Papers by Richard van Kranenburg
Trends in Biotechnology, Jul 1, 2016
Catalytically inactive variants of CRISPR-Cas systems are used for transcriptional control in bac... more Catalytically inactive variants of CRISPR-Cas systems are used for transcriptional control in bacteria with great potential for fundamental research and applications.
Plasmid, 2000
The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopoly... more The complete 42180-bp nucleotide sequence of the mobilization plasmid pNZ4000, coding for exopolysaccharide (EPS) production in Lactococcus lactis, was determined. This plasmid contains a region involved in EPS biosynthesis, four functional replicons, a region containing mobilization genes, and three origin of transfer (oriT ) sequences. Sequences identical to these oriT sequences were also found on two other lactococcal plasmids and a plasmid from Lactobacillus helveticus. Several complete and partial IS elements were identified on pNZ4000, including iso-ISS1, iso-IS946, and iso-IS982 sequences. Furthermore, pNZ4000 contains a gene cluster that may encode a cobalt transport system and a gene that encodes a CorA homologue which may function as a magnesium transporter.
Journal of Biological Chemistry, 1996
Carbon Monoxide Dehydrogenas e from Methanosarcin a frisia Gol CHARACTERIZATION OF THE ENZ\1ME AN... more Carbon Monoxide Dehydrogenas e from Methanosarcin a frisia Gol CHARACTERIZATION OF THE ENZ\1ME AND THE REGULATED EXPRESSION OF TWO OPERON-LIKE cdh GENE CLUSTERS*
Frontiers in Microbiology, Oct 20, 2022
Parageobacillus thermoglucosidasius is a thermophilic bacterium of interest for lignocellulosic b... more Parageobacillus thermoglucosidasius is a thermophilic bacterium of interest for lignocellulosic biomass fermentation. However, carbon catabolite repression (CCR) hinders co-utilization of pentoses and hexoses in the biomass substrate. Hence, to optimize the fermentation process, it is critical to remove CCR in the fermentation strains with minimal fitness cost. In this study, we investigated whether CCR could be removed from P. thermoglucosidasius DSM by mutating the Ser regulatory sites on HPr and Crh to a non-reactive alanine residue. It was found that neither the ptsH (HPr-S A) nor the crh (Crh-S A) mutation individually eliminated CCR in P. thermoglucosidasius DSM . However, it was not possible to generate a ptsH crh double mutant. While the Crh-S A mutation had no obvious fitness e ect in DSM , the ptsH mutation had a negative impact on cell growth and sugar utilization under fermentative conditions. Under these conditions, the ptsH mutation was associated with the production of a brown pigment, believed to arise from methylglyoxal production, which is harmful to cells. Subsequently, a less directed adaptive evolution approach was employed, in which DSM was grown in a mixture of -deoxy-D-glucose( -DG) and xylose. This successfully removed CCR from P. thermoglucosidasius DSM . Two selection strategies were applied to optimize the phenotypes of evolved strains. Genome sequencing identified key mutations a ecting the PTS components PtsI and PtsG, the ribose operon repressor RbsR and adenine phosphoribosyltransferase APRT. Genetic complementation and bioinformatics analysis revealed that the presence of wild type rbsR and apt inhibited xylose uptake or utilization, while ptsI and ptsG might play a role in the regulation of CCR in P. thermoglucosidasius DSM .
The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcription... more The genes for DNA uptake and recombination in Bacilli are commonly regulated by the transcriptional factor ComK. We have identified a ComK homologue in Bacillus coagulans, an industrial relevant organism that is recalcitrant for transformation. Introduction of B. coagulans comK gene under its own promoter region into Bacillus subtilis comK strain results in low transcriptional induction of the late competence gene comGA, but lacking bistable expression. The promoter regions of B. coagulans comK and the comGA genes are recognized in B. subtilis and expression from these promoters is activated by B. subtilis ComK. Purified ComK protein of B. coagulans showed DNA-binding ability in gel retardation assays with B. subtilis-and B. coagulans-derived probes. These experiments suggest that the function of B. coagulans ComK is similar to that of ComK of B. subtilis. When its own comK is overexpressed in B. coagulans the comGA gene expression increases 40-fold, while the expression of another late competence gene, comC is not elevated and no reproducible DNAuptake could be observed under these conditions. Our results demonstrate that B. coagulans ComK can recognize several B. subtilis comK-responsive elements, and vice versa, but indicate that the activation of the transcription of complete sets of genes coding for a putative DNA uptake apparatus in B. coagulans might differ from that of B. subtilis.
Microorganisms
Pseudoclostridium thermosuccinogenes is a thermophilic bacterium capable of producing succinate f... more Pseudoclostridium thermosuccinogenes is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a platform organism. However, exploitation of P. thermosuccinogenes has been limited partly due to the genetic inaccessibility and lack of genome engineering tools. In this study, we established the genetic accessibility for P. thermosuccinogenes DSM 5809. By overcoming restriction barriers, transformation efficiencies of 102 CFU/µg plasmid DNA were achieved. To this end, the plasmid DNA was methylated in vivo when transformed into an engineered E. coli HST04 strain expressing three native methylation systems of the thermophile. This protocol was used to introduce a ThermodCas9-based CRISPRi tool targeting the gene encoding malic enzyme in P. thermosuccinogenes, demonstrating the principle of gene silencing. This resulted in 75% downregulation of its expression and had an impact on the strain’s fermentation profi...
Communications Biology
Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inh... more Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr an...
Microbial Cell Factories, Nov 21, 2012
Background: Bacterial cell surface-associated polysaccharides are involved in the interactions of... more Background: Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action. Results: The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (Δcps1A-I, Δcps2A-J, Δcps3A-J and Δcps4A-J) or combinations of cps clusters (Δcps1A-3J and Δcps1A-3I, Δcps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up-and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The Δcps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-κB activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the Δcps1A-I and Δcps3A-J mutants but appeared slightly increased after stimulation with the Δcps2A-J and Δcps4A-J mutants, while the Δcps1A-3J and Δcps1A-3J, Δcps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling.
Background Pseudomonas putida as a cell factory is increasingly being used due to its remarkable ... more Background Pseudomonas putida as a cell factory is increasingly being used due to its remarkable features such as fast growth, versatile and robust metabolism, extensive genetic toolbox and high tolerance to oxidative stress and toxic compounds. This interest is driven by the need to improve microbial performance to a level that enables biologically possible processes to become economically feasible and thereby foster the transition from an oil-based economy to a more sustainable bio-based one. To this end, one of the current strategies is to maximize the product-substrate yield of an aerobic biocatalyst such as P. putida during growth on glycolytic carbon sources, such as glycerol and xylose. We demonstrate that this can be achieved by implementing the phosphoketolase shunt, through which pyruvate decarboxylation is prevented, and thus carbon loss is minimized. ResultsIn this study, we introduced the phosphoketolase shunt in the metabolism of P. putida KT2440. To maximize the effec...
Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular... more Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcal metC-cysK operon, encoding a cystathionine -lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect on metC-cysK gene expression. In addition we found that O-acetyl-L-serine, the substrate of cysteine synthase, was an inducer of the metC-cysK operon. Using a random mutagenesis approach, we identified two genes, cmbR and cmbT, involved in regulation of metC-cysK expression. The cmbT gene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption of cmbT resulted in a two-to threefold reduction of metC-cysK transcription. A 5.7-kb region containing the cmbR gene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of the metC-cysK operon. In analogy to CysB from Escherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of the metC-cysK operon.
ACS Synthetic Biology, 2021
Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassignin... more Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9mediated counterselection built upon the minimal PAM 5′-NNG-3′ of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond.
Anaerobe, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Journal of Bacteriology, 1999
Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemic... more Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with pri...
Journal of Biological Chemistry, 2019
The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Ther... more The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, wh...
Applied and Environmental Microbiology, 2018
Clostridium thermosuccinogenes is a thermophilic anaerobic bacterium able to convert various carb... more 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 C 5 with the C 3 /C 6 metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic sp...
Frontiers in Microbiology, 2019
Pseudoclostridium thermosuccinogenes and Hungateiclostridium thermocellum are being studied for t... more 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.
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Papers by Richard van Kranenburg