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Assessing cofactor usage in Pseudoclostridium thermosuccinogenes via heterologous expression of central metabolic enzymes
Koendjbiharie, Jeroen Girwar ; Wevers, Kimberly ; Kranenburg, Richard van - \ 2019
Frontiers in Microbiology 10 (2019)May. - ISSN 1664-302X
6-phosphofructokinase - Cofactor specificity - Energy charge - Glyceraldehyde 3-phosphate dehydrogenase - GTP - Pseudoclostridium thermosuccinogenes - Pyrophosphate
Pseudoclostridium thermosuccinogenes and Hungateiclostridium thermocellum are being studied for their potential to contribute to a more sustainable bio-based economy. Both species were shown previously to rely on GTP or pyrophosphate instead of ATP as cofactors in specific reactions of central energy metabolism for reasons that are not well understood yet. Since it is often impossible to predict cofactor specificity from the primary protein structure, thirteen enzymes from P. thermosuccinogenes were cloned and heterologous expressed in Escherichia coli to assess the cofactor usage in vitro and paint a more complete picture of the cofactor usage in the central metabolism of P. thermosuccinogenes. The assays were conducted with heat-treated E. coli cell-free extract devoid of background activity to allow the quick assessment of a relatively large number of (thermophilic) enzymes. Selected enzymes were also purified to allow the determination of the enzyme kinetics for competing cofactors. Following the results of the glucokinase (GK), galactokinase, xylulokinase (XK), and ribokinase assays, it seems that phosphorylation of monosaccharides by and large is mainly GTP-dependent. Some possible implications of this relating to the adenylate/guanylate energy charge are discussed here. Besides the highly expressed pyrophosphate-dependent 6-phosphofructokinase, another 6-phosphofructokinase was found to be equally dependent on ATP and GTP, while no 6-phosphofructokinase activity could be demonstrated for a third. Both type I glyceraldehyde 3-phosphate dehydrogenases were found to be NAD+-dependent, and further, acetate kinase, isocitrate dehydrogenase, and three enzymes predicted to be responsible for the interconversion of phosphoenolpyruvate and pyruvate (i.e., pyruvate kinase; pyruvate, phosphate dikinase; phosphoenolpyruvate synthase), were also assessed.
Thermostable Cas9 nucleases with reduced off-target activity
Oost, J. van der; Kranenburg, R. van; Bosma, E.F. ; Mougiakos, I. ; Mohanraju, P. - \ 2019
Octrooinummer: WO2019072596, verleend: 2019-04-18.
ThermoCas9 is identified and characterized from the thermophilic bacterium Geobacillus thermodenitriﬁcans T12. Experiments show how in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. Described are ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 5 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida
Thermostable Cas9 nucleases
Oost, J. van der; Kranenburg, R. van; Bosma, E.F. ; Mougiakos, I. ; Mohanraju, P. - \ 2018
Octrooinummer: WO2018108339, verleend: 2018-06-21.
The present invention relates to the field of genetic engineering and more particularly to nucleic acid editing and genome modification. The present invention provides an isolated Cas protein or polypeptide fragment thereof having an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 77% identity therewith. The Cas protein or polypeptide is capable of binding, cleaving, marking or modifying a double stranded target polynucleotide at a temperature in the range 20°C and 100°C inclusive. The invention further provides isolated nucleic acid molecules encoding said Cas9 nucleases, expression vectors and host cells. The invention also provides PAM sequences recognized by the Cas protein or polypeptide, The Cas9 nucleases disclosed herein provide novel tools for genetic engineering in general, in particular at elevated temperatures.
Thermostable Cas9 nucleases
Oost, J. van der; Kranenburg, R. van; Bosma, E.F. ; Mougiakos, I. ; Mohanraju, P. - \ 2018
Octrooinummer: WO2018109101, verleend: 2018-06-21.
A polynucleotide encoding a ThermoCas9protein from Geobacillus thermodenitrificans and a constitutive promoter are used to engineer eukaryotic cells, e.g. fungi, yeast or algae, so that the ThermoCas9 endonuclease is integrated and expressed from the genome of the cell. Then, a second expression plasmid is used to transfect these ThermoCas9 expressing cells, the second plasmid containing an inducible promoter and a polynucleotide encoding a guide RNA. The guide RNA combines with the ThermoCas9 to provide the targeted endonuclease activity to cleave the cell DNA at a desired locus or gene of interest. A repair-oligo is also provided to the cell whereby following DNA cleavage, homologous recombination takes place in the cell with the repair-oligo so that either a deletion or substitution of nucleotides in the locus or gene of interest is achieved. Expression vectors and methods of using the vectors to achieve ThermoCas9 mediated gene editing are described whereby higher temperatures, e.g. greater than 30 °C, are used.
Microbial genome editing
Oost, J. van der; Kranenburg, R. van; Bosma, E.F. ; Mougiakos, I. - \ 2018
Octrooinummer: WO2018083128, verleend: 2018-05-11.
In an absence of efficient non-homologous end joining (NHEJ) repair mechanisms in the majority of microbes, double stranded DNA break (DSDB) typically leads to cell death. In methods of microbial gene editing using plasmid transformation, both homologous recombination and Cas9 site-specific gene editing events can be used together. Single or multiple plasmid approaches are used. In a method of counter-selection of microbes for a desired genetic change, a two-phase approach is used whereby a switch is made from a higher growth temperature phase favouring homologous recombination (HR) - as opposed to a Cas9 site-directed nuclease activity- to a lower growth temperature phase at which the Cas9 site directed nuclease activity takes place. This has the effect whereby the Cas9 site-directed nuclease activity has counter selecting activity, removing microbes which do not have a desired modification introduced beforehand by HR. The population of microbes surviving after the temperature switch counter selection is thereby enhanced for the desired modification.
Investigating the central metabolism of Clostridium thermosuccinogenes
Koendjbiharie, Jeroen Girwar ; Wiersma, Kilian ; Kranenburg, Richard van - \ 2018
Applied and Environmental Microbiology 84 (2018)13. - ISSN 0099-2240
Clostridium thermosuccinogenes - Malate dehydrogenase - Succinate - Xylulokinase
Clostridium thermosuccinogenes is a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced, and the genome of the type strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine cofactor specificity. The genes were identified for the pathways to all fermentation products, as well as for the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking, and transcriptomics during growth on glucose versus that on xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C5 with the C3/C6 metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression in Escherichia coli and subsequent assays with the cell extract, which has proven to be a simple and powerful method for the basal characterization of thermophilic enzymes.
Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes
Daas, Martinus J.A. ; Nijsse, Bart ; Weijer, Antonius H.P. van de; Groenendaal, Bart W.A.J. ; Janssen, Fons ; Oost, John van der; Kranenburg, Richard van - \ 2018
BMC Biotechnology 18 (2018)1. - ISSN 1472-6750
CBP - Cellulase - Geobacillus - Metagenome - β-Xylosidase
Background: Consolidated bioprocessing (CBP) is a cost-effective approach for the conversion of lignocellulosic biomass to biofuels and biochemicals. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to harbor desired features for CBP, although it lacks the desired endo and exoglucanases required for the conversion of cellulose. Here, we report the expression of both endoglucanase and exoglucanase encoding genes by G. thermodenitrificans T12, in an initial attempt to express cellulolytic enzymes that complement the enzymatic machinery of this strain. Results: A metagenome screen was performed on 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 (GH5) were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-β-glucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having pronounced activity towards p-nitrophenyl-β-d-xylopyranoside. Structure modelling of GE40 revealed its protein architecture to be similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic activity against 2-hydroxyethylcellulose, carboxymethylcellulose and barley β-glucan. Additionally, we introduced expression constructs into T12 containing Geobacillus sp. 70PC53 endoglucanase gene celA and both endoglucanase genes (M1 and M2) from Geobacillus sp. WSUCF1. Finally, we introduced expression constructs into T12 containing the C. thermocellum exoglucanases celK and celS genes and the endoglucanase celC gene. Conclusions: We identified a novel G. thermodenitrificans β-xylosidase (GE39) and a novel endoglucanase (GE40) using a metagenome screen based on multiple HMM profiles. We successfully expressed both genes in E. coli and functionally expressed the GE40 endoglucanase in G. thermodenitrificans T12. Additionally, the heterologous production of active CelK, a C. thermocellum derived exoglucanase, and CelA, a Geobacillus derived endoglucanase, was demonstrated with strain T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of G. thermodenitrificans T12 as a host for consolidated bioprocessing.
In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria
Frenzel, Elrike ; Legebeke, Jelmer ; Stralen, Atze Van; Kranenburg, Richard Van; Kuipers, Oscar P. - \ 2018
Biotechnology for Biofuels 11 (2018)1. - ISSN 1754-6834
Biotechnology - Cyan - FACS - Geobacillus sp. - GFP - In vivo application - Parageobacillus sp. - Protein engineering - sfGFP - Thermophilic bacteria - Thermostability - Yellow
Background: Fluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius, an in vivo screening approach based on a mutational library of superfolder GFP was applied. Results: Flow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 °C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 °C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 °C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability. Conclusions: This study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest.
Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering : advances and prospects
Mougiakos, Ioannis ; Bosma, Elleke F. ; Ganguly, Joyshree ; Oost, John van der; Kranenburg, Richard van - \ 2018
Current Opinion in Biotechnology 50 (2018). - ISSN 0958-1669 - p. 146 - 157.
High engineering efficiencies are required for industrial strain development. Due to its user-friendliness and its stringency, CRISPR-Cas-based technologies have strongly increased genome engineering efficiencies in bacteria. This has enabled more rapid metabolic engineering of both the model host Escherichia coli and non-model organisms like Clostridia, Bacilli, Streptomycetes and cyanobacteria, opening new possibilities to use these organisms as improved cell factories. The discovery of novel Cas9-like systems from diverse microbial environments will extend the repertoire of applications and broaden the range of organisms in which it can be used to create novel production hosts. This review analyses the current status of prokaryotic metabolic engineering towards the production of biotechnologically relevant products, based on the exploitation of different CRISPR-related DNA/RNA endonuclease variants.
Complete Genome Sequence of Geobacillus thermodenitrificans T12, A Potential Host for Biotechnological Applications
Daas, Tijn ; Vriesendorp, Bastienne ; Weijer, Tom van de; Oost, John van der; Kranenburg, Richard van - \ 2018
Current Microbiology 75 (2018)1. - ISSN 0343-8651 - p. 49 - 56.
In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes are similar to those of the denitrification pathways found in strain NG80-2. The hemicellulose utilization (HUS) locus was identified based on sequence homology against G. stearothermophilus T-6. It appeared that T12 has all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. Strain T12 has complete pathways for the synthesis of purine and pyrimidine, all 20 amino acids and several vitamins except D-biotin. The host-defense systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system. It is concluded that G. thermodenitrificans T12 is a potentially interesting candidate for industrial applications.
Characterizing a thermostable Cas9 for bacterial genome editing and silencing
Mougiakos, Ioannis ; Mohanraju, Prarthana ; Bosma, Elleke F. ; Vrouwe, Valentijn ; Finger Bou, Max ; Naduthodi, Mihris I.S. ; Gussak, Alex ; Brinkman, Rudolf B.L. ; Kranenburg, Richard Van; Oost, John Van Der - \ 2017
Nature Communications 8 (2017)1. - ISSN 2041-1723
CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.
Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
Mougiakos, Ioannis ; Bosma, Elleke F. ; Weenink, Koen ; Vossen, Eric ; Goijvaerts, Kirsten ; Oost, John van der; Kranenburg, Richard van - \ 2017
ACS synthetic biology 6 (2017)5. - ISSN 2161-5063 - p. 849 - 861.
Bacillus smithii - bacteria - CRISPR/Cas9 - genome editing - homologous recombination - thermophiles
Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.
Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12
Daas, Tijn ; Martínez, Patricia Murciano ; Weijer, Tom van de; Oost, John van der; Vos, Willem M. de; Kabel, Mirjam A. ; Kranenburg, Richard van - \ 2017
BMC Biotechnology 17 (2017)1. - ISSN 1472-6750
Biotechnology - Endo-xylanase - Geobacillus - Thermophile - Xylan
Background: Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential. Results: A HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 °C and 80 °C, with an optimum activity at 60 °C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 °C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. Conclusions: The G. thermodenitrificans T12 endo-xylanase, GtXynA1, shows temperature tolerance up to 80 °C and high activity to a variety of xylans. The mode of action of GtXynA1 reveals that arabinose substituents do not hamper substrate degradation by GtXynA1. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates.
Bacterial cell factoriest : applying thermophiles to fuel the biobased economy
Kranenburg, Richard van - \ 2017
Wageningen : Wageningen University & Research - ISBN 9789463431750 - 19
industriële microbiologie - chemie op basis van biologische grondstoffen - biobased economy - bacteriën - genetische modificatie - thermofiele bacteriën - biomassaconversie - industrial microbiology - biobased chemistry - bacteria - genetic engineering - thermophilic bacteria - biomass conversion
The research of Bacterial Cell Factories aims to apply bacteria for production of biobased chemicals from renewable resources. The focus lies on thermophilic Gram-positives. This group of relatively unexplored thermophiles has many relevant characteristics that make them attractive as production organism for green chemicals. Development of genetic tools is a requirement for high-throughput engineering. The scientific challenge lies in exploring and exploiting the microbial physiology of the selected production organisms, involving an integrated approach of various disciplines. Successful development of such Bacterial Cell Factories is crucial for establishing the biobased economy.
Prof. Richard Kranenburg: Bacteriën als fabriekjes van de bio-economie
Kranenburg, R. van - \ 2017
Wageningen University & Research
industriële microbiologie - bacteriën - biobased economy - microbiële afbraak - enzymen - genetische verandering - onderzoek - industrial microbiology - bacteria - microbial degradation - enzymes - genetic change - research
Video over micro-organismen in de biobased economy
Isolation of a genetically accessible thermophilic xylan degrading bacterium from compost
Daas, Tijn ; Weijer, Tom van de; Vos, Willem M. de; Oost, John van der; Kranenburg, Richard van - \ 2016
Biotechnology for Biofuels 9 (2016). - ISSN 1754-6834
CMC - Compost - Electroporation - Fermentation - Geobacillus - Lactic acid - Thermophile - Xylan
Background: Due to the finite nature of global oil resources we are now faced with the challenge of finding renewable resources to produce fuels and chemicals in the future. Lactic acid has great potential as a precursor for the production of bioplastics alternatives to conventional plastics. Efficient lactic acid fermentation from non-food lignocellulosic substrates requires pretreatment and saccharification to generate fermentable sugars. A fermentation process that requires little to no enzyme additions, i.e. consolidated bioprocessing would be preferred and requires lactic acid-producing organisms that have cellulolytic and/or hemicellulolytic activity. Results: To obtain candidate production strains we have enriched and isolated facultative anaerobic (hemi) cellulolytic bacterial strains from compost samples. By selecting for growth on both cellulose and xylan, 94 Geobacillus strains were isolated. Subsequent screening for lactic acid production was carried out from C6 and C5 sugar fermentations and a selection of the best lactic acid producers was made. The denitrifying Geobacillus thermodenitrificans T12 was selected for further research and was rendered genetically accessible. In fermentations on a mixture of glucose and xylose, a total of 20.3 g of lactic acid was produced with a yield of 0.94 g product/g sugar consumed. In addition, strain T12 is capable of direct conversion of beech wood xylan to mainly lactic acid in minimal media. Conclusions: We have demonstrated that G. thermodenitrificans T12 is genetically accessible and produces lactic acid as its main fermentation product on glucose, xylose and a mixture thereof. Strain T12 was additionally used for the direct conversion of xylan to lactic acid. The genetic accessibility of the T12 strain provides a solid basis for the development of this strain into a host for consolidated bioprocessing of biomass to lactic acid.
Complete genome sequence of thermophilic Bacillus smithii type strain DSM 4216T
Bosma, Elleke F. ; Koehorst, Jasper J. ; Hijum, Sacha A.F.T. van; Renckens, Bernadet ; Vriesendorp, Bastienne ; Weijer, Tom van de; Schaap, Peter J. ; Vos, Willem M. de; Oost, John van der; Kranenburg, Richard van - \ 2016
Standards in Genomic Sciences 11 (2016). - ISSN 1944-3277 - 11 p.
Bacillus smithii - Biotechnology - Genome sequence - Lactic acid - Thermophile - Thermophilic bacillus
Bacillus smithii is a facultatively anaerobic, thermophilic bacterium able to use a variety of sugars that can be derived from lignocellulosic feedstocks. Being genetically accessible, it is a potential new host for biotechnological production of green chemicals from renewable resources. We determined the complete genomic sequence of the B. smithii type strain DSM 4216T, which consists of a 3,368,778 bp chromosome (GenBank accession number CP012024.1) and a 12,514 bp plasmid (GenBank accession number CP012025.1), together encoding 3880 genes. Genome annotation via RAST was complemented by a protein domain analysis. Some unique features of B. smithii central metabolism in comparison to related organisms included the lack of a standard acetate production pathway with no apparent pyruvate formate lyase, phosphotransacetylase, and acetate kinase genes, while acetate was the second fermentation product.
Next Generation Prokaryotic Engineering : The CRISPR-Cas Toolkit
Mougiakos, Ioannis ; Bosma, Elleke F. ; Vos, Willem M. de; Kranenburg, Richard van; Oost, John van der - \ 2016
Trends in Biotechnology 34 (2016)7. - ISSN 0167-7799 - p. 575 - 587.
Archaea - Bacteria - Cas9 - CRISPR-Cas - Genome editing - Recombineering
The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering. SpyCas9 has recently been established as an efficient counterselection system in combination with homologous recombination-based strategies for bacterial genome editing.Besides the traditionally used SpyCas9, other CRISPR-Cas systems (both heterologous and native) are currently being evaluated in bacteria for their editing potential.Catalytically inactive variants of CRISPR-Cas systems are used for transcriptional control in bacteria with great potential for fundamental research and applications.
Analysis of acid-stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation
Mols, J.M. ; Kranenburg, R. van; Melis, C.C.J. van; Moezelaar, R. ; Abee, T. - \ 2010
Environmental Microbiology 12 (2010)4. - ISSN 1462-2912 - p. 873 - 885.
arginine deiminase system - food poisoning toxins - nitric-oxide - listeria-monocytogenes - staphylococcus-aureus - superoxide-dismutase - tolerance response - lactococcus-lactis - genome sequence - low-ph
Acid stress resistance of the food-borne human pathogen Bacillus cereus may contribute to its survival in acidic environments, such as encountered in soil, food and the human gastrointestinal tract. The acid stress responses of B. cereus strains ATCC 14579 and ATCC 10987 were analysed in aerobically grown cultures acidified to pH values ranging from pH 5.4 to pH 4.4 with HCI. Comparative phenotype and transcriptome analyses revealed three acid stressinduced responses in this pH range: growth rate reduction, growth arrest and loss of viability. These physiological responses showed to be associated with metabolic shifts and the induction of general stress response mechanisms with a major oxidative component, including upregulation of catalases and superoxide dismutases. Flow cytometry analysis in combination with the hydroxyl (OH center dot) and peroxynitrite (ONOO-)-specific fluorescent probe 3'-(phydroxyphenyl) fluorescein (HPF) showed excessive radicals to be formed in both B. cereus strains in bactericidal conditions only. Our study shows that radicals can indicate acid-induced malfunctioning of cellular processes that lead to cell death.
Comparative analysis of transcriptional and physiological responses of Bacillus cereus to organic and inorganic acid shocks
Mols, J.M. ; Kranenburg, R. van; Tempelaars, M.H. ; Schaik, W. van; Moezelaar, R. ; Abee, T. - \ 2010
International Journal of Food Microbiology 137 (2010)1. - ISSN 0168-1605 - p. 13 - 21.
gram-positive bacteria - staphylococcus-aureus - lactobacillus-plantarum - tolerance response - gene-expression - nitric-oxide - low-ph - subtilis - stress - growth
Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to pH 5.5 set with different acidulants including hydrochloric acid (HCl), lactic acid (HL) and acetic acid (HAc). Phenotypes observed included a decreased growth rate (with HCl), bacteriostatic and bactericidal conditions, with 2 mM undissociated HAc or HL, and 15 mM undissociated HAc, respectively. In the latter condition a concomitant decrease in intracellular ATP levels was observed. The transcriptome analyses revealed general and specific responses to the acidulants used. The general acid stress response includes modulation of pyruvate metabolism with activation of the butanediol fermentation pathway, and an oxidative stress response that was, however, more extensive in the bacteriostatic and bactericidal conditions. HL-specific and HAc-specific responses include modulation of metabolic pathways for amino acid metabolism. Activation of lactate, formate, and ethanol fermentation pathways, alternative electron-transport chain components and fatty acid biosynthesis genes was noted in the presence of 15 mM undissociated HAc. In conclusion, our study has provided insights in phenotype-associated, and general and acidulant-specific responses in B. cereus.