences of many contributing elements are intertwined, which complicates a full understanding of the individual factors. In natural genes, a functional balance between these factors has been obtained in the course of evolution, whereas for genetic-engineering projects, our incomplete understanding still limits optimal design of synthetic genes. However, notable advances have recently been made, supported by high-throughput analysis of synthetic gene libraries as well as by state-of-the-art biomolecular techniques. We discuss here how these advances further strengthen understanding of the gene expression process and how they can be harnessed to optimize protein production.
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http://dx.doi.org/10.1016/j.molcel.2020.09.014DOI Listing
October 2020

Growth-uncoupled isoprenoid synthesis in .

Biotechnol Biofuels 2020 13;13:123. Epub 2020 Jul 13.

Bioprocess Engineering, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Background: Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis.

Results: This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways.

Conclusions: Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.
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http://dx.doi.org/10.1186/s13068-020-01765-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359475PMC
July 2020

Guide-free Cas9 from pathogenic bacteria causes severe damage to DNA.

Sci Adv 2020 Jun 17;6(25):eaaz4849. Epub 2020 Jun 17.

Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.
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http://dx.doi.org/10.1126/sciadv.aaz4849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299616PMC
June 2020

CRISPR with a Happy Ending: Non-Templated DNA Repair for Prokaryotic Genome Engineering.

Biotechnol J 2020 Jul 29;15(7):e1900404. Epub 2020 Jun 29.

Laboratory of Microbiology, Wageningen University and Research, Wageningen, 6708 WE, The Netherlands.

The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR-Cas and other nucleases mediate double-strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template-independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non-homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.
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http://dx.doi.org/10.1002/biot.201900404DOI Listing
July 2020

From Eat to trEat: engineering the mitochondrial Eat1 enzyme for enhanced ethyl acetate production in .

Biotechnol Biofuels 2020 19;13:76. Epub 2020 Apr 19.

2Bioprocess Engineering, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Background: Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in . Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as , expression of heterologous proteins with eukaryotic signal sequences may not be optimal.

Results: Unprocessed and synthetically truncated eat1 variants of and have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in

Conclusion: By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in . Truncated variants of could therefore benefit future engineering approaches towards efficient ethyl acetate production.
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http://dx.doi.org/10.1186/s13068-020-01711-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168974PMC
April 2020

Multilevel optimisation of anaerobic ethyl acetate production in engineered .

Biotechnol Biofuels 2020 7;13:65. Epub 2020 Apr 7.

1Bioprocess Engineering, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Background: Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable.

Results: We engineered an strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Eat1 from and Eat1 from were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/ and XylS/ promoters allowed optimisation of their expression levels.

Conclusion: Engineering efforts on protein and fermentation level resulted in an strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.
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http://dx.doi.org/10.1186/s13068-020-01703-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137189PMC
April 2020

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT.

Nano Lett 2020 04 20;20(4):2264-2270. Epub 2020 Mar 20.

Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Super-resolution imaging allows for the visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA point accumulation in nanoscale topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), which allows for faster binding. Ago preorders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude, while maintaining the high spatial resolution. We envision this tool to be useful for super-resolution imaging and other techniques that rely on nucleic acid interactions.
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http://dx.doi.org/10.1021/acs.nanolett.9b04277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146856PMC
April 2020

First structural insights into CRISPR-Cas-guided DNA transposition.

Cell Res 2020 03;30(3):193-194

Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE, Wageningen, the Netherlands.

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http://dx.doi.org/10.1038/s41422-020-0284-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054401PMC
March 2020

Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a.

Nucleic Acids Res 2020 04;48(6):3228-3243

Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands.

Genome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.
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http://dx.doi.org/10.1093/nar/gkz1240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102956PMC
April 2020

Editor's cut: DNA cleavage by CRISPR RNA-guided nucleases Cas9 and Cas12a.

Biochem Soc Trans 2020 02;48(1):207-219

Laboratory of Microbiology, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, Netherlands.

Discovered as an adaptive immune system of prokaryotes, CRISPR-Cas provides many promising applications. DNA-cleaving Cas enzymes like Cas9 and Cas12a, are of great interest for genome editing. The specificity of these DNA nucleases is determined by RNA guides, providing great targeting adaptability. Besides this general method of programmable DNA cleavage, these nucleases have different biochemical characteristics, that can be exploited for different applications. Although Cas nucleases are highly promising, some room for improvement remains. New developments and discoveries like base editing, prime editing, and CRISPR-associated transposons might address some of these challenges.
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http://dx.doi.org/10.1042/BST20190563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054755PMC
February 2020

Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants.

Nat Rev Microbiol 2020 02 19;18(2):67-83. Epub 2019 Dec 19.

National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, USA.

The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.
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http://dx.doi.org/10.1038/s41579-019-0299-xDOI Listing
February 2020

Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics.

Nucleic Acids Res 2020 02;48(4):e19

Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia PA, USA.

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.
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http://dx.doi.org/10.1093/nar/gkz1165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038991PMC
February 2020

Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering.

Microb Cell Fact 2019 Nov 25;18(1):204. Epub 2019 Nov 25.

Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.

Background: Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides.

Results: Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected.

Conclusions: In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.
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http://dx.doi.org/10.1186/s12934-019-1255-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876111PMC
November 2019

Harnessing type I CRISPR-Cas systems for genome engineering in human cells.

Nat Biotechnol 2019 12 18;37(12):1471-1477. Epub 2019 Nov 18.

Caribou Biosciences, Inc., Berkeley, CA, USA.

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea. Target interference relies on a multi-subunit, RNA-guided complex called Cascade, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.
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http://dx.doi.org/10.1038/s41587-019-0310-0DOI Listing
December 2019

Medium-throughput in vitro detection of DNA cleavage by CRISPR-Cas12a.

Methods 2020 02 11;172:27-31. Epub 2019 Nov 11.

Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands. Electronic address:

Quantifying DNA cleavage by CRISPR-Cas nucleases is usually done by separating the cleaved products from the non-cleaved target by agarose gel electrophoresis. We devised a method that eliminates the quantification from band intensity on agarose gel, and uses a target with a fluorescent dye on the one end and a biotin on the other. Cleavage of the target will separate the dye from the biotin, and cause the dye to stay in solution when streptavidin beads are introduced. All non-cleaved target will be eliminated from solution and no longer contribute to detectable fluorescence. Cleavage will therefore increase the fluorescent signal. A control, which has no streptavidin treatment, is taken along to correct for any errors that might have been introduced by pipetting, inactivation of the fluorescent dye or release of the biotin during several steps of the procedure. With this method we were able to quantify the fraction of active Cas12a in a purification sample and assess the cleavage rate.
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http://dx.doi.org/10.1016/j.ymeth.2019.11.005DOI Listing
February 2020

Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome.

Science 2019 11;366(6465):606-612

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, Netherlands.

Microorganisms living inside plants can promote plant growth and health, but their genomic and functional diversity remain largely elusive. Here, metagenomics and network inference show that fungal infection of plant roots enriched for Chitinophagaceae and Flavobacteriaceae in the root endosphere and for chitinase genes and various unknown biosynthetic gene clusters encoding the production of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). After strain-level genome reconstruction, a consortium of and was designed that consistently suppressed fungal root disease. Site-directed mutagenesis then revealed that a previously unidentified NRPS-PKS gene cluster from was essential for disease suppression by the endophytic consortium. Our results highlight that endophytic root microbiomes harbor a wealth of as yet unknown functional traits that, in concert, can protect the plant inside out.
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http://dx.doi.org/10.1126/science.aaw9285DOI Listing
November 2019

Argonaute bypasses cellular obstacles without hindrance during target search.

Nat Commun 2019 09 26;10(1):4390. Epub 2019 Sep 26.

Kavli Institute of Nanoscience and Department of Bionanoscience, Delft University of Technology, Delft, The Netherlands.

Argonaute (Ago) proteins are key players in both gene regulation (eukaryotes) and host defense (prokaryotes). Acting on single-stranded nucleic-acid substrates, Ago relies on base pairing between a small nucleic-acid guide and its complementary target sequences for specificity. To efficiently scan nucleic-acid chains for targets, Ago diffuses laterally along the substrate and must bypass secondary structures as well as protein barriers. Using single-molecule FRET in conjunction with kinetic modelling, we reveal that target scanning is mediated through loose protein-nucleic acid interactions, allowing Ago to slide short distances over secondary structures, as well as to bypass protein barriers via intersegmental transfer. Our combined single-molecule experiment and kinetic modelling approach may serve as a platform to dissect search processes and study the effect of sequence on search kinetics for other nucleic acid-guided proteins.
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http://dx.doi.org/10.1038/s41467-019-12415-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763497PMC
September 2019

Nature of Amorphous Hydrophilic Block Affects Self-Assembly of an Artificial Viral Coat Polypeptide.

Biomacromolecules 2019 10 29;20(10):3641-3647. Epub 2019 Aug 29.

Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences (UIPS), Faculty of Science , Utrecht University , Universiteitsweg 99 , 3584 CG Utrecht , The Netherlands.

Consensus motifs for sequences of both crystallizable and amorphous blocks in silks and natural structural analogues of silks vary widely. To design novel silklike polypeptides, an important question is therefore how the nature of either the crystallizable or the amorphous block affects the self-assembly and resulting physical properties of silklike polypeptides. We address herein the influence of the amorphous block on the self-assembly of a silklike polypeptide that was previously designed to encapsulate single DNA molecules into rod-shaped viruslike particles. The polypeptide has a triblock architecture, with a long N-terminal amorphous block, a crystallizable midblock, and a C-terminal DNA-binding block. We compare the self-assembly behavior of a triblock with a very hydrophilic collagen-like amorphous block (GXaaYaa) to that of a triblock with a less hydrophilic elastin-like amorphous block (GSGVP). The amorphous blocks have similar lengths and both adopt a random coil structure in solution. Nevertheless, atomic force microscopy revealed significant differences in the self-assembly behavior of the triblocks. If collagen-like amorphous blocks are used, there is a clear distinction between very short polypeptide-only fibrils and much longer fibrils with encapsulated DNA. If elastin-like amorphous blocks are used, DNA is still encapsulated, but the polypeptide-only fibrils are now much longer and their size distribution partially overlaps with that of the encapsulated DNA fibrils. We attribute the difference to the more hydrophilic nature of the collagen-like amorphous block, which more strongly opposes the growth of polypeptide-only fibrils than the elastin-like amorphous blocks. Our work illustrates that differences in the chemical nature of amorphous blocks can strongly influence the self-assembly and hence the functionality of engineered silklike polypeptides.
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http://dx.doi.org/10.1021/acs.biomac.9b00512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794640PMC
October 2019
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