Metabolic Pathway Of Crude Agar-degrading In Flammeovirga Pacifica WPAGA1 And A Novel Way For Producing Agaro-olisaccharides

Agaro-oligosaccharides are hydrosoluble sugar and the products of the degradation of agarose.Comparing with agarose,agaro-oligosaccharides were reported to hold more potential applications in food,cosmetic and medical industries.Flammeovirga pacifica WPAGA1 was isolated from the West Pacific's deep-sea sediments in our previous study,which showed excellent ability to degrade crude agarose of Gracilaria lemameiformis and to produce agaro-saccharides.Because of the high percentage of SO42-in crude agarose,it can't be degraded by agarases,and the metabolic mechanism of crude agarose in bacteria is still unknown.Herein,we investigate the metabolic pathway of crude agarose-degrading in strain WPAGA1.The main results are listed as follow:(1)Strain WPAGA1 could utilized many polysaccharides as sole carbon sources,including agar,carrageenan,amylum,sodium alginate,colloidal chitin,carboxymethyl cellulose,fucoidan,porphyran,arabinogalactan and xylan.And these polysaccharides were degraded by the extracellular enzymes of strain WPAGA1 to product reducing sugar.The enzymatic activity of agarose was highest,followed by carrageenan.Meanwhile,there were at least six agarases secreted by strain WPAGA1.(2)To reveal the strain WPAGA1's mechanism of polysaccharides-degrading in genomic level,the genome of Flammeovirga pacifica WPAGA1 was sequenced.These results presented the fine genome of strain WPAGA1 consisting of 6.6 Mb bases with 32.89%G+C content,5036 encoding genes,and rich genetic elements including genomic islands,ncRNA,CRISPRs genes.A total of 1022 carbohydrate-related genes and other rich genes were annotated by the KEGG,COG,GO,CAZy and other databases.To further investigate the metabolic pathway,these agarose-related genes in strain WPAGA1 genome were identified and analyzed.A total of 16 putative genes of agarase and 4 putative genes of glucoside hydrolase belonged to GH117 family(NABH4900,NABH4454,NABH4989 and NABH4302).And one putative gene of 3,6-anhydrogalactonate cycloisomerase(AHGAC4986)and two putative genes of 3,6-anhydro-L-galactose dehydrogenase(AHGD4985 and AHGD4649)were also found.And 10 putative agarases belong to GH86 family,4 putative agarases belong to GH16 family and 2 putative agarases belong to GH50 family based on amino acid sequences of these genes.In this study,the whole metabolic pathway of agarose in strain WPAGA1 was predicated based on these agarose-related genes.(3)To further investigate the enzymatic activities in vitro,the corresponding recombinant proteins were expressed in Escherichia coli BL21(DE3).These results showed that agarose were hydrolyzed by agarase Aga4007,Aga2593,Aga4779,Aga950 and Aga1974 to generate neagarotetraose and neoagarohexaose and then further degraded into neoagarobiose by GH50-dependent agarase Aga2660.Neoagarobiose was then hydrolyzed by the GH117 family glycoside hydrolase NABH4454 into D-galactose and 3,6-anhydro-L-galactose.Finally,D-galactose entered the galactose metabolic pathway,and AHGD4985 and AHGAC4986 catalyzed 3,6-anhydro-L-galactose conversion to 2-keto-3-deoxy-galactonate,which was utilized by the TCA cycle.Meanwhile,the sulfatase Su11971 could release free SO42-from the crude agarose,and the reducing sugar from hydrolyzing of crude agarose by agarases when Su11971 was present,furthermore,these results indicated the key role of sulfatases in metabolism of crude agarose in bacteria for the first time.(4)To produce the sole neoagarooligosaccharides,these enzymes were co-expressed in E.coli BL21(DE3)in different combinations based on the metabolic pathway of crude agarose.500 mg/L neoagarobiose was produced in E.coli BL21(DE3)which harbor recombinant plasmid pACY-NAB1 containing Aga4007 and Aga2660 using agarose as materials.While there was only 50 mg/L neoagarobiose detected when crude agarose was used as materials for fermentation.To decrease the harm to environments and reduce the cost,the crude agarose in Gracilaria lemameiformis as materials for producing of neoagarooligosaccharides is suggested.A recombinant plasmid pET-Sull containing sulfatase Su11971 was transformed into E.coli BL21(DE3)which harbored plasmid pACY-NAB1.Comparing with controls without Sul 1971,the concentration of neoagarobiose was increased 9 fold when using crude agarose as materials,and the concentration of neoagarobiose was 450 mg/L.This novel way for producing of neoagarooligosaccharides provides theoretical basis for exploring the applications of this strain.