Dual Function of a Novel Bacterium, Slackia sp. D-G6: Detoxifying Deoxynivalenol and Producing the Natural Estrogen Analogue, Equol

Abstract

Deoxynivalenol (DON) is a highly abundant mycotoxin that exerts many adverse effects on humans and animals. Much effort has been made to control DON in the past, and bio-transformation has emerged as the most promising method. However, useful and effective application of bacterial bio-transformation for the purpose of inhibiting DON remains urgently needed. The current study isolated a novel DON detoxifying bacterium, Slackia sp. D-G6 (D-G6), from chicken intestines. D-G6 is a Gram-positive, non-sporulating bacterium, which ranges in size from 0.2-0.4 μm × 0.6-1.0 μm. D-G6 de-epoxidizes DON into a non-toxic form called DOM-1. Optimum conditions required for degradation of DON are 37-47 °C and a pH of 6-10 in WCA medium containing 50% chicken intestinal extract. Besides DON detoxification, D-G6 also produces equol (EQL) from daidzein (DZN), which shows high estrogenic activity, and prevents estrogen-dependent and age-related diseases effectively. Furthermore, the genome of D-G6 was sequenced and characterized. Thirteen genes that show potential for DON de-epoxidation were identified via comparative genomics. In conclusion, a novel bacterium that exhibits the dual function of detoxifying DON and producing the beneficial natural estrogen analogue, EQL, was identified.

Keywords: DON; Slackia sp. D-G6; de-epoxidize; equol.

Figure 1

Screening procedure and HPLC analysis of DON and DOM-1 after co-culture of DON with D-G6. (a). Procedure for screening DON transforming bacterium. (b). HPLC chromatography of standard DON and DOM-1. (c). The products of DON transformed by D-G6. DON and DOM-1 standards are indicated at 10.1 min and 15.6 min, respectively.

Figure 2

The phylogenetic tree and morphology of Slackia sp. D-G6. (a). The phylogenetic tree of D-G6 based on 16S rDNA genes. The GenBank accession numbers of the sequences are shown in parentheses. The bar indicates 0.01 substitutions per nucleotide position. (b). Gram staining of D-G6. (c). Microscopic images of D-G6 under TEM.

Figure 3

Metabolic characteristics of Slackia sp. D-G6. (a). Transforming efficiency under different media. (b). Transforming efficiency under different medias plus chicken intestine extracts (Ext). (c). The efficiency of DON de-epoxidation of D-G6 at different temperatures. The experiments were performed in WCA modified medium at pH 6.5. (d). The effects of varying pH on DON de-epoxidation. The experiments were performed in WCA modified medium and the temperature was set at 37 °C. (e). The growth curve and metabolic curve of D-G6 against time. Inoculation density was approximately 100 CFU/mL, the temperature was 37 °C, and pH was 6.5. All experiments were replicated three times biologically. The error bars represent the standard deviations. * indicate significantl difference (* P < 0.05, *** P < 0.001), “NS” means “no significance”.

Figure 4

Slackia sp. D-G6 is capable of transforming daidzein (DZN). (a,b). DZN standards (a) and equol (EQL) (b). The retention time of DZN and EQL were approximately 5.2 min and 6.2 min, respectively. (c,d). HPLC analysis of the products after 2 d of incubation of DZE^T (c) or D-G6 (d) with DZN. (e,f). Mass spectrometry (e) and mass/mass spectrometry (f) analysis of the product at 4.2 min. g,h. HPLC analysis of the products after 3 d of incubation with DZE^T (g) or D-G6 (h) with DZN.

Figure 5

The genome of Slackia sp. D-G6 and comparative analysis of four genomes, D-G6, DZE^T, DII-9, and P3277. (a). The genome circle map was drawn using Circos (version 0.69) software. Seven types of information are contained in the circle diagram, from outside to inside: the first circle shows the size of the genome, the second one is GC content, the third one are coding genes on the positive chain (red), the fourth one are the coding genes on the negative chain (green), the fifth and sixth ones are ncRNA on positive strand (blue) and negative strand (purple) respectively, and the seventh one are the repeating sequences of long segments within the genome (orange). (b). Venn diagram showing the core genes and genes shared among D-G6, DII-9, DZE^T, and P3277.