Molecular Mechanism Of Reduction Of Selenite By Bacillus Subtilis To Synthesize Nano-Selenium

High concentrations of selenite are seriously toxic to organisms,and the biotransformation of selenite by microorganisms is an effective detoxification and assimilation process,and they can reduce selenite to low-toxic nano-selenium.Over the past decade,this phenomenon has received increasing attention from researchers,not only because of the widespread use of nanoparticles in nanobiotechnology,but also because "green synthetic" nanomaterials are increasingly in line with green development.idea.Bacillus subtilis is an aerobic bacterium widely distributed in soil and decaying organic matter.It grows fast,has low nutritional requirements,can efficiently secrete many proteins and metabolites,and does not produce toxins.A nonpathogenic safe microorganism.B.subtilis can reduce selenite and produce nanoselenium under aerobic conditions,and the process of reducing selenite does not pose an environmental safety risk.However,the current understanding of the molecular mechanism of selenite reduction by B.subtilis is still limited.In this study,the reduction of selenite by B.subtilis 168 was systematically analyzed,and the molecular mechanism of selenite reduction was elaborated.The specific research results are as follows:(1)B.subtilis can grow normally in 5 mM selenite,and can synthesize nanoselenium particles with a particle size of about 180 nm in vitro and in vivo.Fourier transform infrared spectroscopy(FTIR)analysis results showed that some organic substances such as proteins,lipids and sugars were attached to the surface of the Bacillus subtilis synthetic nano-selenium.These organic matters may provide some help for the stability of nano-selenium.(2)The molecular mechanism of sodium selenite reduction by B.subtilis was studied by transcriptomic method.KEGG pathway enrichment analysis enriched 29 KEGG pathways.Significantly enriched pathways in the selenite-treated group included pentose phosphate cycle pathway,pyruvate metabolism pathway,proline metabolic pathway and riboflavin metabolic pathway.(3)The effect of sulfite reductase CysJI on selenite reduction was studied,and the cysJI mutant strain ΔcysJI and the ΔcysJI mutant strain cysJI complementing strainΔcysJI-C were constructed.The reduction rate of selenate was significantly reduced,and the reduction rate of the complementing strain was not significantly different from that of the wild type.CysJI was recombinantly expressed and purified,and it was found that with NADPH as the electron donor,CysJI could reduce selenite in vitro.It was confirmed that sulfite reductase indeed plays an important role in the reduction of selenite.(4)The effects of two important dehydrogenases(PutC and GabD)in the proline metabolic pathway on selenite reduction were explored.The reduction rate of selenite was significantly reduced after the gene deletion mutation of these two enzymes.When the expression of the relevant genes was complemented in the mutant strain,the reduction rate of the complemented strain was comparable to that of the wild type.The results of in vitro enzyme activity experiments showed that all of them could reduce selenite with NADPH as electron donor.It was confirmed that the proline metabolic pathway was involved in the reduction of selenite.In this paper,we report a selenite reduction mechanism mediated by multiple pathways—sulfite metabolism pathway and proline metabolism pathway-mediated selenite reduction mechanism,revealing a new type of selenite reduction mechanism in aerobic bacteria.Molecular mechanism of selenite reduction.