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

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 lign...

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Main Author:	Abdullah, Noradilin
Format:	Thesis
Language:	English
Published:	2022
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/102863/1/NoradilinAbdullahPSChE2022.pdf.pdf
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spelling	my-utm-ep.1028632023-09-26T06:01:50Z Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars 2022 Abdullah, Noradilin TP Chemical technology 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. 2022 Thesis http://eprints.utm.my/102863/ http://eprints.utm.my/102863/1/NoradilinAbdullahPSChE2022.pdf.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:150683 phd doctoral Universiti Teknologi Malaysia Faculty of Engineering - School of Chemical & Energy Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Abdullah, Noradilin Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars
description	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.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Abdullah, Noradilin
author_facet	Abdullah, Noradilin
author_sort	Abdullah, Noradilin
title	Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars
title_short	Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars
title_full	Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars
title_fullStr	Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars
title_full_unstemmed	Construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars
title_sort	construction and characterization of recombinant escherichia coli for production of xylitol from mixed sugars
granting_institution	Universiti Teknologi Malaysia
granting_department	Faculty of Engineering - School of Chemical & Energy Engineering
publishDate	2022
url	http://eprints.utm.my/102863/1/NoradilinAbdullahPSChE2022.pdf.pdf
_version_	1783729227099537408

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

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