Publications by authors named "Bastien Chevreux"

9 Publications

  • Page 1 of 1

Rhodoxanthin synthase from honeysuckle; a membrane diiron enzyme catalyzes the multistep conversation of β-carotene to rhodoxanthin.

Sci Adv 2020 Apr 22;6(17):eaay9226. Epub 2020 Apr 22.

DSM Nutritional Products, 60 Westview St, Lexington, MA 02421, USA.

Rhodoxanthin is a vibrant red carotenoid found across the plant kingdom and in certain birds and fish. It is a member of the atypical retro class of carotenoids, which contain an additional double bond and a concerted shift of the conjugated double bonds relative to the more widely occurring carotenoid pigments, and whose biosynthetic origins have long remained elusive. Here, we identify LHRS ( hydroxylase rhodoxanthin synthase), a variant β-carotene hydroxylase (BCH)-type integral membrane diiron enzyme that mediates the conversion of β-carotene into rhodoxanthin. We identify residues that are critical to rhodoxanthin formation by LHRS. Substitution of only three residues converts a typical BCH into a multifunctional enzyme that mediates a multistep pathway from β-carotene to rhodoxanthin via a series of distinct oxidation steps in which the product of each step becomes the substrate for the next catalytic cycle. We propose a biosynthetic pathway from β-carotene to rhodoxanthin.
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http://dx.doi.org/10.1126/sciadv.aay9226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176425PMC
April 2020

Genetic Competence Drives Genome Diversity in Bacillus subtilis.

Genome Biol Evol 2018 01;10(1):108-124

Instituto Gulbenkian de Ciência, Oeiras, Portugal.

Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43% of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them.
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http://dx.doi.org/10.1093/gbe/evx270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765554PMC
January 2018

Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine.

Metab Eng 2015 May 14;29:196-207. Epub 2015 Mar 14.

DSM Nutritional Products Ltd., Nutrition Innovation Center R&D Biotechnology, Wurmisweg 576, CH-4303 Kaiseraugst, Switzerland. Electronic address:

Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5'-phosphate-(DXP)-dependent vitamin B6 pathway. Here we report the optimization of the condensing reaction of this pathway that consists of the 4-hydroxy-l-threonine-phosphate dehydrogenase PdxA, the pyridoxine 5'-phosphate synthase PdxJ and the native DXP synthase, Dxs. To allow feeding of high amounts of 4-hydroxy-threonine (4-HO-Thr) that can be converted to PN by B. subtilis overexpressing PdxA and PdxJ, we first adapted the bacteria to tolerate the antimetabolite 4-HO-Thr. The adapted bacteria produced 28-34mg/l PN from 4-HO-Thr while the wild-type parent produced only 12mg/l PN. Moreover, by expressing different pdxA and pdxJ alleles in the adapted strain we identified a better combination of PdxA and PdxJ enzymes than reported previously, and the resulting strain produced 65mg/l PN. To further enhance productivity mutants were isolated that efficiently take up and convert deoxyxylulose (DX) to DXP, which is incorporated into PN. Although these mutants were very efficient to convert low amount of exogenous DX, at higher DX levels they performed only slightly better. The present study uncovered several enzymes with promiscuous activity and it revealed that host metabolic pathways compete with the heterologous pathway for 4-HO-Thr. Moreover, the study revealed that the B. subtilis genome is quite flexible with respect to adaptive mutations, a property, which is very important for strain engineering.
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http://dx.doi.org/10.1016/j.ymben.2015.03.007DOI Listing
May 2015

Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine.

Metab Eng 2014 Sep 24;25:38-49. Epub 2014 Jun 24.

DSM Nutritional Products Ltd., P.O. Box 2676, CH-4002 Basel, Switzerland. Electronic address:

Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5'-phosphates. Pyridoxal 5'-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5'-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti, were assembled to two expression cassettes and introduced into the B. subtilis chromosome. in vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis.
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http://dx.doi.org/10.1016/j.ymben.2014.06.007DOI Listing
September 2014

Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads--a baiting and iterative mapping approach.

Nucleic Acids Res 2013 Jul 9;41(13):e129. Epub 2013 May 9.

Natural History Museum, University of Oslo, Oslo N-0318, Norway.

We present an in silico approach for the reconstruction of complete mitochondrial genomes of non-model organisms directly from next-generation sequencing (NGS) data-mitochondrial baiting and iterative mapping (MITObim). The method is straightforward even if only (i) distantly related mitochondrial genomes or (ii) mitochondrial barcode sequences are available as starting-reference sequences or seeds, respectively. We demonstrate the efficiency of the approach in case studies using real NGS data sets of the two monogenean ectoparasites species Gyrodactylus thymalli and Gyrodactylus derjavinoides including their respective teleost hosts European grayling (Thymallus thymallus) and Rainbow trout (Oncorhynchus mykiss). MITObim appeared superior to existing tools in terms of accuracy, runtime and memory requirements and fully automatically recovered mitochondrial genomes exceeding 99.5% accuracy from total genomic DNA derived NGS data sets in <24 h using a standard desktop computer. The approach overcomes the limitations of traditional strategies for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information at hand and represents a fast and highly efficient in silico alternative to laborious conventional strategies relying on initial long-range PCR. We furthermore demonstrate the applicability of MITObim for metagenomic/pooled data sets using simulated data. MITObim is an easy to use tool even for biologists with modest bioinformatics experience. The software is made available as open source pipeline under the MIT license at https://github.com/chrishah/MITObim.
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http://dx.doi.org/10.1093/nar/gkt371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711436PMC
July 2013

Sequence verification of synthetic DNA by assembly of sequencing reads.

Nucleic Acids Res 2013 Jan 4;41(1):e25. Epub 2012 Oct 4.

Virginia Bioinformatics Institute, Virginia Tech, Washington Street MC 0477, Blacksburg, VA 24061, USA.

Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org.
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http://dx.doi.org/10.1093/nar/gks908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592409PMC
January 2013

Small genes under sporulation control in the Bacillus subtilis genome.

J Bacteriol 2010 Oct 13;192(20):5402-12. Epub 2010 Aug 13.

The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138, USA.

Using an oligonucleotide microarray, we searched for previously unrecognized transcription units in intergenic regions in the genome of Bacillus subtilis, with an emphasis on identifying small genes activated during spore formation. Nineteen transcription units were identified, 11 of which were shown to depend on one or more sporulation-regulatory proteins for their expression. A high proportion of the transcription units contained small, functional open reading frames (ORFs). One such newly identified ORF is a member of a family of six structurally similar genes that are transcribed under the control of sporulation transcription factor σ(E) or σ(K). A multiple mutant lacking all six genes was found to sporulate with slightly higher efficiency than the wild type, suggesting that under standard laboratory conditions the expression of these genes imposes a small cost on the production of heat-resistant spores. Finally, three of the transcription units specified small, noncoding RNAs; one of these was under the control of the sporulation transcription factor σ(E), and another was under the control of the motility sigma factor σ(D).
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http://dx.doi.org/10.1128/JB.00534-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2950494PMC
October 2010

The origins of 168, W23, and other Bacillus subtilis legacy strains.

J Bacteriol 2008 Nov 22;190(21):6983-95. Epub 2008 Aug 22.

Bacillus Genetic Stock Center, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210, USA.

Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.
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http://dx.doi.org/10.1128/JB.00722-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580678PMC
November 2008

Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs.

Genome Res 2004 Jun 12;14(6):1147-59. Epub 2004 May 12.

Department of Molecular Biophysics, German Cancer Research Centre Heidelberg, 69120 Heidelberg, Germany.

We present an EST sequence assembler that specializes in reconstruction of pristine mRNA transcripts, while at the same time detecting and classifying single nucleotide polymorphisms (SNPs) occuring in different variations thereof. The assembler uses iterative multipass strategies centered on high-confidence regions within sequences and has a fallback strategy for using low-confidence regions when needed. It features special functions to assemble high numbers of highly similar sequences without prior masking, an automatic editor that edits and analyzes alignments by inspecting the underlying traces, and detection and classification of sequence properties like SNPs with a high specificity and a sensitivity down to one mutation per sequence. In addition, it includes possibilities to use incorrectly preprocessed sequences, routines to make use of additional sequencing information such as base-error probabilities, template insert sizes, strain information, etc., and functions to detect and resolve possible misassemblies. The assembler is routinely used for such various tasks as mutation detection in different cell types, similarity analysis of transcripts between organisms, and pristine assembly of sequences from various sources for oligo design in clinical microarray experiments.
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http://dx.doi.org/10.1101/gr.1917404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC419793PMC
June 2004