{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/82402"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/82402","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Protein and Pathway Engineering for Biosynthesis or Aromatic Compounds","abstract":"A type I fatty acid synthase, FAS-B from Brevibacterium ammoniagenes, and a type I polyketide synthase, 6-MSAS from Penicillium patulum, were successfully engineered via rational pathway design to synthesize TAL in vivo with a maximal yield of 1.7 g/L. The direct biosynthesis of phloroglucinol from D-glucose was achieved by the discovery of a novel type III polyketide synthase, PhlD, from Pseudomonas fluorescens. Expression of PhlD in E. coli led to the production of phloroglucinol in vivo with an estimated yield of 0.7 g/L under the shake flask condition and 20 g/L using a continuous fermentation for 5 days. In vitro assay revealed that PhlD catalyzed the synthesis of phloroglucinol from three molecules of malonyl-CoA, and exhibited broad substrate specificity, which was successfully altered via saturation mutagenesis guided by a homology structural model of PhlD. To improve phloroglucinol production for industrial manufacturing, a two-prone approach was undertaken. Firstly, directed evolution was carried out to improve the poor properties of PhlD. Family shuffling of 52 PhlD homologous genes using synthetic DNA shuffling technique gave two improved PhlD mutants, which showed 4 fold increased enzymatic catalytic efficiency and/or thermostability. Secondly, the availability of substrate for phloroglucinol synthesis, malonyl-CoA in the host E. coli, was improved via metabolic engineering. The combination of various metabolic engineering strategies led to a total of 15 fold elevated malonyl-CoA level in E. coli. Ultimately, when combined, these two manipulations would translate synergistically into improvement in phloroglucinol production.","abstract_html":"A type I fatty acid synthase, FAS-B from Brevibacterium ammoniagenes, and a type I polyketide synthase, 6-MSAS from Penicillium patulum, were successfully engineered via rational pathway design to synthesize TAL in vivo with a maximal yield of 1.7 g/L. The direct biosynthesis of phloroglucinol from D-glucose was achieved by the discovery of a novel type III polyketide synthase, PhlD, from Pseudomonas fluorescens. Expression of PhlD in E. coli led to the production of phloroglucinol in vivo with an estimated yield of 0.7 g/L under the shake flask condition and 20 g/L using a continuous fermentation for 5 days. In vitro assay revealed that PhlD catalyzed the synthesis of phloroglucinol from three molecules of malonyl-CoA, and exhibited broad substrate specificity, which was successfully altered via saturation mutagenesis guided by a homology structural model of PhlD. To improve phloroglucinol production for industrial manufacturing, a two-prone approach was undertaken. Firstly, directed evolution was carried out to improve the poor properties of PhlD. Family shuffling of 52 PhlD homologous genes using synthetic DNA shuffling technique gave two improved PhlD mutants, which showed 4 fold increased enzymatic catalytic efficiency and/or thermostability. Secondly, the availability of substrate for phloroglucinol synthesis, malonyl-CoA in the host E. coli, was improved via metabolic engineering. The combination of various metabolic engineering strategies led to a total of 15 fold elevated malonyl-CoA level in E. coli. Ultimately, when combined, these two manipulations would translate synergistically into improvement in phloroglucinol production.","abstract_has_math":false,"creators":["Zha, Wenjuan"],"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"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-25T20:43:31Z","date_published":"2015-09-25T20:43:31Z","updated_at":"2026-07-22T22:26:18Z","subjects":["Engineering, Chemical"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI3290452"],"render_values":[{"text":"(MiAaPQ)AAI3290452","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/82402","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhao, Huimin"]},{"key":"dc:creator","label":"Author","values":["Zha, Wenjuan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-25T20:43:31Z","10000-01-01","2007"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"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":["Engineering, Chemical"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/82402","(MiAaPQ)AAI3290452"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["A type I fatty acid synthase, FAS-B from Brevibacterium ammoniagenes, and a type I polyketide synthase, 6-MSAS from Penicillium patulum, were successfully engineered via rational pathway design to synthesize TAL in vivo with a maximal yield of 1.7 g/L. The direct biosynthesis of phloroglucinol from D-glucose was achieved by the discovery of a novel type III polyketide synthase, PhlD, from Pseudomonas fluorescens. Expression of PhlD in E. coli led to the production of phloroglucinol in vivo with an estimated yield of 0.7 g/L under the shake flask condition and 20 g/L using a continuous fermentation for 5 days. In vitro assay revealed that PhlD catalyzed the synthesis of phloroglucinol from three molecules of malonyl-CoA, and exhibited broad substrate specificity, which was successfully altered via saturation mutagenesis guided by a homology structural model of PhlD. To improve phloroglucinol production for industrial manufacturing, a two-prone approach was undertaken. Firstly, directed evolution was carried out to improve the poor properties of PhlD. Family shuffling of 52 PhlD homologous genes using synthetic DNA shuffling technique gave two improved PhlD mutants, which showed 4 fold increased enzymatic catalytic efficiency and/or thermostability. Secondly, the availability of substrate for phloroglucinol synthesis, malonyl-CoA in the host E. coli, was improved via metabolic engineering. The combination of various metabolic engineering strategies led to a total of 15 fold elevated malonyl-CoA level in E. coli. Ultimately, when combined, these two manipulations would translate synergistically into improvement in phloroglucinol production.","Made available in DSpace on 2015-09-25T20:43:31Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 3290452.pdf: 3201987 bytes, checksum: 922e9300a3eac66ceffe34eefe3d9da3 (MD5) Previous issue date: 2007","Embargo set by: Seth Robbins for item 83683 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","155 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007."]},{"key":"dc:title","label":"Title","values":["Protein and Pathway Engineering for Biosynthesis or Aromatic Compounds"]}]}],"canonical_facts":{"dc:contributor":["Zhao, Huimin"],"dc:creator":["Zha, Wenjuan"],"dc:date":["2015-09-25T20:43:31Z","10000-01-01","2007"],"dc:description":["A type I fatty acid synthase, FAS-B from Brevibacterium ammoniagenes, and a type I polyketide synthase, 6-MSAS from Penicillium patulum, were successfully engineered via rational pathway design to synthesize TAL in vivo with a maximal yield of 1.7 g/L. The direct biosynthesis of phloroglucinol from D-glucose was achieved by the discovery of a novel type III polyketide synthase, PhlD, from Pseudomonas fluorescens. Expression of PhlD in E. coli led to the production of phloroglucinol in vivo with an estimated yield of 0.7 g/L under the shake flask condition and 20 g/L using a continuous fermentation for 5 days. In vitro assay revealed that PhlD catalyzed the synthesis of phloroglucinol from three molecules of malonyl-CoA, and exhibited broad substrate specificity, which was successfully altered via saturation mutagenesis guided by a homology structural model of PhlD. To improve phloroglucinol production for industrial manufacturing, a two-prone approach was undertaken. Firstly, directed evolution was carried out to improve the poor properties of PhlD. Family shuffling of 52 PhlD homologous genes using synthetic DNA shuffling technique gave two improved PhlD mutants, which showed 4 fold increased enzymatic catalytic efficiency and/or thermostability. Secondly, the availability of substrate for phloroglucinol synthesis, malonyl-CoA in the host E. coli, was improved via metabolic engineering. The combination of various metabolic engineering strategies led to a total of 15 fold elevated malonyl-CoA level in E. coli. Ultimately, when combined, these two manipulations would translate synergistically into improvement in phloroglucinol production.","Made available in DSpace on 2015-09-25T20:43:31Z (GMT). No. of bitstreams: 2 license.txt: 4848 bytes, checksum: 96035ab3f5e1c23cc7138a224ce498bd (MD5) 3290452.pdf: 3201987 bytes, checksum: 922e9300a3eac66ceffe34eefe3d9da3 (MD5) Previous issue date: 2007","Embargo set by: Seth Robbins for item 83683 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","155 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007."],"dc:identifier":["http://hdl.handle.net/2142/82402","(MiAaPQ)AAI3290452"],"dc:language":["eng"],"dc:subject":["Engineering, Chemical"],"dc:title":["Protein and Pathway Engineering for Biosynthesis or Aromatic Compounds"],"dc:type":["text"],"thesis:degree_discipline":["Chemical Engineering"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:18Z"}