Reseach Of Mannan Biosynthesis By Recombinant Bacillus Subtilis 168

Mannan,a hemicellulose polysaccharide,is widely distributed in plant cells and microbial cell walls without color,toxicity and odor.Mannan can not only effectively prevent food spoilage,mold and insect damage,but also has unique biological activity,which can stimulate the body's immune response.Therefore,mannan has important application prospects in the fields of food and medicine.Due to the complex extraction process of plant mannan,especially many problems in the following purification by physical and chemical methods,lead to unstable product quality.Meantime,the yield of mannan extract from the microbial cell wall is low.These factors have led to high production costs for pharmaceutical and food-grade mannans.The rapid developments of metabolic engineering and synthetic biology techologies have been applied in engineered microbial cell for the production of high value-added products.This provides a theoretical and technical reference for the mannan biosynthesis.The main research contents are as follows:?1?The mannan synthase gene?ctmanS?from the guar seed was screened and analyzed through genome database,and then artificially synthesized with codon optimization.The synthetic ctmanS DNA fragment was ligated into Escherichia coli expression plasmid pET21a and transferred into E.coli BL21?DE3?,the positive recombinant strain was screened with ampicillin at 100?g/?L,and then induced the expression of ctmanS by adding IPTG?Isopropyl?-D thiogalactopy ranoside?for 30 h at30°C.The effects of xylose concentration,carbon source concentration and induction temperature on the production of mannan in recombinant strain were investigated.The optimal shake-flashing culture conditions were determined with 0.4 mmol/L IPTG and 20 g/L glucose at 37°C.The mannan yield reached 0.69 g/L in recombinant E.coli BL21?DE3?under shaking flask conditions.?2?The three encoding genes of mannosidase,GDP-mannose pyrophosphorylase and mannan synthase were not present in Bacillus subtilis 168 through genomic information and metabolic analysis.Therefore,to reconstitute the mannan synthesis pathway in Bacillus subtilis 168,the mannan synthase encoding gene ctmanS was cloned into the E.coli-B.subtilis shuttle integration vector pAX01 and then homologously recombined into the Bacillus subtilis 168 genome,which was controlled under the xylose inducible promoter P_xyl.The GDP-mannose pyrophosphorylase-encoding gene manC and the phosphomannosidase-encoding gene manB from E.coli BL21?DE3?genome were cloned and integrated into the Bacillus subtilis 168 genome to obtain the recombinant strain Bacillus subtilis 168M.The recombinant Bacillus subtilis 168M was cultured in the shaking flask,resulting in 1.69 g/L of mannan.Furthermore,the inducer xylose concentration,carbon source concentration,induction temperature and induction time were investigated,and the optimal shaking flask conditions was obtained with 10 g/L xylose,20 g/L glucose and induction temperature of 30°C.The mannan yield of recombinant Bacillus subtilis 168M strain reached a maximum of 2.68 g/L?3?To further investigate the effects of the upstream pathway genes manC,manB and manA on mannan,the operons manC,manC-manB and manC-manB-manA were constructed and recombined into the Bacillus subtilis constitutive plasmid pP43NMK respectively,generated the recombinant plasmids pP43-C,pP43-CB and pP43-CBA.The above three recombinant plasmids were separately transferred into Bacillus subtilis 168M strain to obtain recombinant strains Bacillus subtilis168M/pP43-C,Bacillus subtilis 168M/pP43-CB and Bacillus subtilis 168M/pP43-CBA,respectively.Four recombinant strains Bacillus subtilis 168M,Bacillus subtilis 168M/pP43-C,Bacillus subtilis168M/pP43-CB and Bacillus subtilis 168M/pP43-CBA were cultured in shaking flask culture conditions,and the yields were 2.67,3.12,2.99 and 4.21 g/L,respectively.These results showed that the up-regulation of the expression levels of manC and manA genes significantly enhanced the increase of mannan yield,indicating that manC and manA are the key rate-limiting enzymes for mannan synthesis.In this study,a mannan biosynthesis pathway was successfully constructed in the food-grade host Bacillus subtilis 168.The engineered strain for efficiently producing mannan from low-cost glucose as a carbon source was obtained by overexpression the upstream pathway genes and optimizing the culture condition.The maximal yield of mannan was 4.21 g/L under shaking flask contidions.