Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars

• Main Author: Abdullah, Noradilin
• Format: Thesis
• Language: English
• Published: 2022
• Subjects: TP Chemical technology
• Online Access: http://eprints.utm.my/id/eprint/102863/1/NoradilinAbdullahPSChE2022.pdf.pdf; http://eprints.utm.my/id/eprint/102863/; http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:150683

Development of a microbial system for production of value-added chemicals has garnered interest for its benefits of low operational cost and greater substrate specificity. Microbial production of xylitol is highly desired for its ability to produce xylitol from unpure carbon sources mainly from lignocellulosic waste. While recent studies on xylitol production focused on utilizing mixed sugars for xylitol production, this required the expression of multiple genes to enable simultaneous xylitol conversion from glucose, xylose and arabinose which leads to cell‘s metabolic burden. Moreover, manipulation on the cell is also needed to remove catabolite repression present in the cell. This is the first study that describes xylitol production from multiple sugars (glucose, xylose and arabinose) in Escherichia coli BL21 expressing only a single gene, xylitol 5-phosphate dehydrogenase (XPDH). XPDH converts D-xylulose-5-phosphate, an intermediate in E. coli pentose phosphate pathway to D-xylitol-5-phosphate which is then hydrolyzed to D-xylitol by phosphatase. XPDH from Clostridium difficile was cloned into E. coli and screened for xylitol production using high pressure liquid chromatography analysis. Then, xylitol production was improved through metabolic engineering by deleting competing pathways and process optimized using one factor-at-a-time (OFAT) method. Initial screening of xylitol production revealed that E. coli BL21 expressing XPDH (NA116) was able to produce xylitol from each sugar, glucose, xylose and arabinose (supplied at 10 g/L) with final xylitol of 0.283 g/L, 0.518 g/L and 2.09 g/L respectively. Metabolic manipulation of the E. coli was made by deleting competing pathways in glycolysis and pentose phosphate pathway, namely phospoglucose isomerase (pgi), ribose isomerase A (rpiA), and ribose isomerase B (rpiB) genes. Screening of the mutants revealed highest xylitol production from arabinose by NA207 (ΔrpiA) mutant, with final xylitol produced of 3.91 g/L. Further manipulation of NA207 strain was made by introducing ptsG deletion to allow simultaneous carbon uptake in the presence of glucose for mixed sugars fermentation, yielding NA223 (ΔrpiAΔptsG) strain. The result revealed that NA223 showed 4 times more arabinose uptake in the mixed sugar culture compared to NA116, with final xylitol production of 1.18 g/L and 0.815 g/L respectively. Optimization of NA223 mutant was done by using OFAT method manipulating several parameters; inducer concentrations, temperature, media type and initial pH. The final parameters manipulation showed to have improved xylitol production to 5 times compared to initial conditions with 1.674 g/L in 0.05mM IPTG, 3.687 g/L in 25 °C, 3.95 g/L in buffered YT broth (BYT) medium, and 5.216 g/L in initial pH 8.5 of BYT. This study shows that xylitol conversion from mixed sugars is possible by expressing only a single heterologous gene, XPDH in E. coli while ptsG deletion alleviates carbon catabolite repression by allowing simultaneous arabinose uptake in the presence of glucose.