Genome Sequence of EU-Unauthorized Genetically Modified Bacillus subtilis Strain 2014-3557 Overproducing Riboflavin, Isolated from a Vitamin B2 80% Feed Additive.

This paper announces the genome sequence and annotation of the genetically modified (GM) Bacillus subtilis strain 2014-3557 overproducing riboflavin (vitamin B2). This GM-strain is unauthorized in the European Union. Nevertheless, it has been isolated from a lot of vitamin B2 (riboflavin) 80% feed grade imported to Europe from China.

GENOME ANNOUNCEMENT

Riboflavin (vitamin B2) cannot be biosynthesized by vertebrates, whereas plant and most microorganisms are able to do so (1). Therefore, vitamin B2 is used as a food and feed additive. As an alternative to costly chemical synthesis, microbial fermentation processes of riboflavin were developed for industrial production. Naturally producing or riboflavin-overproducing microorganisms transformed by a genetic engineering, chemical, or physical process can be used. Bacillus subtilis, a Gram-positive, rod-shaped bacterium, is exploited in industry for this purpose (1–3).

In July 2014, Germany detected a viable Bacillus subtilis strain which harbored a non-naturally occurring combination of DNA sequences in a lot of vitamin B2 feed additive imported from China. This strain was considered as genetically modified and unknown; therefore it is unauthorized in the European Union. In September 2014, the European Rapid Alert System for Food and Feed (RASFF) created a notification to alert the other European countries about the presence of the unauthorized GM-Bacillus subtilis in this particular vitamin B2 feed additive (https://webgate.ec.europa.eu/rasff-window/portal/ [enter reference 2014.1249]).

Consequently, the French competent authorities investigated this kind of product imported in France. A French National Reference Laboratory (NRL) for GMO isolated a yellow substance (presumably overproduction of riboflavin)-secreting bacterial strain from three samples of vitamin B2 feed additives imported from China.

To further characterize this finding, whole-genome sequencing was performed on one of the three isolates with an Illumina HiSeq2500 run using a paired-end library. Sequencing yielded 10,914,314 paired-end reads (350-fold coverage), which were assembled de novo using CLC Genomics Workbench version 7.5.1 (CLC Bio). The resulting draft genome was further linked into scaffolds with SSPACE (4) based on paired-end read linkage. Finally, a total of 39 gap-closed scaffolds were generated consisting of 143 contigs with a maximum gap-closed scaffold size of 1,018,461 bp and a minimum size of 370 bp. After filtering of these 39 scaffolds, three were discarded as two matched with Homo sapiens and one with Haemonchus placei. The total sequence length post-filtering is 4,175,764 bp, and has a G+C content of 44.32%.

Genome annotation was performed on the assembled scaffold sequences using the BaseClear annotation pipeline which is based on the Prokka Prokaryotic Genome Annotation System. This annotation confirmed the organism as Bacillus subtilis with 100% identity. 4,233 genes were predicted, of which 4,144 have a known function, including several encoding for proteins involved in riboflavin synthesis and transport. The genome sequence of this Bacillus subtilis strain will be useful for further characterization of its GM nature and in developing a specific method for its detection in food and feed additives by enforcement laboratories.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. JYFL00000000. The version described in this paper is version JYFL01000000.