Key Genes Analysis Of Polysaccharide Synthesis In Bacillus Licheniformis And Construction Of Recombinant Strains With High Flocculating Activities

As two common bioflocculants,polysaccharide and gamma-poly glutamic acid are getting more and more attention in the field of wastewater treatment,drinking water purification,food and fermentation industry because of their numerous advantages such as biodegradability,non-toxicity,high efficiency and no secondary pollution.Bacillus licheniformis CGMCC 2876,screened by our laboratory,is a strain that can secrete two extracellular polymers polysaccharides and y-PGA with high flocculating acitivities.This research aims to explore the specific impact of the key genes that involved in the polysaccharide metabolic pathways in B.licheniformis.We try to use genetic engineering to construct recombinant strains to improve the industrial production of flocculants.Firstly,several key genes included pgcA,crr,gtaBl,epsA and epsB were amplified by PCR and cloned into the expression vector pHY300PLK-PamyL-TTamyL that constructed in our previous studies.Then the recombinant expression vectors were expressed in B.licheniformis using the method of electrotransformation and six gene overexpression recombinant strains were constructed successfully.The crude yield and flocculating activity of the bioflocculant produced by recombinant strains were determined and compared with the original strain.Among them the crude yield of bioflocculants produced by B.licheniformis-pHY300-epsA and B.licheniformis-pHY300-pgcA-gtaBl was enhanced by 23.70%and 20.77%respectively.But solely overexpressing pgcA and gtaBl did not have an obvious impact on the polysaccharide biofocculant production.So improving the expression level of glycosyltransferase gene epsA and tandem gene pgcA-gtaBl can promote the production of polysaccharide bioflocculant,but regulating the expression of phosphoglucomutase gene pgcA and UDP-glucose pyrophosphorylase gene gtaBl alone have little influence on the polysaccharide synthesis.Besides,overexpressing epsB from eps gene cluster not only improved the bioflocculant crude yield by 13.98%,reaching 8.56 g·L-1,but also largely enhanced the flocculating activity by 117.92%to 5100.50 U·mL-1.Therefore,B.licheniformis-pHY300-epsB is a high-yield bioflocculant strain compared with the original strain,which has improved potential capacity for industrial production.Then we selected B.licheniformis-pHY300-epsB as the main research object,and analyzed the the compositions of biofocculants and the transcriptional level of several key genes that involved in the synthesis of polysaccharide and y-PGA.The compositions of the bioflocculant from B.licheniformis-pHY300-epsB were 28.95%total sugar and 44.03%y-PGA,while in the original strain,the contents were 53.67%and 34.13%,respectively.At the same time,the transcriptional level of several key genes that involved in the synthesis of polysaccharide and y-PGA has changed in B.licheniformis-pHY300-epsB.The transcriptional level of carbon metabolism regulation protein genes ccpA and ccpN declined,while that of nitrogen metabolism regulation protein genes nrgB,?-PGA synthetase gene pgsA and pgsB,isocitrate dehydrogenase gene icd and 1-pyrroline-5-carboxylate dehydrogenase gene rocA had different degrees of increase.Thus epsB played an important role in the synthesis of polysaccharides and y-PGA simultaneously.Regulating the expression of epsB can change the direction of carbon metabolism flow in the bacteria,thereby affecting the synthesis level of polysaccharides and y-PGA.The bioflocculant production of recombinant strain B.licheniformis-pHY300-epsB was further evaluated with a batch fermentation in a 2 L fermentor.Results showed that the flocculating activity and yield increased by 224%and 36.62%respectively compared with the original strain.