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

Abstract

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.

Use of next generation sequencing data to develop a qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin

“…The first step in the development of the specific qPCR assay consisted of identifying the junction of the GM-insert into the Bacillus subtilis GM-strains extracted from vitamin B2 samples 2014–3557 [Genbank: JYFL01000000] [ 21 ]. As elaborated in Methods, a BLAST study of the contigs of the previously published sequence of the GM- Bacillus subtilis 2014–3557 isolated from vitamin B2 80 % [Genbank:JYFL01000000.1] [ 21 ] was used to identify the contigs containing the riboflavin biosynthesis operon ribGBAH , with a focus on locating the ribA gene as it has been previously reported that additional expression of this gene encoding the rate limiting enzyme in riboflavin synthesis increases riboflavin synthesis even more as compared to only overexpression of ribGBAH [ 7 , 11 , 24 ]. The overlapping contigs Contig0022 [Genbank:JYFL01000022.1] and Contig0016 [Genbank:JYFL01000016.1] were retrieved as containing genes having 100 % similarity with B. subtilis subsp.…”

“…subtilis str. 168 genes ribH, ribBA, ribE and ribD , involved in riboflavin biosynthesis [ 7 , 25 ], as it could also be deduced from the annotation of the GM- B. subtilis genome sequence [Genbank:JYFL01000000] [ 21 ] (Fig. 1 ).…”

“…The dynamic range of a qPCR assay is the range of concentrations where the assay performs linearly. This was assessed for the VitB2-UGM qPCR assay by the analysis in duplicate of a serial dilution of gDNA (10,000 to 0.01 theoretical genomic copies) of the GM- Bacillus subtilis strain 2014 – 3557 overproducing riboflavin [ 21 ]. In addition, this analysis allowed for the assessment of the coefficient of determination (R 2 ) and the PCR efficiency (E).…”

“…Next generation sequencing (NGS) was performed on the genomic DNA extracted from one of the isolates. The sequencing reads were de novo assembled and genome annotation was performed on the contig sequences [ 21 ]. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not always present in the average enforcement laboratory.…”

“…In this study, the DNA sequences previously generated by the NGS approach [ 21 ] were used to develop a TaqMan® qPCR method targeting the junction between the B. subtilis riboflavin operon and the vector used to construct this GM-strain. Different performance criteria of the developed qPCR method such as specificity, sensitivity, PCR efficiency and repeatability were evaluated according to the GMO guidelines [ 22 , 23 ].…”