{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/18812"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/18812","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Engineering a Fungal L-Arabinose Pathway Towards the Co-Utilization of Hemicellulosic Sugars for Production of Xylitol","abstract":"The biosynthesis of value-added products has shown great promise in recent years due to the advances in molecular biology and protein engineering. Many advantages over chemical synthesis include high selectivity and specificity, increased yield, and function under milder conditions without the need for toxic metals, organic solvents, strong acids or bases, or high pressures and temperatures. However, naturally occurring enzymatic pathways are often times not particularly well suited towards obtaining high product yield due to the organism’s evolution not dependent on the production of such high levels of desired product. Xylitol is an attractive pentose sugar alcohol that has many applications in the food, pharmaceutical, and high-value based biochemical product industries. It is still, however, relatively expensive to produce, which prevents its economical integration into the marketplace. To expand the possible routes of xylitol synthesis, and provide a process which can utilize both hemicellulosic pentose sugars D-xylose and L-arabinose from renewable plant biomass as feed substrates, my thesis research focused on utilizing a fungal-derived biosynthetic pathway for production of xylitol. The pathway consists of two NADPH-dependent reductases, xylose reductase (XR) and L-xylulose reductase (LXR), and an NAD+-dependent L-arabinitol 4-dehydrogenase (LAD). The XR enzyme converts D-xylose directly to xylitol, while the three enzyme in tandem convert L-arabinose to xylitol. However, the cofactor imbalance presented with the pathway potentially makes nicotinamide regeneration difficult for production in vivo. After cloning and characterization of a highly active and stable NAD+-dependent L-arabinitol 4-dehydrogenase (LAD) from Neurospora crassa, subsequent engineering via rational design and directed evolution resulted in the isolation of the first known NADP+-dependent LAD enzyme. This novel engineered LAD was then introduced into the fungal xylitol pathway and expressed in a model organism, E. coli, and the effect of the cofactor specificity alteration was evaluated in the conversion of L-arabinose to xylitol. Further investigation of the cofactor balancing benefits were applied to xylitol dehydrogenase (XDH) for full redox balancing of the initial steps of the L-arabinose pathway, and investigations of the limiting steps in L-arabinose utilization conducted in S. cerevisiae and E. coli led to further proposed engineering of LAD.","abstract_html":"The biosynthesis of value-added products has shown great promise in recent years due to the advances in molecular biology and protein engineering. Many advantages over chemical synthesis include high selectivity and specificity, increased yield, and function under milder conditions without the need for toxic metals, organic solvents, strong acids or bases, or high pressures and temperatures. However, naturally occurring enzymatic pathways are often times not particularly well suited towards obtaining high product yield due to the organism’s evolution not dependent on the production of such high levels of desired product. Xylitol is an attractive pentose sugar alcohol that has many applications in the food, pharmaceutical, and high-value based biochemical product industries. It is still, however, relatively expensive to produce, which prevents its economical integration into the marketplace. To expand the possible routes of xylitol synthesis, and provide a process which can utilize both hemicellulosic pentose sugars D-xylose and L-arabinose from renewable plant biomass as feed substrates, my thesis research focused on utilizing a fungal-derived biosynthetic pathway for production of xylitol. The pathway consists of two NADPH-dependent reductases, xylose reductase (XR) and L-xylulose reductase (LXR), and an NAD+-dependent L-arabinitol 4-dehydrogenase (LAD). The XR enzyme converts D-xylose directly to xylitol, while the three enzyme in tandem convert L-arabinose to xylitol. However, the cofactor imbalance presented with the pathway potentially makes nicotinamide regeneration difficult for production in vivo. After cloning and characterization of a highly active and stable NAD+-dependent L-arabinitol 4-dehydrogenase (LAD) from Neurospora crassa, subsequent engineering via rational design and directed evolution resulted in the isolation of the first known NADP+-dependent LAD enzyme. This novel engineered LAD was then introduced into the fungal xylitol pathway and expressed in a model organism, E. coli, and the effect of the cofactor specificity alteration was evaluated in the conversion of L-arabinose to xylitol. Further investigation of the cofactor balancing benefits were applied to xylitol dehydrogenase (XDH) for full redox balancing of the initial steps of the L-arabinose pathway, and investigations of the limiting steps in L-arabinose utilization conducted in S. cerevisiae and E. coli led to further proposed engineering of LAD.","abstract_has_math":false,"creators":["Sullivan, Ryan P."],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemical Engineering","degree_department":null,"school":null,"contributors":["Zhao, Huimin","Pack, Daniel W.","Price, Nathan D.","Blaschek, Hans-Peter M."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2011,"date_issued":"2011-04-11T18:08:49Z","date_published":"2011-04-11T18:08:49Z","updated_at":"2026-07-22T22:25:11Z","subjects":["Xylitol production","L-arabinitol dehydrogenase","cofactor balancing","Neurospora crassa","nicotinamide regeneration","rational design","directed evolution","fungal arabinose pathway"],"languages":["en"],"rights":["Copyright 2009 Ryan Patrick Sullivan"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/18812","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhao, Huimin","Pack, Daniel W.","Price, Nathan D.","Blaschek, Hans-Peter M."]},{"key":"dc:creator","label":"Author","values":["Sullivan, Ryan P."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2011-04-11T18:08:49Z","2013-04-12T10:00:15Z","2009-12"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemical Engineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Xylitol production","L-arabinitol dehydrogenase","cofactor balancing","Neurospora crassa","nicotinamide regeneration","rational design","directed evolution","fungal arabinose pathway"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2009 Ryan Patrick Sullivan"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/18812"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The biosynthesis of value-added products has shown great promise in recent years due to the advances in molecular biology and protein engineering. Many advantages over chemical synthesis include high selectivity and specificity, increased yield, and function under milder conditions without the need for toxic metals, organic solvents, strong acids or bases, or high pressures and temperatures. However, naturally occurring enzymatic pathways are often times not particularly well suited towards obtaining high product yield due to the organism’s evolution not dependent on the production of such high levels of desired product. Xylitol is an attractive pentose sugar alcohol that has many applications in the food, pharmaceutical, and high-value based biochemical product industries. It is still, however, relatively expensive to produce, which prevents its economical integration into the marketplace. To expand the possible routes of xylitol synthesis, and provide a process which can utilize both hemicellulosic pentose sugars D-xylose and L-arabinose from renewable plant biomass as feed substrates, my thesis research focused on utilizing a fungal-derived biosynthetic pathway for production of xylitol. The pathway consists of two NADPH-dependent reductases, xylose reductase (XR) and L-xylulose reductase (LXR), and an NAD+-dependent L-arabinitol 4-dehydrogenase (LAD). The XR enzyme converts D-xylose directly to xylitol, while the three enzyme in tandem convert L-arabinose to xylitol. However, the cofactor imbalance presented with the pathway potentially makes nicotinamide regeneration difficult for production in vivo. After cloning and characterization of a highly active and stable NAD+-dependent L-arabinitol 4-dehydrogenase (LAD) from Neurospora crassa, subsequent engineering via rational design and directed evolution resulted in the isolation of the first known NADP+-dependent LAD enzyme. This novel engineered LAD was then introduced into the fungal xylitol pathway and expressed in a model organism, E. coli, and the effect of the cofactor specificity alteration was evaluated in the conversion of L-arabinose to xylitol. Further investigation of the cofactor balancing benefits were applied to xylitol dehydrogenase (XDH) for full redox balancing of the initial steps of the L-arabinose pathway, and investigations of the limiting steps in L-arabinose utilization conducted in S. cerevisiae and E. coli led to further proposed engineering of LAD.","Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2009-12-04T21:35:56Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 1 Sullivan_Ryan.pdf: 2483877 bytes, checksum: 081a5b5e24afe93a96fccaaaec741f79 (MD5)","Made available in DSpace on 2011-04-11T18:08:49Z (GMT). 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Many advantages over chemical synthesis include high selectivity and specificity, increased yield, and function under milder conditions without the need for toxic metals, organic solvents, strong acids or bases, or high pressures and temperatures. However, naturally occurring enzymatic pathways are often times not particularly well suited towards obtaining high product yield due to the organism’s evolution not dependent on the production of such high levels of desired product. Xylitol is an attractive pentose sugar alcohol that has many applications in the food, pharmaceutical, and high-value based biochemical product industries. It is still, however, relatively expensive to produce, which prevents its economical integration into the marketplace. To expand the possible routes of xylitol synthesis, and provide a process which can utilize both hemicellulosic pentose sugars D-xylose and L-arabinose from renewable plant biomass as feed substrates, my thesis research focused on utilizing a fungal-derived biosynthetic pathway for production of xylitol. The pathway consists of two NADPH-dependent reductases, xylose reductase (XR) and L-xylulose reductase (LXR), and an NAD+-dependent L-arabinitol 4-dehydrogenase (LAD). The XR enzyme converts D-xylose directly to xylitol, while the three enzyme in tandem convert L-arabinose to xylitol. However, the cofactor imbalance presented with the pathway potentially makes nicotinamide regeneration difficult for production in vivo. After cloning and characterization of a highly active and stable NAD+-dependent L-arabinitol 4-dehydrogenase (LAD) from Neurospora crassa, subsequent engineering via rational design and directed evolution resulted in the isolation of the first known NADP+-dependent LAD enzyme. This novel engineered LAD was then introduced into the fungal xylitol pathway and expressed in a model organism, E. coli, and the effect of the cofactor specificity alteration was evaluated in the conversion of L-arabinose to xylitol. Further investigation of the cofactor balancing benefits were applied to xylitol dehydrogenase (XDH) for full redox balancing of the initial steps of the L-arabinose pathway, and investigations of the limiting steps in L-arabinose utilization conducted in S. cerevisiae and E. coli led to further proposed engineering of LAD.","Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2009-12-04T21:35:56Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 1 Sullivan_Ryan.pdf: 2483877 bytes, checksum: 081a5b5e24afe93a96fccaaaec741f79 (MD5)","Made available in DSpace on 2011-04-11T18:08:49Z (GMT). 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