{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/125795"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/125795","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Exploring Issatchenkia orientalis’ potential as a platform organism for the production of organic acids","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","abstract_has_math":false,"creators":["Tran, Vinh Gia"],"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","Rao, Christopher V","Shukla, Diwakar","Lu, Ting"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-07-09","date_published":"2024-07-09","updated_at":"2026-07-22T22:25:02Z","subjects":["Metabolic Engineering","Synthetic Biology"],"languages":["en","eng"],"rights":["Copyright 2024 Vinh Tran"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/125795","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhao, Huimin","Rao, Christopher V","Shukla, Diwakar","Lu, Ting"]},{"key":"dc:creator","label":"Author","values":["Tran, Vinh Gia"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2024-07-09","2024-08"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"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":["Metabolic Engineering","Synthetic Biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2024 Vinh Tran"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/125795"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","The student, Vinh Tran, accepted the attached license on 2024-07-08 at 01:56.","The student, Vinh Tran, submitted this Dissertation for approval on 2024-07-08 at 01:59.","This Dissertation was approved for publication on 2024-07-09 at 13:29.","DSpace SAF Submission Ingestion Package generated from Vireo submission #20976 on 2025-02-04 at 21:25:35","With advances in synthetic biology and metabolic engineering, microbial cell factories are being extensively developed as sustainable alternatives to the traditional petroleum-based production processes, enabling the conversion of renewable biomass into beneficial chemicals relevant chemical, agriculture, food, pharmaceutical, and energy industries. A large group of chemicals that can be produced from engineered microorganisms is organic acids. As highly valued products, organic acids have been broadly applied in a wide range of industries. For efficient and economical microbial production of organic acids, the use of chassis with superior tolerance to low pH conditions and high concentrations of organic acids are preferred to reduce the use of neutralizing agents during fermentation and to simplify downstream separation and purification processes. Nevertheless, bacteria, such as Escherichia coli, can only grow well at neutral pH conditions, while organic acid production by engineered yeasts suffers from low titers, yields, and productivities. Thus, non-model organisms that can tolerate highly acidic conditions have been increasingly explored as platforms for the production of organic acids. Issatchenkia orientalis is a non-conventional yeast that can be engineered to produce organic acids. I. orientalis is tolerant to several stresses, such as high osmotic pressure, high temperature, and high salt concentration. However, the most attractive feature of this species is its ability to survive and proliferate at extremely low pH. It was used in ethanol fermentation at pH of 2 and in the saccharification of lignocellulosic biomasses hydrolyzed by sulfuric acid. Moreover, I. orientalis was also engineered to produce organic acids, such as lactic acid, malic acid, and citramalic acid. Nevertheless, the lack of efficient genetic tools and its poorly characterized metabolism have significantly prohibited extensive metabolic engineering efforts in I. orientalis. Therefore, much remains to be explored and investigated to further employ I. orientalis as a platform organism to produce organic acids at industrially relevant scales. We have aimed to establish efficient genetic tools to facilitate the genetic manipulation of I. orientalis and to employ them in the metabolic engineering of this non-conventional yeast for the production of organic acids. First, we report that the autonomously replicating sequence from Saccharomyces cerevisiae was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1′-tRNA promoter for single guide RNA expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. Next, we further improved the production of succinic acid (SA), a top value-added chemical, in I. orientalis by deletion of byproduct pathways, transport engineering, and expanding the substrate scope. The resulting strains could produce SA at a titer of 109.5 g/L, a yield of 0.63 g/g glucose equivalent, and a productivity of 0.54 g/L/h using minimal medium containing glucose and glycerol as well as at a titer of 104.6 g/L, a yield of 0.63 g/g glucose equivalent, and a productivity of 1.25 g/L/h using sugarcane juice medium in fed-batch fermentations in bench-top reactors at pH 3. We also scaled up our fermentation process to an industrial pilot scale in batch mode with a scaling factor of 300× and achieve SA production at a titer of 63.1 g/L, a yield of 0.50 g/g glucose equivalent, and a productivity of 0.66 g/L/h using sugarcane juice medium at pH 3. Furthermore, we performed biorefinery design, simulation, techno-economic analysis, and life cycle assessment under uncertainty to characterize the financial viability and environmental benefits of the developed SA production pathway. Sensitivity analyses were also conducted to identify key drivers of production costs and environmental impacts for prioritization of future research, development, and deployment directions. While our engineered I. orientalis strains could produce more than 100 g/L of SA in fed-batch fermentations, the yields achieved by our strains were lower than those obtained by engineered bacteria, such as E. coli and Mannheimia succiniciproducens. The production of highly reduced molecules, such as SA, and acetyl-CoA-derived molecules are particularly more challenging in yeasts compared to bacteria due to compartmentation of NADH and acetyl-CoA metabolisms. To leverage the high pyruvate oxidation capability of Issatchenkia orientalis, we localized its pyruvate dehydrogenase to the cytosol to enable higher provisions of cytosolic NADH and acetyl-CoA. The production of SA was improved by 1.19-fold, while the production of acetyl-CoA-derived citramalic acid and triacetic acid lactone was enhanced by 1.22- and 4.35-folds, respectively. Further cofactor engineering was performed by coupling a reductive tricarboxylic acid pathway with a glyoxylate shunt, which bypasses the NADH-dependent malate dehydrogenase and supplies the former pathway with more reducing power. SA production reached a titer of 104 g/L and yield of 0.82 g/g glucose, exceeding the yield of 0.66 g/g glucose that is biologically limited by the shortage of cytosolic NADH. Finally, we further attempted to expand the genetic toolbox in I. orientalis. First, I. orientalis was found to harbor endogenous genes for RNA interference that enabled up to 67% repression in GFP expression. Next, the GAL regulon from S. cerevisiae was expressed in I. orientalis, enabling galactose-induced gene expression from a hybrid promoter containing the upstream activating sequence of the promoter of GAL1 of S. cerevisiae and the core region of FBA1 promoter of I. orientalis. Finally, a 3-hydroxypropionic acid biosensor was developed by using the g1655 promoter, which was highly upregulated in cells exposed to high concentration of 3-hydroxypropionic acid, to drive the expression of the transcription factor mmsR. The 3-HP biosensor, which contained GFP expressed by the ∆UAS-O1-FBA1p hybrid promoter and mmsR-VPR driven by the g1655 promoter, could respond to both 3-HP supplemented extracellularly to a medium and 3-HP produced intracellularly by a 3-HP-producing I. orientalis strain."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Exploring Issatchenkia orientalis’ potential as a platform organism for the production of organic acids"]}]}],"canonical_facts":{"dc:contributor":["Zhao, Huimin","Rao, Christopher V","Shukla, Diwakar","Lu, Ting"],"dc:creator":["Tran, Vinh Gia"],"dc:date":["2024-07-09","2024-08"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","The student, Vinh Tran, accepted the attached license on 2024-07-08 at 01:56.","The student, Vinh Tran, submitted this Dissertation for approval on 2024-07-08 at 01:59.","This Dissertation was approved for publication on 2024-07-09 at 13:29.","DSpace SAF Submission Ingestion Package generated from Vireo submission #20976 on 2025-02-04 at 21:25:35","With advances in synthetic biology and metabolic engineering, microbial cell factories are being extensively developed as sustainable alternatives to the traditional petroleum-based production processes, enabling the conversion of renewable biomass into beneficial chemicals relevant chemical, agriculture, food, pharmaceutical, and energy industries. A large group of chemicals that can be produced from engineered microorganisms is organic acids. As highly valued products, organic acids have been broadly applied in a wide range of industries. For efficient and economical microbial production of organic acids, the use of chassis with superior tolerance to low pH conditions and high concentrations of organic acids are preferred to reduce the use of neutralizing agents during fermentation and to simplify downstream separation and purification processes. Nevertheless, bacteria, such as Escherichia coli, can only grow well at neutral pH conditions, while organic acid production by engineered yeasts suffers from low titers, yields, and productivities. Thus, non-model organisms that can tolerate highly acidic conditions have been increasingly explored as platforms for the production of organic acids. Issatchenkia orientalis is a non-conventional yeast that can be engineered to produce organic acids. I. orientalis is tolerant to several stresses, such as high osmotic pressure, high temperature, and high salt concentration. However, the most attractive feature of this species is its ability to survive and proliferate at extremely low pH. It was used in ethanol fermentation at pH of 2 and in the saccharification of lignocellulosic biomasses hydrolyzed by sulfuric acid. Moreover, I. orientalis was also engineered to produce organic acids, such as lactic acid, malic acid, and citramalic acid. Nevertheless, the lack of efficient genetic tools and its poorly characterized metabolism have significantly prohibited extensive metabolic engineering efforts in I. orientalis. Therefore, much remains to be explored and investigated to further employ I. orientalis as a platform organism to produce organic acids at industrially relevant scales. We have aimed to establish efficient genetic tools to facilitate the genetic manipulation of I. orientalis and to employ them in the metabolic engineering of this non-conventional yeast for the production of organic acids. First, we report that the autonomously replicating sequence from Saccharomyces cerevisiae was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1′-tRNA promoter for single guide RNA expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. Next, we further improved the production of succinic acid (SA), a top value-added chemical, in I. orientalis by deletion of byproduct pathways, transport engineering, and expanding the substrate scope. The resulting strains could produce SA at a titer of 109.5 g/L, a yield of 0.63 g/g glucose equivalent, and a productivity of 0.54 g/L/h using minimal medium containing glucose and glycerol as well as at a titer of 104.6 g/L, a yield of 0.63 g/g glucose equivalent, and a productivity of 1.25 g/L/h using sugarcane juice medium in fed-batch fermentations in bench-top reactors at pH 3. We also scaled up our fermentation process to an industrial pilot scale in batch mode with a scaling factor of 300× and achieve SA production at a titer of 63.1 g/L, a yield of 0.50 g/g glucose equivalent, and a productivity of 0.66 g/L/h using sugarcane juice medium at pH 3. Furthermore, we performed biorefinery design, simulation, techno-economic analysis, and life cycle assessment under uncertainty to characterize the financial viability and environmental benefits of the developed SA production pathway. Sensitivity analyses were also conducted to identify key drivers of production costs and environmental impacts for prioritization of future research, development, and deployment directions. While our engineered I. orientalis strains could produce more than 100 g/L of SA in fed-batch fermentations, the yields achieved by our strains were lower than those obtained by engineered bacteria, such as E. coli and Mannheimia succiniciproducens. The production of highly reduced molecules, such as SA, and acetyl-CoA-derived molecules are particularly more challenging in yeasts compared to bacteria due to compartmentation of NADH and acetyl-CoA metabolisms. To leverage the high pyruvate oxidation capability of Issatchenkia orientalis, we localized its pyruvate dehydrogenase to the cytosol to enable higher provisions of cytosolic NADH and acetyl-CoA. The production of SA was improved by 1.19-fold, while the production of acetyl-CoA-derived citramalic acid and triacetic acid lactone was enhanced by 1.22- and 4.35-folds, respectively. Further cofactor engineering was performed by coupling a reductive tricarboxylic acid pathway with a glyoxylate shunt, which bypasses the NADH-dependent malate dehydrogenase and supplies the former pathway with more reducing power. SA production reached a titer of 104 g/L and yield of 0.82 g/g glucose, exceeding the yield of 0.66 g/g glucose that is biologically limited by the shortage of cytosolic NADH. Finally, we further attempted to expand the genetic toolbox in I. orientalis. First, I. orientalis was found to harbor endogenous genes for RNA interference that enabled up to 67% repression in GFP expression. Next, the GAL regulon from S. cerevisiae was expressed in I. orientalis, enabling galactose-induced gene expression from a hybrid promoter containing the upstream activating sequence of the promoter of GAL1 of S. cerevisiae and the core region of FBA1 promoter of I. orientalis. Finally, a 3-hydroxypropionic acid biosensor was developed by using the g1655 promoter, which was highly upregulated in cells exposed to high concentration of 3-hydroxypropionic acid, to drive the expression of the transcription factor mmsR. The 3-HP biosensor, which contained GFP expressed by the ∆UAS-O1-FBA1p hybrid promoter and mmsR-VPR driven by the g1655 promoter, could respond to both 3-HP supplemented extracellularly to a medium and 3-HP produced intracellularly by a 3-HP-producing I. orientalis strain."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/125795"],"dc:language":["en","eng"],"dc:rights":["Copyright 2024 Vinh Tran"],"dc:subject":["Metabolic Engineering","Synthetic Biology"],"dc:title":["Exploring Issatchenkia orientalis’ potential as a platform organism for the production of organic acids"],"dc:type":["text","Thesis"],"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":"2026-07-22T22:25:02Z"}