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Dioxygenases in the catabolism of syringols in Pseudomonas putida KT2440 Dumalo, Linda
Abstract
Biomass is a potential source for many of the fuels, chemicals, and materials that are needed by society and that are currently derived from petroleum. However, the sustainable biorefining of lignocellulosic biomass requires the valorization of all three of its components: cellulose, hemicellulose and lignin. Lignin has proven particularly difficult to valorize due to its recalcitrance. One approach has involved developing whole cell bacterial biocatalysts to upgrade the p-hydroxyphenyl, guaiacyl, and syringyl compounds resulting from lignin depolymerisation. Pseudomonas putida KT2440 is arguably the best characterized bacterial strain being developed as a biocatalyst, and is being engineered to transform syringyl monoaromatics. In this thesis, I investigated the role of two ring-cleavage dioxygenases, GalA and PcaHG, in the conversion of syringaldehyde-derived catechols to 2-pyrone-4,6-dicarboxylic acid (PDC), a precursor for biodegradable polyesters. PcaHG, protocatechuate 3,4-dioxygenase, cleaved gallate with an apparent specificity 20% that for protocatechuate. UV-vis spectroscopy and liquid chromatography-coupled mass spectrometry revealed that PcaHG cleaved gallate to 4-oxalomesaconate (OMA) and that this was subsequently transformed non-enzymatically to PDC. GalA cleaved 3-O-methyl gallate (3-MGA) to 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD), which cyclized non-enzymatically to PDC. However, 3-MGA potently inactivated GalA with a partition ratio >500-fold less than the partition ratio for gallate. Further analysis revealed that while GalA was inactivated by gallate and 3-MGA at similar rates, 3-MGA was cleaved several orders of magnitude less efficiently than gallate. The enzyme’s specificity for gallate was three-fold higher than previously reported. Overall, these results indicate that GalA is highly susceptible to inactivation and suggest that PcaHG is a better choice in engineering KT2440 to transform syringyl monoaromatics to PDC.
Item Metadata
Title |
Dioxygenases in the catabolism of syringols in Pseudomonas putida KT2440
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2020
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Description |
Biomass is a potential source for many of the fuels, chemicals, and materials that are needed by society and that are currently derived from petroleum. However, the sustainable biorefining of lignocellulosic biomass requires the valorization of all three of its components: cellulose, hemicellulose and lignin. Lignin has proven particularly difficult to valorize due to its recalcitrance. One approach has involved developing whole cell bacterial biocatalysts to upgrade the p-hydroxyphenyl, guaiacyl, and syringyl compounds resulting from lignin depolymerisation. Pseudomonas putida KT2440 is arguably the best characterized bacterial strain being developed as a biocatalyst, and is being engineered to transform syringyl monoaromatics. In this thesis, I investigated the role of two ring-cleavage dioxygenases, GalA and PcaHG, in the conversion of syringaldehyde-derived catechols to 2-pyrone-4,6-dicarboxylic acid (PDC), a precursor for biodegradable polyesters. PcaHG, protocatechuate 3,4-dioxygenase, cleaved gallate with an apparent specificity 20% that for protocatechuate. UV-vis spectroscopy and liquid chromatography-coupled mass spectrometry revealed that PcaHG cleaved gallate to 4-oxalomesaconate (OMA) and that this was subsequently transformed non-enzymatically to PDC. GalA cleaved 3-O-methyl gallate (3-MGA) to 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD), which cyclized non-enzymatically to PDC. However, 3-MGA potently inactivated GalA with a partition ratio >500-fold less than the partition ratio for gallate. Further analysis revealed that while GalA was inactivated by gallate and 3-MGA at similar rates, 3-MGA was cleaved several orders of magnitude less efficiently than gallate. The enzyme’s specificity for gallate was three-fold higher than previously reported. Overall, these results indicate that GalA is highly susceptible to inactivation and suggest that PcaHG is a better choice in engineering KT2440 to transform syringyl monoaromatics to PDC.
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Genre | |
Type | |
Language |
eng
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Date Available |
2020-09-10
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Provider |
Vancouver : University of British Columbia Library
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Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
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DOI |
10.14288/1.0394310
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2020-11
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Campus | |
Scholarly Level |
Graduate
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Rights URI | |
Aggregated Source Repository |
DSpace
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Rights
Attribution-NonCommercial-NoDerivatives 4.0 International