Biosynthesis Of Xylitol In Escherichia Coli By Xylan

As an important industrial fine chemical and sugar-based chemical with manyoutstanding features, xylitol has a wide range of applications in chemical, pharmaceutical,food and feed industries. The traditional method of xylitol production is to degradehemicellulose into xylose, then convert pure xylose to xylitol with the chemical catalyst or thewhole cell. Owing to the use of high pressure and temperature, as well as the need ofexpensive separation and purification steps in the chemical reduction process, xylitolproduction through bioconversion has been proposed as an alternative process. This researchinvestigated the feature of promoters and constructed a new engineered Escherichia coliwhich could produce xylitol by xylan directly with the technology of Synthetic Biology. It’s anew cleaning approach to produce xylitol. The main topics and results of this paper werelisted as follow:The constitutive Promoters (Ppgi, PgapA, PpykA, PpykF) and inducible promoter PT7werecharacterized with enhanced green fluorescent protein (eGFP) as report gene. The fluorescentstrength of promoters were determined: Ppgi102.18, PgapA784.12, PpykA94.04, PpykF163.20,PT78785.87. PgapAwas the strongest one in the constitutive Promoters, and PT7’s fluorescentstrength was ten times more than PgapA’s.The pathway hydrolyzing xylan into xylose was constructed using PgapAwith the methodof Synthetic Biology, then the expressing plasmid pRS42K-xyn-xyl was obtained. Theengineered strain BL21-xyn-xyl which succeeded in hydrolyzing xylan into xylose wasobtained after the plasmid pRS42K-xyn-xyl was transformed into the strain BL21(DE3).The expressing plasmid pET-28a(+)-T7-xr and pET-28a(+)-gapA-xr were constructedusing PgapAand PT7respectively. The engineered strains BL21-T7-xr and BL21-gapA-xr whichsucceeded in converting xylose into xylitol were obtained after the two expressing plasmidswere transformed into the strain BL21(DE3) separately. Under the optimal fermentationconditions (IPTG concentration0.2mM, induction temperature25℃, initial xyloseconcentration10g/L), the best xylitol production of strain BL21-T7-xr was2.92g/L. Thexylitol production of strain BL21-gapA-xr was up to8.48g/L, when the optimal initial xyloseconcentration was15g/L. Compared the xylitol production between the two strains, the factthat strain BL21-gapA-xr was better indicated PgapAbenefited for xylitol production more. Inaddition, the optimal feeding concentration of xylose (8g/L) in the fermentation of strainBL21-gapA-xr was confirmed and the xylitol production reached40.6g/L.After the expressing plasmid pRS42K-xyn-xyl and pET-28a(+)-gapA-xr weretransformed into the engineered strain BL21-T at the same time, the integrated strainBL21-xyn-xyl-xr-T was obtained.