Progress 01/01/22 to 12/31/24
Outputs
Target Audience:• Potential future collaborators in crop sciences • Undergraduate and graduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has allowed for both training activities and professional development. Training activities have included various lab techniques with most notable being specific training on HPLC operation, microbalance operation, primer design, plasmid construction, and proper sterile technique. Additionally, training has included use of computational tools: KBase and the Holland Computing Center at UNL. Professional development has included attending and presenting at the Metabolic Engineering 15 conference. How have the results been disseminated to communities of interest?Results to this point have been disseminated to communities of interest through conference presentations. A final dissertation has also been published but is being held for one year while a manuscript is published. 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 world is in need of sustainable chemical production rather than relying on non-sustainable sources such as petroleum. A solution can be found in bioproduction which can produce target chemicals from lignocellulosic biomass, thus adding the benefit of boosting the value of agricultural feedstocks. In order to increase the application of bioproduction, further research is needed to expand the repertoire of organisms available for industrial use. This project seeks to develop the non-model bacterium, Paraburkholderia sacchari, as a bioproduction platform for biodegradable plastic production. This would replace petroleum-based plastic. This bacterium is already a natural producer of two bioplastics- polyhydroxybutyrate (PHB) and polybydroxyvalerate (PHV). When both of these are produced at the same time, a co-polymer, PHBV, is formed. Previous studies have shown that PHBV has characteristics that make it more competitive with petroleum-based plastics including high tensile strength, malleability, and thermal working window. Therefore, the specific goal of this project is to improve the PHBV yield possible when growing P. sacchari on a cheap feedstock, corn kernel fiber. This would provide a source of biodegradable plastic and simultaneously boost the value of currently low value agricultural byproducts. Dianna Morris, the principal investigator on this project, used two main strategies to improve bioplastic production by this bacterium. The first was to overexpress a gene which should increase the efficiency with which P. sacchari can convert a key growth medium component, propionate, into PHV. This strategy has shown a 45% improvement in PHV production. While this is a significant result, the propionate in the medium is expensive when considering large scale production, and does show a negative impact on cell growth. Therefore, the second strategy has been to introduce a gene set from E. coli which would remove the need for propionate by making PHV through a different metabolic route that starts with sugars instead. This strategy alone has not worked to produce PHV, which led Morris to explore the thermodynamic characteristics of the metabolic pathways by using a max-min driving force (MDF) model. The mdf model incorporates all the metabolic pathways that lead from glucose and propionate to PHB and PHV (35 reactions total), ranges of concentrations for each metabolite, as well as the delta G values for each reaction which represent if the reaction is favorable in the forward or reverse direction and HOW favorable. By running an optimization algorithm on this model, the output shows the most likely path of metabolites through the pathway. Morris was able to elucidate that internally sourced propionyl-coA was being recycled back to acetyl-CoA and subsequently PHB rather than progressing through the PHV production pathway. This finding supports the proposed plan to knock out the competing methyl citrate cycle and remove the ability to recycle the propionyl-CoA to PHB. Unfortunately, attempts to create a knockout were unsuccessful. In parallel with this work, Morris has also worked more on a genome-scale metabolic model of P. sacchari. This is a computational model that takes all the genes known to be in P. sacchari and predicts metabolic pathway structure and capabilities. To date, a draft model has been made and all the predicted metabolic pathways, however many of the pathways were unable to be validated. These results do show some promise in this for turning P. sacchari into a PHBV production platform and demonstrates the potential for the use of non-model organisms in bioproduction. However, the difficulties encountered in this projectalso demonstrates the need for more research to further develop this bacterium.This has great importance as harnessing non-model organisms would vastly increase the range of metabolic capabilities that could be used for industrial purposes. Objective 1: Develop a genome scale model of P. sacchari and predict metabolic engineering strategies for improved PHBV production 1. The annotated genome of P. sacchari was imported into KBase, annotated with RASTtk annotation, and an initial model was created. 2. This currated model has 1424 reactions and 1441 compounds, and produces biomass 3. These results mean the there are the necessary reactions included that allow for all components of biomass. However, this does not mean all reactions should be included, or that this is a complete picture of the existing reactions in P. sacchari. Manual curration is needed to assess the validity of reactions and to add any missing reactions that have been shown to be present in literature. 4. The key outcomes have been changes in knowledge as Morris has learned how to correctly import a genome into KBase, construct this initial model and is learning the programming language, GAMS, which is being used for the gap-filling stage. Morris also learned that many of the reactions cannot be validated in this non-model bacterium. Objective 2: Engineer P. sacchari to have improved PHBV production by both altering existing metabolic pathways and introducing heterologous genes 1. A strain transformed with the sbm gene was grown in 4 mL closed batch fermentation using media containing 1.83 g/L ammonium sulfate as the nitrogen source and run for 24 hours. The PHB and PHV measurements were accomplished using an alkaline digestion method for extraction and then quantified via HPLC. These demonstrated no production of PHV, but a 14% increase in PHB production. 2. A max-min driving force (MDF) model was created to assess the most thermodynamically favorable route of metabolitesthrough the core metabolism and PHB and PHV producttion pathways. Results suggest that the precursor to PHV is often recycled through the methyl citrate pathway toward PHB production. 3. A potential knockout strain was generated by transforming wild type P. sacchari with a suicide plasmid which included kanamycin resistance gene surrounded by gene regions homologous to sequences surrounding the prpC gene in P. sacchari's genome. This should allow for transformation to occur if P. sacchari is capable of this genome editing method. The homologous regions on the plasmid should bind to the genome and the natural mechanisms present should swap the prpC gene with the kan resistance gene. The suicide plasmid is then lost after generations as it does not contain an origin of replication. After growth in the absence of antibiotics to allow time for heterologous transformation to occur, cells were plated on kanamycin plates to select for those that had swapped the prpC gene for kanamycin. One colony grew, however PCR and gel visualization showed that the prpC gene was still present. This suggests that the kanamycin resistance gene was incorporated elsewhere on the genome and the knockout was unsuccessful. This route to creating a knockout wasabandoned.Morris reached out to the company "Creative Biogene" for assistance on generating a knockout using CRISPR, however the attempts were unsuccessful as well. 4. Data collected included gel electrophoresis images to confirm proper gene product size, sequencing data to confirm plasmid construction and insertion, and HPLC data to quantify PHB and PHV produced. Data generated included driving force predictions from the MDF model. 5. To date, key outcomes have been both changes in knowledge and changes in action. Changes in knowledge have included but are not limited to Morris learning how to design primers to extract target genes, how to trouble-shoot pcr parameters, necessary skills for sterile growth and extraction of cell samples, and how to operate the HPLC. Changes in action have included applying published media recipes and methods for PHBV extraction.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2024
Citation:
Morris, D. (2024). "Successes and Challenges Developing Non-Model Bacteria for Bioproduction Platforms" [Doctoral Dissertation, University of Nebraska-Lincoln]. Proquest.
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:• Potential future collaborators in crop sciences • Undergraduate and graduate students Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has allowed for both training activities and professional development. Training activities have included various lab techniques with most notable being specific training on HPLC operation, microbalance operation, primer design, plasmid construction, and proper sterile technique. Additionally, training has included use of computational tools: KBase and the Holland Computing Center at UNL. Professional development has included attending and presenting at the Metabolic Engineering 15 conference. How have the results been disseminated to communities of interest?Results to this point have been disseminated to communities of interest through conference presentations. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period I will: Knockout a metabolic pathway that competes for resources (methylcitrate cycle). Overexpress the sbm gene in the knockout strain. Collect HPLC data on the created strains to assess PHBV production. Gap-fill and further currate the metabolic model to then use to predict other metabolic engineering strategies I could try to improve PHBV production. Publish a research article on the findings to date.
Impacts
What was accomplished under these goals?
The world is in need of sustainable chemical production rather than relying on non-sustainable sources such as petroleum. A solution can be found in bioproduction which can produce target chemicals from lignocellulosic biomass, thus adding the benefit of boosting the value of agricultural feedstocks. In order to increase the application of bioproduction, further research is needed to expand the repertoire of organisms available for industrial use. This project seeks to develop the non-model bacterium, Paraburkholderia sacchari, as a bioproduction platform for biodegradable plastic production. This would replace petroleum-based plastic. This bacterium is already a natural producer of two bioplastics- polyhydroxybutyrate (PHB) and polybydroxyvalerate (PHV). When both of these are produced at the same time, a co-polymer, PHBV, is formed. Previous studies have shown that PHBV has characteristics that make it more competitive with petroleum-based plastics including high tensile strength, malleability, and thermal working window. Therefore, the specific goal of this project is to improve the PHBV yield possible when growing P. sacchari on a cheap feedstock, corn kernel fiber. This would provide a source of biodegradable plastic and simultaneously boost the value of currently low value agricultural byproducts. To date, Dianna Morris, the principal investigator on this project, has used two main strategies to improve bioplastic production by this bacterium. The first is to overexpress a gene which should increase the efficiency with which P. sacchari can convert a key growth medium component, propionate, into PHV. This strategy has shown a 45% improvement in PHV production. While this is a significant result, the propionate in the medium is expensive when considering large scale production, and does show a negative impact on cell growth. Therefore, the second strategy has been to introduce a gene set from E. coli which would remove the need for propionate by making PHV through a different metabolic route that starts with sugars instead. This strategy alone has not worked to produce PHV, which led Morris to explore the thermodynamic characteristics of the metabolic pathways by using a max-min driving force (MDF) model.The mdf model incorporates all the metabolic pathways that lead from glucose and propionate to PHB and PHV (35reactions total), ranges of concentrations for each metabolite,as well as the delta G values for each reaction which represent if the reaction is favorable in the forward or reverse direction and HOW favorable. By running an optimization algorithm on this model, the output shows the most likely path of metabolites through the pathway. Morris was able to elucidate that internally sourcedpropionyl-coA was being recycled back to acetyl-CoA and subsequently PHB rather than progressing through the PHV production pathway. This finding supports the proposed plan to knock out the competing methyl citrate cycle and remove the ability to recycle the propionyl-CoA to PHB. This is currently in progress. In parallel with this work, Morris has also worked more on a genome-scale metabolic model of P. sacchari. This is a computational model that takes all the genes known to be in P. sacchari and predicts metabolic pathway structure and capabilities. To date, a draft model has been made and all the predicted metabolic pathways are being validated. These results not only show the promise in this ongoing project for turning P. sacchari into a PHBV production platform, but, as this work is disseminated, demonstrates the potential for the use of non-model organisms in bioproduction. This has great importance as harnessing non-model organisms would vastly increase the range of metabolic capabilities that could be used for industrial purposes. Objective 1: Develop a genome scale model of P. sacchari and predict metabolic engineering strategies for improved PHBV production 1. The annotated genome of P. sacchari was imported into KBase, annotated with RASTtk annotation, and an initial model was created. 2. This curratedmodel has 1424reactions and 1441compounds, and produces biomass 3. These results mean the there are the necessary reactions included that allow for all components of biomass. However, this does not mean all reactions should be included, or that this is a complete picture of the existing reactions in P. sacchari. Manual curration is needed to assess the validity of reactions and to add any missing reactions that have been shown to be present in literature. 4. To date, the key outcomes have been changes in knowledge as Morris has learned how to correctly import a genome into KBase, construct this initial model and is learning the programming language, GAMS, which is being used for the gap-filling stage. Objective 2: Engineer P. sacchari to have improved PHBV production by both altering existing metabolic pathways and introducing heterologous genes 1. A strain transformed with the sbm gene was grown in 4 mL closed batch fermentationusing media containing 1.83g/L ammonium sulfate as the nitrogen source and run for 24 hours. The PHB and PHV measurements were accomplished using an alkaline digestion method for extraction and then quantified via HPLC. These demonstrated no production of PHV, but a 14% increase in PHB production. 2. A max-min driving force (MDF) model was created to assess the most thermodynamically favorable route of metabolites through the core metabolism and PHB and PHV producttion pathways. Results suggest that the precursor to PHV is often recycled through the methyl citrate pathway toward PHB production. 3. A potential knockout strain was generated by transforming wild type P. sacchari with a suicideplasmid which included kanamycin resistance gene surrounded by gene regions homologous to sequences surrounding the prpC gene in P. sacchari's genome. This should allow for transformation to occur if P. sacchari is capable of this genome editing method. The homologous regions on the plasmid should bind to the genome and the natural mechanisms present should swap the prpC gene with the kan resistance gene. The suicide plasmid is then lost after generations as it does not contain an origin of replication.After growth in the absence of antibiotics to allow time for heterologous transformation to occur, cells were plated on kanamycin plates to select for those that had swapped the prpC gene for kanamycin. One colony grew, however PCR and gel visualization showed that the prpC gene was still present. This suggests that the kanamycin resistance gene was incorporated elsewhere on the genome and the knockout was unsuccessful. This route to creating a knockout has been abandoned and CRISPR is now being explored. 4. Data collected included gel electrophoresis images to confirm proper gene product size, sequencing data to confirm plasmid construction and insertion, and HPLC data to quantify PHB and PHV produced. Data generated included driving force predictions from the MDF model. 5. To date, key outcomes have been both changes in knowledge and changes in action. Changes in knowledge have included but are not limited to Morris learning how to design primers to extract target genes, how to trouble-shoot pcr parameters, necessary skills for sterile growth and extraction of cell samples, and how to operate the HPLC. Changes in action have included applying published media recipes and methods for PHBV extraction.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Morris, D. (2023, November 11-15). Metabolic Engineering of Paraburkholderia Sacchari for Improved Bioplastic Production. [Poster]. Metabolic Engineering 15, Singapore.
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience: Potential future collaborators in crop sciences Undergraduate and graduate students Changes/Problems:I changed from taking PHBV measurements on GCMS to using HPLC instead. This is due to equipment availability issues. The GCMS I had planned to use broke down. However, I found a method to measure PHB and PHBV via HPLC instead and I have that equipment readily available. This does not change the overall plan ofthe project. What opportunities for training and professional development has the project provided?This project has allowed for both training activities and professional development. Training activities have included various lab techniques with most notable being specific training on HPLC operation, microbalance operation, primer design, plasmid construction, and proper sterile technique. Additionally, training has included use of computational tools: KBase and the Holland Computing Center at UNL. Professional development has included attending and presenting at the Central US Synthetic Biology Workshop and attending and presenting at the AIChE Annual Meeting. How have the results been disseminated to communities of interest?Results to this point have been disseminated to communities of interest through conference presentations. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period I will: Determine growth conditions and measure PHBV production in the sbm strain Knockout a metabolic pathway that competes for resources (methylcitrate cycle) Gap-fill and further currate the metabolic model to then use to predict other metabolic engineering strategies I could try to improve PHBV production
Impacts
What was accomplished under these goals?
The world is in need of sustainable chemical production rather than relying on non-sustainable sources such as petroleum. A solution can be found in bioproduction which can produce target chemicals from lignocellulosic biomass, thus adding the benefit of boosting the value of agricultural feedstocks. In order to increase the application of bioproduction, further research is needed to expand the repertoire of organisms available for industrial use. This project seeks to develop the non-model bacterium, Paraburkholderia sacchari, as a bioproduction platform for biodegradable plastic production. This would replace petroleum-based plastic. This bacterium is already a natural producer of two bioplastics- polyhydroxybutyrate (PHB) and polybydroxyvalerate (PHV). When both of these are produced at the same time, a co-polymer, PHBV, is formed. Previous studies have shown that PHBV has characteristics that make it more competitive with petroleum-based plastics including high tensile strength, malleability, and thermal working window. Therefore, the specific goal of this project is to improve the PHBV yield possible when growing P. sacchari on a cheap feedstock, corn kernel fiber. This would provide a source of biodegradable plastic and simultaneously boost the value of currently low value agricultural byproducts. To date, Dianna Morris, the principal investigator on this project, has used two main strategies to improve bioplastic production by this bacterium. The first is to overexpress a gene which should increase the efficiency with which P. sacchari can convert a key growth medium component, propionate, into PHV. This strategy has shown a 45% improvement in PHV production. While this is a significant result, the propionate in the medium is expensive when considering large scale production, and does show a negative impact on cell growth. Therefore, the second strategy has been to introduce a gene set from E. coli which would remove the need for propionate by making PHV through a different metabolic route that starts with sugars instead. Currently, this strategy is still being tuned, however the genes have been successfully inserted. In parallel with this work, Morris has also initiated a genome-scale metabolic model of P. sacchari. This is a computational model that takes all the genes known to be in P. sacchari and predicts metabolic pathway structure and capabilities. To date, a draft model has been made and all the predicted metabolic pathways are being validated. These results not only show the promise in this ongoing project for turning P. sacchari into a PHBV production platform, but, as this work is disseminated, demonstrates the potential for the use of non-model organisms in bioproduction. This has great importance as harnessing non-model organisms would vastly increase the range of metabolic capabilities that could be used for industrial purposes. Objective 1: Develop a genome scale model ofP. sacchariand predict metabolic engineering strategies for improved PHBV production The annotated genome of P. sacchari was imported into KBase and an initial model was created. This initial model has 146 reactions and 297 compounds, but cannot produce biomass. These results mean there are missing pathways that must be identified and added manually. This process is currently underway. To date, the key outcomes have been changes in knowledge as Morris has learned how to construct this initial model and is learning the programming language, GAMS, which is being used for the gap-filling stage. Objective 2: EngineerP. saccharito have improved PHBV production by both altering existing metabolic pathways and introducing heterologous genes An initial investigation of growth conditions was performed on wild type P. sacchari to generate a reasonable set of parameters for small scale fermentations. This was done by comparing levels of nitrogen starvation and fermentation durations as well as treatment and amount of cells needed for PHB extraction. At this stage, propionate was not added to the media, so just PHB was measured. Then target genes, pct from Cupriavidus necator and the sbm operon from E. coli, were extracted and inserted independently into plasmids. These were then transformed into P. sacchari and maintained using kanamycin as a selection antibiotic. The transformed cells, as well as a wild type control were grown in glucose and propionate as appropriate and PHB and PHV yields were measured. The measurements were accomplished using an alkaline digestion method for extraction and then quantified via HPLC. Data collected included gel electrophoresis images to confirm proper gene product size, sequencing data to confirm plasmid construction and insertion, growth data to compare transformed cell lines and wild type, and HPLC data to quantify PHB and PHV produced. In determining fermentation conditions, results indicate 4 mL closed batch fermentations using media containing 1.83 g/L ammonium sulfate as the nitrogen source and run for 24 hours yielded sufficient PHB following extraction from either fresh or frozen samples. Under these conditions, the titer was 3.6 g/L PHB which, although not as high as previously reported in bioreactor work with this bacterium, is high enough amount for comparison between planned engineered strains. Gene product length, plasmid sequences, and transformation into P. sacchari were all confirmed. Growth data demonstrated no significant difference between wild type and transformed cell lines. However, growth data did show an effect of propionate addition, with anything above 1 g/L having significant inhibition. In comparing wild type and transformed cell lines, PHBV for the strain with pct showed a 45% increase in PHV production. Data for the sbm strain is still being collected. To date, key outcomes have been both changes in knowledge and changes in action. Changes in knowledge have included but are not limited to Morris learning how to design primers to extract target genes, how to trouble-shoot pcr parameters, necessary skills for sterile growth and extraction of cell samples, and how to operate the HPLC. Changes in action have included applying published media recipes and methods for PHBV extraction.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Morris, D. & Saha, R. (2022, October 6-7). Use of Anderson promoters for improved bioplastic production in the non?model, Paraburkholderia sacchari. [Poster]. Central US Synthetic Biology Workshop, Madison, WI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Morris, D. (2022, November 13-18). Metabolic Engineering of Paraburkholderia Sacchari for Improved Bioplastic Co-Polymer Production. [Session Talk]. AIChE Annual Meeting, Phoenix, AZ.
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