## 3 Results
### In Silico Analysis of EPS Biosynthetic Pathways  
In silico genome analysis revealed that _B. velezensis_ VTX20 contains two EPS clusters an EPS cluster and a levan cluster. The _eps_ cluster consists of 16 open reading frames, _epsA_, _epsB_, _epsC_, _epsD_, _epsE_, _epsF_, _epsG_, _epsH_, _epsI_, _epsJ_, _epsK_, _epsN_, _epsM_, _epsO_, and _slrR_. All _eps_ genes are located in the same orientation with a size of 16,414 bp. _B. velezensis_ VTX20 presented around 99% sequence similarity to _B. velezensis_ LPL061, _B. velezensis_ FZB42, _B. velezensis_ L-S60, indicating that the _eps_ cluster is conserved across _B. velezensis_ strains (Figure 5A). Surprisingly, _eps_ genes observed in _B. velezensis_ VTX20 are located on the plus strands, holding an opposite orientation compared to that of other compared strains.  
The _epsA-O_ operon is shown to be negatively regulated by the transcriptional regulation factor _slrR_ (_orf_\(\_\)_720) (Figure 5A). Both _epsA_ (_orf_\(\_\)721) and _epsB_ (_orf_\(\_\)722) function in determining polysaccharide chain length; however, _epsA_ is also responsible for the export of polysaccharides [37]. The _epsL_ (_orf_\(\_\)732), _epsD_ (_orf_\(\_\)724), _epsE_ (_orf_\(\_\)725), _epsF_ (_orf_\(\_\)726), _epsH_ (_orf_\(\_\)728), and _epsJ_ (_orf_\(\_\)730) genes all encoded glycosyltransferases, participating in the biosynthesis of the repeating units. _epsL_ transfers UDP-glucose or UDP-galactose onto lipid carriers, while _epsD_ (_orf_\(\_\)724) functions in transporting UDP-GlcNAc. Both _epsE_ (_orf_\(\_\)725) and _epsF_ (_orf_\(\_\)726) are responsible for UDP-GlcNAc transport, and _epsH_ (_orf_\(\_\)728) and _epsJ_ (_orf_\(\_\)730) are responsible for amino sugars transport. _epsG_ (_orf_\(\_\)727) functions in the polymerization of EPS repeating units, and _epsK_ (_orf_\(\_\)731) transports polysaccharide repeating units into periplasmic space [10]. Moreover, the _epsC_ (_orf_\(\_\)723), _epsJ_ (_orf_\(\_\)729) and _epsO_ (_orf_\(\_\)725), _epsM_ (_orf_\(\_\)734), and _epsN_ (_orf_\(\_\)733) are involved in the modification of polysaccharide repeating units.  
Other bacterial EPSs, such as levan, the fructan polysaccharide, were found in the genome of VTX20. Levan-type fructooligosaccharide biosynthesis includes levansucrase _ls_ (EC 2.4.1.10), 2,6-beta-fructan 6-levanbiohydrolase _levB_ (EC 3.2.1.64), and levanase _sacA_ (EC 3.2.1.26) (Figure 5B). _ls_ and _levB_ genes are clustered together in an operon, while _sacA_ is located on a different operon. Although these genes are conserved among _B. velezensis_ strains, the direction of all three genes in the VTX20 genome is opposite to those of the compared strains (Figure 5B). Levan is synthesized outside cells by an action Ls enzyme that converts disaccharide sucrose into levan, leading to the release of glucose [14].  
Figure 4: The cluster of orthologous group (COG) categories of the strain VTX20.  
The released glucose is then employed for metabolic purposes and partly inhibits levan biosynthesis [18]. Moreover, levan is hydrolyzed to levan-type fructoligosaccharides, for example, levanbiose, by LevB and SacA. In addition, SacA is also responsible for cleaving sucrose into glucose and fructose [38].