Development of a recombinant Sphingomonas paucimobilis ATCC 31461 with multiple expression of gellan gum biosynthetic genes gelC gelD and gelE Production Characterization and Application

Abstract

Gellan gum is a linear, high molecular weight, anionic exopolysaccharide newlinecomprising variable units of glucose, rhamnose and glucuronic acid. Sphingomonas newlinespecies are the primary source of gellan producers for various industrial applications. At newlinepresent, the overall cost of gellan production is at a higher end due to the cumulative newlineeffect of the high price of substrates, media and downstream strategies used and newlinetherefore, the development of novel methods to improve the yield of gellan in terms of newlineproduction is needed to bring down the total cost of developed biopolymer from the newlinecommercial point of view. The objective of the present research was to produce gellan newlinein a cost-effective approach. Since the wild-type production is deficient, recombinant newlineexpression is necessitated. newlineIn this study, gellan gum genes gelC, gelD and gelE of Sphingomonas newlinePaucimobilis ATCC 31461 encoding the polymerization and export of gellan were newlinecloned into the pBBR122 vector. The recombinant plasmids were transformed into newlineSphingomonas Paucimobilis ATCC 31461 using the electroporation technique. The newlinerecombinant gellan gum proteins were purified by affinity chromatography using Ni- newlineNTA resin. SDS-PAGE analysis revealed the presence of a single major band of gelC newline(110 KDa), gelD (74 KDa) and gelE (60 KDa) protein. The results showed that the newlinerecombinant Sphingomonas paucimobilis with multiple expressions of cloned gellan newlinegum biosynthetic genes of gelC, gelD and gelE were successfully constructed. newlineHomology modeling was used to construct the 3D structure of gellan gum newlineproteins gelC, gelD and gelE. The protein modeling of gellan gum genes was examined newlineusing SWISS-MODEL, Phyre2 and I-TASSER. The reliability of the modeled newlinestructures was validated by tools like RAMPAGE, ProSA, Q-MEAN, ERRAT and newlineVERIFY3D. The computational studies of the gellan gum proteins revealed that protein