Progress 06/15/22 to 06/14/24
Outputs
Target Audience: Scientists and engineers in the academic community, primarily specialists in metabolic engineering, synthetic biology, and fermentation science pursuing the redesign of biological systems for useful purposes. Industry in the fields of foods and bioproducts developing bioprocesses for food ingredients or value-added chemicals with increased yield and productivity. Students and scientists in the Department of Food Science at Purdue University. Students and scientists in the Schoolof Chemical Engineeringat Purdue University. Industry upcycling agricultural residuesand biomasses to produce value-added products. Changes/Problems: To expand the utilization of the engineered acid-tolerant I. orientalis strain from this study, we conducted research to produce not only 3-HP but also L-lactic acid as value-added products, as outlined in Objective 2 of the accomplishments. Additionally, we utilized hemp stalk, an acetate-rich biomass, along with CPW to expand the diversity of low-cost biomass in Objective 2. What opportunities for training and professional development has the project provided? During the development of Cas9-based genetic tools, four graduatestudents effectively learned CRISPR/Cas9 technology, providing an excellent educational opportunity. We hosted high school students for the Agriculture Science Research Institute (ASRI) program. ASRI, a week-long residential program, engages students interested in STEM fields by offering an immersive experience in Purdue's campus life while also providing an opportunity to earn college credit. High school students participated in hands-on experiments based on this project throughout the week (June, 2023). How have the results been disseminated to communities of interest?The outcomes of this project were disseminated through seven poster presentations and four seminars, showcasing the excellence of the research conducted. Five posters were presented at conferences at Purdue University, and two were presented at the Society for Industrial Microbiology and Biotechnology (SIMB). Additionally, four seminars were hosted at Kyungpook National University in South Korea and at Purdue University's Whistler Center for Carbohydrate Research. The research results from this project are being prepared for the publication of three papers this year. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The proposed project is innovative in linking non-glucose fermenting pathways and diversified final bioproducts to stress-tolerant non-conventional microbes using CRISPR/Cas9 genome engineering. Ultimately, the reconfigured central metabolism in stress-tolerant yeast, Issatchenkia orientalis,will significantly expand the versatility of renewable resource systems for innovative biotechnological applications. Objective 1.Develop a stress-tolerant yeast I. orientalis platform for the simultaneous co-utilization of non-glucose carbon sources. Task 1.1: Constructing a platform strain utilizing multiple substrates simultaneously 1)Major activities completed / experiments conducted Establish an engineered yeast platform using Saccharomyces cerevisiae as the primary model We ultimately incorporated the pathways for xylose, arabinose, cellobiose, and galacturonic acid into a Saccharomycescerevisiae platform. Optimum pH levels for galacturonic acid (pH 3.5) and cellobiose (pH 6) differ, affecting their consumption rates in a mixture of carbon sources. To enhance cellobiose consumption at low pH (3.5), we conducted adaptive laboratory evolution experiments. We identified two beneficial mutations (M363I, L402H) in the cellodextrin transporter (cdt-1) gene by comparing the DNA sequences of beta-glucosidase (gh1-1) and cdt-1 with the wild-type strain. The engineered S. cerevisiae strain expressing the mutant cdt-1 exhibited a 20-fold increase (0.791 g/L/h) in cellobiose consumption in the presence of galacturonic acid, compared to the parental strain (0.026 g/L/h) at 48 hours. Construct engineered I. orientalis capable of metabolizing of non-glucose carbon sources We selected four strong promoters (IoTDH3p, IoGPM1p, IoTEF1p, IoFBA1p) and four terminators (IoMDH1t, IoPDC1t, IoINO1t, IoENO2t) from I. orientalis, as referenced in VG Tran et al., 2019, mSphere. These selected promoters and terminators were obtained through PCR amplification from the genomic DNA of I. orientalis NRRL Y-27441. Essential genes for the metabolic pathways of xylose, L-arabinose, D-galacturonic acid, and cellobiose were amplified from other microorganisms (Pichia stipitis, Ambrosiozyma monospora, Aspergillus niger, Trichoderma reesei, Neurospora crassa) through PCR. 2)Key outcomes or other accomplishments realized We developed a platform using S. cerevisiae as the primary model organism, enabling the simultaneous utilization of multiple substrates (xylose, arabinose, galacturonic acid, and cellobiose) derived from both lignocellulosic and pectin-rich biomass Drawing from our results with S. cerevisiae, we constructed an engineered I. orientalis strain capable of consuming four non-conventional carbon sources: xylose, arabinose, galacturonic acid and cellobiose. Task 1.2:Comparative analysis of extracellular and intracellular metabolite profiles 1) Major activities completed / experiments conducted Conduct flask fermentation experiments to quantify extracellular metabolites When cultured with mixed carbon sources (40 g/L xylose, 40 g/L L-arabinose, 20 g/L D-galacturonic acid), the engineered I. orientalis strain consumed all of the xylose but showed significantly slower consumption of D-galacturonic acid (7.7 g/L) and L-arabinose (4.2 g/L). YP medium containing 40 g/L cellobiose was used to confirm that the I. orientalis strainharboring the expression vector could consume cellobiose. The engineered I. orientalis consumed 11.8 g/L of cellobiose over 60 hours. 2) Discussion of results We observed that galacturonic acid consumption by the engineered I. orientalisstrain was significantly low when cultured on D-galacturonic acid as the sole carbon source. Since NADPH is required for D-galacturonic acid metabolism, we hypothesize that this issue may be due to a redox imbalance within the cells. To test this hypothesis, we plan to assess the accumulation of 2-keto-3-deoxy-L-galactonate, an indicator of D-galacturonic acid metabolism, via intracellular metabolite analysis using GC/MS. Since cellobiose consumption by I. orientalis was lower than that of the S. cerevisiae strain, we plan to conduct adaptive laboratory evolution using cellobiose as the sole carbon source. Objective 2: Validate the effect of multiplex metabolic pathways on bioproducts in an engineered I. orientalis platform. Task 2.1: Validating co-utilization of mixed non-glucose carbon sources in I. orientalis for enhanced acetyl-CoA-mediated bioproduction 1) Major activities completed / experiments conducted We selected 3-hydroxypropionic acid (3-HP) and L-lactic acid as target products to produce value-added compounds from non-conventional carbon sources in the engineered I. orientalis strain. Production of 3-HP from xylose and acetate in the engineered I. orientalis strain Three essential genes (panD, BAPAT, ydfG) synthesized were introduced into the genome of the I. orientalisstrain, creating the IoDY01H strain. The 3-HP production was measured in YP medium with 40 g/L xylose and varying concentrations of acetate (0 to 15 g/L). The engineered strain showed inhibited cell growth starting at 7.5 g/L acetate and did not grow at 15 g/L acetate. However, 3-HP production increased at 5 g/L acetate (5.6 g/L) compared to without acetate (4.8 g/L). GC-MS analysis confirmed that introducing the 3-HP metabolic pathway in the I. orientalis strain increased the accumulation of intracellular β-alanine and citrate, enhancing the metabolic flux related to 3-HP with acetate as a substrate. Production of L-lactic acid from non-conventional carbon sources (glucose, xylose, and D-galacturonic acid) in the engineered I. orientalis strain To produce L-lactic acid, a PCR-amplified LDH gene from Lactobacillus acidophilus was introduced into the genome of the I.orientalisstrain, creating the IoDY03L strain. Under sole carbon conditions, the IoDY03L strain produced L-lactic acid from glucose (14.1 g/L) and xylose (16.5 g/L), but not from D-galacturonic acid. However, under mixed sugar conditions with D-galacturonic acid and another carbon source (glucose or xylose), the substrates were consumed simultaneously, producing L-lactic acid at 15.6 g/L and 18.0 g/L, respectively. 2) Key outcomes or other accomplishments realized We developed an engineered I. orientalisstrain that produces high-value products, such as L-lactic acid and 3-HP, from non-conventional carbon sources. Task 2.2: Fermentation of carbon sources in biomass hydrolysates by engineered I. orientalis 1) Major activities completed / experiments conducted Production of 3-HP from acetate-rich biomass in the engineered I. orientalis strain We used hemp stalk as a substrate to produce 3-HP from acetate-rich biomass. The industrial raw hemp stalk, composed of 59.4% cellulose, 18.9% hemicellulose, and 20.2% lignin, was harvested at the Throckmorton Purdue Agricultural Center, then dried and milled. To determine the optimal fermentation process for 3-HP production, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were performed, using cellulase (30 units/g biomass) and hemicellulase (30 FBGU/g biomass) as saccharification enzymes. Results showed that SHF produced more 3-HP (8.7 g/L) than SSF (7.7 g/L). Production of L-lactic acid from pectin-rich biomass in the engineered I. orientalis strain We used citrus peel waste (CPW) as a substrate to produce L-lactic acid from pectin-rich biomass. The 10% (w/v) CPW hydrolysate containing distilled water was sterilized by autoclaving and saccharified using cellulase (C, 30 units/g biomass), hemicellulase (H, 30 FBGU/g biomass), and pectinase (P, 30 IU/g biomass). SSF was performed with four combinations of saccharification enzymes (P, CP, HP, and CHP). As a result, 8.3, 8.2, 13.1, and 14.2 g/L of L-lactic acid were produced under P, CP, HP, and CHP conditions over 72 hours, respectively.
Publications
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Jeong D, Kim SR, Oh EJ. 2023. Metabolic engineering of yeast for fermentation of L-rhamnose. Whistler Center for Carbohydrate Research Update Series, Purdue University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Jeong, D., Kuanyshev, N., Jin, Y. S., Oh, E. J. 2023. Development of acid-tolerant yeast Issatchenkia orientalis for the production of biofuel from lignocellulosic biomass. Society for Industrial Microbiology and Biotechnology Annual Meeting, Minneapolis, MN, July 30-August 2.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Lee, D., Oh, E. J., 2023. Development of microbial platforms capable of fermenting non-conventional substrates for enhanced production of value-added chemicals. Society for Industrial Microbiology and Biotechnology Annual Meeting, Minneapolis, MN, July 30-August 2.
- Type:
Other
Status:
Published
Year Published:
2024
Citation:
Jeong D, Park S, Evelina G, Kim S, Park H, Lee JM, Kim SK, Kim IJ, Kim SR, Oh EJ. 2024. Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae. Fermentation Frenzy, Purdue University Networking Session.
- Type:
Other
Status:
Published
Year Published:
2024
Citation:
Jeong D, Oh EJ. 2024. Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae. Whistler Center for Carbohydrate Research Update Series, Purdue University.
- Type:
Other
Status:
Published
Year Published:
2024
Citation:
Jeong D, Park S, Evelina G, Kim S, Park H, Lee JM, Kim SK, Kim IJ, Kim SR, Oh EJ. 2024. Bioconversion of citrus waste into mucic acid by xylose-fermenting Saccharomyces cerevisiae. Whistler Center for Carbohydrate Research annual Board Meeting, Purdue University.
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Progress 06/15/22 to 06/14/23
Outputs
Target Audience:Target Audiences Scientists and engineers in the academic community, primarily specialists in metabolic engineering, synthetic biology, and fermentation science pursuing the redesign of biological systems for useful purposes. Industry in the fields of foods and bioproducts developing bioprocesses for food ingredients or value-added chemicals with increased yield and productivity. Students and scientistsin the Department of Food Science at Purdue University. Efforts The outcomes of this project were disseminated through threeposter presentations and two seminars, showcasing the excellence of the research conducted. Specifically, the threeposters were presented at conferences at Purdue University. In addition, two seminars were hosted at Kyungpook National University in South Korea and at Purdue University's Whistler Center for Carbohydrate Research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the development of Cas9-based genetic tools, two newly admitted doctoral students effectively learned CRISPR/Cas9 technology, providing an excellent educational opportunity. How have the results been disseminated to communities of interest?The outcomes of this project were disseminated through threeposter presentations and two seminars, showcasing the excellence of the research conducted. Specifically, the threeposters were presented at conferences at Purdue University. In addition, two seminars were hosted at Kyungpook National University in South Korea and at Purdue University's Whistler Center for Carbohydrate Research. What do you plan to do during the next reporting period to accomplish the goals?We plan to engineer I.orientalis strains that can simultaneously consume four types of non-conventional carbon sources (cellobiose, xylose, L-arabinose, and D-galacturonic acid) and introduce additional heterologous pathways for the production of high-value chemicals, such as 3-Hydroxypropionic acid (3-HP). Additionally, we will investigate the production of 3-HP using biomass hydrolysates, such as those derived from citrus peel waste, as a substrate. To understand the issue of reduced D-galacturonic acid consumption, we intend to carry out intracellular metabolite analysis and apply metabolic flux analysis strategies. We will further scrutinize flux maps of strains, specifically engineered for target bioproduct production, when grown on mixtures of carbon sources representative of biomass hydrolysates, using metabolic flux analysis.
Impacts
What was accomplished under these goals?
Traditional strain-based metabolic engineering often results in uncompetitive bioprocessing due to high energy and water use, low productivity in hydrolysates, and significant downstream separation costs. To address this, the engineering of non-conventional microbes has recently emerged as a promising strategy. The native stress tolerance of these non-conventional strains is being leveraged to establish new, high-performing chassis for biosynthesis. While previous studies suggest that the co-expression of heterologous metabolic pathways can function synergistically in model microbial platforms, these platforms face two fundamental limitations: 1) the presence of inhibitors in biomass hydrolysates impedes the bioconversion efficiency of model microbial platforms, and 2) such studies typically target bioethanol production, which accumulates significantly as a byproduct during sugar fermentation. The synergistic combination of metabolic pathways is key to successful bioprocessing of renewable biomass, but its operation under industrially relevant conditions requiring stress tolerance, high productivity, and diverse target bioproducts is poorly understood. Thus, the proposed project is innovative in linking non-glucose fermenting pathways and diversified final bioproducts to stress-tolerant non-conventional microbes using CRISPR/Cas9 genome engineering. The successful completion of our proposal will establish a first-of-its-kind, versatile non-model yeast platform for next-generation chemical biosynthesis. Ultimately, the reconfigured central metabolism in stress-tolerant yeast will significantly expand the versatility of renewable resource systems for innovative biotechnological applications. The objective for the first year is to develop a stress-tolerant yeast Issatchenkia orientalis platform capable of simultaneously co-utilizing non-glucose carbon sources. To meet this annual goal, we accomplished two detailed tasksand also carried out preliminary research in preparation for the upcoming year. Objective 1: Develop a stress-tolerant yeast I. orientalis platform for the simultaneous co-utilization of non-glucose carbon sources. Task 1.1: Constructing a platform strain utilizing multiple substrates simultaneously. 1) Major activities completed / experiments conducted Establish an engineered yeast platform using Saccharomyces cerevisiae as the primary model We ultimately incorporated the pathways for xylose, arabinose, cellobiose, and galacturonic acid into a S. cerevisiae platform. Optimum pH levels for galacturonic acid (pH 3.5) and cellobiose (pH 6) differ, which can affect their consumption rates in a mixture of various carbon sources. To enhance cellobiose consumption rates under low pH conditions (pH 3.5), we carried out adaptive laboratory evolution experiments. This involved continuous serial subcultures in YP medium containing 40 g/L of cellobiose and 20 g/L galacturonic acid intended to introduce random genetic mutations into the engineered S. cerevisiae. The evolution process was driven by selective pressure based on mutations associated with improved growth and consumption rates. After the adaptive laboratory evolution, we identified two beneficial mutations (M363I, L402H) within the cdt-1 gene. These were identified by comparing the DNA sequences of gh1-1 and cdt-1 with those of the wild-type strain. The engineered S. cerevisiae strain, expressing the isolated mutant cdt-1, exhibited a 20-fold increase in the cellobiose consumption rate in the presence of galacturonic acid, compared to the parental strain. Develop Cas9-based genome editing tools for I. orientalis We obtained the pCast plasmid, which is utilized to express Cas9 and gRNA for genome editing in the I. orientalis strain, as reported in YG Lee et al., 2022, J. Agric. Food Chem., from Dr. Yong-Su Jin. We replaced the ClonNAT cassette gene (natMX6) within this plasmid with a hygromycin B cassette gene (HygR) through Gibson assembly, and subsequently named it the pCastH plasmid. We then confirmed that the I. orientalis NRRL Y-27441 strain, when harboring the pCastH, could grow in YPD medium (containing 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) with over 350 ug/ml of hygromycin B antibiotic. As a result, we successfully developed a novel Cas9-based genetic tool that expresses the hygromycin B marker for I. orientalis strains. Construct engineered I. orientalis capable of consuming three non-conventional carbon sources We selected four strong promoters (IoTDH3p, IoGPM1p, IoTEF1p, IoFBA1p) and four terminators (IoMDH1t, IoPDC1t, IoINO1t, IoENO2t) from I. orientalis, as referenced in VG Tran et al., 2019, mSphere. These selected promoters and terminators were obtained through PCR amplification from the genomic DNA of I. orientalis NRRL Y-27441. Essential genes for the metabolic pathways of xylose, L-arabinose, and D-galacturonic acid were amplified from other microorganisms (Pichia stipitis, Ambrosiozyma monospora, Aspergillus niger, and Trichoderma reesei) through PCR. The expression cassettes for these three pathways were integrated into the genome of I. orientalis using the CRISPR/Cas9 system, following the procedure we used with the S. cerevisiae system. 2) Key outcomes or other accomplishments realized We developed a platform using S. cerevisiae as the primary model organism, enabling the simultaneous utilization of multiple substrates (xylose, arabinose, galacturonic acid, and cellobiose) derived from both lignocellulosic and pectin-rich biomass. Drawing from our results with S. cerevisiae, we constructed an engineered I. orientalis strain capable of consuming three non-conventional carbon sources: xylose, arabinose, and galacturonic acid. Task 1.2: Comparative analysis of extracellular and intracellular metabolite profiles. 1) Major activities completed / experiments conducted Conduct flask fermentation experiments to quantify extracellular metabolites We performed fermentation in YP medium containing 40 g/L xylose, 40 g/L L-arabinose, and 20 g/L D-galacturonic acid to confirm that the engineered I. orientalis strains could consume these as sole carbon sources. The wild-type I. orientalis NRRL Y-27441 strain was used as a control. As a result, the control strain was unable to consume xylose, L-arabinose, and D-galacturonic acid as a sole carbon source. However, the engineered I. orientalis IoDY01 strain consumed all of the xylose in 12 hours, producing 6.9 g/L xylitol, 1.2 g/L glycerol, and 10.0 g/L ethanol. In the L-arabinose fermentation, the engineered IoDY02 strain consumed 13.9 g/L L-arabinose over 72 hours but did not produce anything. Furthermore, in the D-galacturonic acid fermentation, the engineered IoDY03 strain consumed 1.8 g/L D-galacturonic acid, but the consumption rate was significantly reduced after 12 hours. 2) Discussion of results We observed that galacturonic acid consumption by the engineered IoDY03 strain was significantly low when cultured on D-galacturonic acid as a sole carbon source. As the NADPH cofactor is required for D-galacturonic acid metabolism, we hypothesize that this observed issue may be due to a redox imbalance within the cells. To test this hypothesis, we plan to assess the accumulation of 2-keto-3-deoxy-L-galactonate, a previously reported indicator of D-galacturonic acid metabolism, via intracellular metabolite analysis using GC/MS. 3) Key outcomes or other accomplishments realized The engineered I. orientalis strains could consume xylose, L-arabinose, and D-galacturonic acid as sole carbon sources. Objective 2:Validate the effect of multiplex metabolic pathways on bioproducts in an engineered I. orientalis platform. We plan to complete objective 2 in year 2.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Lee D, Oh EJ. 2022. Development of microbial platforms capable of fermenting non-conventional substrates for enhanced production of value-added chemicals. Industrial Associates Meeting, Purdue University Poster Competition. (2nd prize)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Lee D, Oh EJ. 2023. Development of microbial platforms capable of fermenting non-conventional substrates for enhanced production of value-added chemicals. Fermentation Frenzy, Purdue University Networking Session.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Jung D, Ye S, Park H, Kim SR, Oh EJ. 2023. Metabolic engineering for the utilization of pectin-rich Biomass by Saccharomyces cerevisiae. Fermentation Frenzy, Purdue University Networking Session.
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Lee D, Oh EJ. 2023. Development of microbial platforms capable of fermenting non-conventional substrates for enhanced production of value-added chemicals. Whistler Center for Carbohydrate Research Update Series, Purdue University.
- Type:
Other
Status:
Other
Year Published:
2023
Citation:
Jung D, Oh EJ. 2023. Development of non-conventional yeast for producing high value-added chemicals from pectin-rich biomass. Kyungpook National University, Daegu, South Korea.
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