Progress 09/01/23 to 08/31/24

Outputs
Target Audience:This study further develops a non-model bacterium for optimized bioplastic production using lignocellulosic biomass as the renewable carbon source. The information gained through these efforts provides insight into R. palustris' PHA metabolism, allowing effective engineering of pathways for industrial applications. R. palustris' flexible metabolism offers many advantages over other organisms as it is one of the most metabolically diverse organisms. In general, identifying the phasins that R. palustris employs for PHA production and cell maintenance would be crucial to further engineering it for a broad range of applications even beyond PHA production, such as enhanced agricultural production or protein purification. Not only does this study provide insight into PHA metabolism, but it also adds revenue diversity for farmers by utilizing lignin as the renewable carbon source. Ultimately, this research aligns with several of AFRI's core priority areas (i.e. (1) Plant health and production and plant products, (4) Bioenergy, natural resources, and environment, (5) Agriculture systems and technology, and (6) Agriculture economics and rural communities) while simultaneously expanding my skills as a researcher and mentor. Changes/Problems:A major hurdle for this project during this period was migrating two labs to a new building at MS State. This set the project behind by at least six months due to having to pack, move, and reestablish the news labs, and thus I requested a no-cost extension. Autoclaves and other equipment had to be recertified for use as well. The large Percival chamber used to grow R. palustris under anaerobic conditions remained in the basement of the old building, so I had to essentially migrate a mini lab on a daily basis to grow the samples for proteomics. The growth chamber was inaccessible by May of 2024 due to the construction, so I had a deadline to grow all of the samplesby this time. It was challenging, and required me to come in to process samples even on the weekends and in the evenings. I am happy to report that I finished this successfully and that the proteomics paid off due to finding the predicted phasins. I also gained a lot of experience establishing labs, which included re-accomplishing the IBC approval for the project, bringing an old lab up to EHS approval, and reestablishing training and protocols for everyone in the new lab. What opportunities for training and professional development has the project provided?As a guest in Professor Shien Lu's laboratory at MS State, I have had the honor of mentoring several graduate students and twoResearch Associates. I have trained graduate students on statistical analyses in Excel and R programming, provided feedback on formal presentations, guided students through the scientific method, assisted with experimental methods, and helped brainstorm about professional decisions in their academic careers. How have the results been disseminated to communities of interest?The results of this first technical report have been briefed at the USDA PD meeting in Kansas City, MO in July, 2023. I have also communicated the project to all my colleagues, including Professor Rajib Saha at the University of Nebraska who specializes in this unique microbe. I also attended the American Institute for Chemical Engineers (AIChE) conference in November, 2023, which allowed me to share these results with conference attendees. What do you plan to do during the next reporting period to accomplish the goals?During this period, proteomics data identified that the four predicted phasins are indeed being produced by R. palustris under certain conditions. To shed more light on how they relate to other proteins involved in bioplastic production by R. palustris, a potential machine learning model is being created in collaboration with Professor Saha's lab from the University of Nebraska.

Impacts
What was accomplished under these goals? The project objectives 2 and 3 were modified to proteomics analysis instead of the immunogold labeling due to an issue with plasmid template mutations. Proteomics analysis serves to identify whether the predicted phasins are indeed being produced across the conditions, just as SDS-PAGE was going to verify. During this period, the PI coordinated with the Institute for Genomics, Biocomputing, and Biotechnology (IGBB) at MS State University to decipher proteomics methods catered to biomining for the predicted phasins. The PIgrew R. palustris across all of the conditions and carbon sources specified in the proposal, collected and washed the samples, and optimized protein extraction, quantification, and filtering protocols to prepare the samples for proteomics analyses.Samples were provided to IGBB in at least biological triplicates. IGBB performed in-solution digestion of the protein samples and subjected each sample to nano-LC -MS analysis via nano-ESI LTQ-Orbitrap Velos (Thermo). Excitingly, all of the predicted phasins were discovered in at least one of the conditions, as verified with at least biological duplicates! Dr. Brown reached out to a former lab at the University of Nebraska, Dr. Rajib Saha's lab, to decipher if a collaboration could happen with the proteomics data to generate a machine learning model for bioplastics production by R. palustris. Using data from this project combined with proteomics efforts from Dr. Saha's lab, a machine model could potentially be generated to better understand how phasins and other proteins are employed by R. palustris for bioplastic production.

Publications

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:This study further develops a non-model bacterium for optimized bioplastic production using lignocellulosic biomass as the renewable carbon source. The information gained through these efforts provides insight into R. palustris' PHA metabolism, allowing effective engineering of pathways for industrial applications. R. palustris' flexible metabolism offers many advantages over other organisms as it is one of the most metabolically diverse organisms. In general, identifying the phasins that R. palustris employs for PHA production and cell maintenance would be crucial to further engineering it for a broad range of applications even beyond PHA production, such as enhanced agricultural production or protein purification. Not only does this study provide insight into PHA metabolism, but it also adds revenue diversity for farmers by utilizing lignin as the renewable carbon source. Ultimately, this research aligns with several of AFRI's core priority areas (i.e. (1) Plant health and production and plant products, (4) Bioenergy, natural resources, and environment, (5) Agriculture systems and technology, and (6) Agriculture economics and rural communities) while simultaneously expanding my skills as a researcher and mentor. Changes/Problems:Due to my family receiving military orders to Mississippi, I coordinated to transfer the project from UNL to MS State University. I am grateful to everyone at NIFA, UNL, and MS State University for helping me achieve this transfer. Although the project start date was September 1, 2022, the department at MS State University did not formally receive approval and funding from the USDA for this project until April 25, 2023. I am thankful that MS State University let me use more than 90 days of overhead funds so that I did not have to wait much longer to begin the project. Moving to a new lab, certifying on new equipment, and performing all necessary required trainings also added more time to initiating the research. I am thankful that the department has entrusted me with being the PI of two labs (Lab 219 and 220), which I had to perform the necessary biosafety review boards to recertify the labs as BSL-2 facilities. I also had to route my USDA project through the biosafety department at MS State University to gain approval to perform the research. Since this project required specialized organisms and plasmids, I performed a Materials Transfer Agreement between UNL and MS State University to obtain the materials. It took a few weeks to regrow these organisms to ship them to MS State, and I am thankful for Professor Rajib Saha's lab at the University of Nebraska for accomplishing this. Dr. Cheryl Immethun was listed as a formal mentor to me on this project regarding the genetic engineering objectives, but she accepted an industry job and I also had to move the project to MS State University. I was able to coordinate for someone else in Professor Saha's Lab to mentor me regarding designing the plasmids for objectives two and three of this project. After we designed one plasmid together, I was confident in designing all others on my own. I will include the researcher on the publication as an author based on their contribution to the project. Since R. palustris requires specific environmental parameters to produce bioplastics (i.e., light and lack of oxygen), I had to engineer a new growth chamber that mimics the same parameters used in the previous lab at UNL. I coordinated with a professor in the department at MS State University to use his Percival growth chamber, and I fitted it with benchtop shakers that could hold anaerobic culture tubes. I also used a photosynthetically active radiation (PAR) meter to pinpoint the exact light intensity configuration that mimics the previously used growth chamber. Through this meticulous approach I was able to grow wild type R. palustris without statistically significant deviations in growth parameters compared to the previous growth chamber. I did incur more project expenses having to buy these benchtop shakers (about $3000), but I am still on track regarding the overall project budget. After further coordination with the Chemistry Department at MS State University regarding PHA quantification via gas chromatography, they do not currently have the specialized column necessary to achieve this analysis. However, I have coordinated with the Industrial Agricultural Products Center at UNL since I worked there previously, and they still have the appropriate GC-MS column to achieve the analysis. Thus, I plan to send my samples to the IAPC at UNL in the future when performing the PHA quantification analysis. I was recently informed that the communal -80°C freezer I store my samples in had completely thawed out, and thus my precious RNA samples were completely ruined. Thankfully, I was finishing up with the first objective of this project regarding the qRT-PCR analysis and already had 95% of the necessary data. I also had some remaining cDNA to finish the analysis. However, the RNA is no longer available should I need it in the future, and I would have to completely redo the entire process from growing the cultures to creating the cDNA. Another huge hurdle I will face during this project is that all faculty in Dorman Hall at MS State University were just informed that the entire building will be undergoing a renovation starting at the end of 2023, and everyone will be required to vacate. The current plan is that the entire department will relocate to the Hill Building at MS State University, but technicians are still deciphering if there will even be enough electricity to power all the equipment. The space will be more limited in the new building as well. It is unclear whether large chambers, like the Percival chamber I am currently using, will be accessible during the move. Thus, I am planning to grow and freeze as many samples as I can from the current growth chamber by the end of the year in case I will not have access to the chamber during the renovation. This will be a lot of work to crank out the samples as quickly as possible, but ultimately it seems like less work than having to completely start over in a new chamber. Despite the hurdles listed above, I am pleased to say that the project is on track, and I have accomplished the first objective. I look forward to completing objectives two and three due to the positive impact this could have on the scientific community and the ability to engineer this industrial microbe more efficiently. What opportunities for training and professional development has the project provided?Having to transfer the project from UNL to MS State gave me more insight into how grants are managed, and this experience will be invaluable for future endeavors. This project has also enabled me to lead two labs as the Principal Investigator in Dorman Hall at Mississippi State University. I successfully accomplished my first biosafety accreditation of the labs, which are Biosafety Level-2 labs due to pathogenic microbes. I'm excited to have passed the annual EHS inspections as well. I adopted a lab that was unorganized due to the former PI leaving very quickly, so I did a lot of work to bring it into good shape. I now also have experience conducting a Materials Transfer Agreement between universities since I had to transfer some materials from UNL to MS State. Having accomplished the first objective of this USDA project, I honed skills in qRT-PCR analysis, including refined methods and data analyses. As a guest in Professor Shien Lu's laboratory at MS State, I have had the honor of mentoring several graduate students and one Research Associate. I have trained graduate students on statistical analyses in Excel and R programming, provided feedback on formal presentations, guided students through the scientific method, assisted with experimental methods, and helped brainstorm about professional decisions in their academic careers. I have also provided insight into experimental designs for Professor Lu's projects based on my own experiences with HPLC and GC-MS analyses. Facilitating this USDA Postdoc Fellowship at MS State enabled me to be an affiliate with the university and take advantage of resources I would not otherwise have access to. I have decided to pursue entrepreneurship after the completion of this USDA project and have been working closely with MS State's Entrepreneurship Center. I intend on applying for the USDA SBIR grant, with an anticipated start date after the completion of this USDA Postdoc project. The Entrepreneurship Center has been invaluable to me regarding training, mentorship, and obtaining connections for potential subcontracts for the SBIR. I am passionate about circular economy, turning agricultural residues into biotechnology, and food security. I'm excited to make this a life-long career through entrepreneurship, and I aim to capitalize on the available resources while I can. How have the results been disseminated to communities of interest?The results of this first technical report have been briefed at the USDA PD meeting in Kansas City, MO this July. I have also communicated the project to all my colleagues, including Professor Rajib Saha at the University of Nebraska who specializes in this unique microbe. I made arrangements to attend the American Institute for Chemical Engineers (AIChE) conference in November, which will allow me to share these results with conference attendees. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting periods I plan to work on objectives two and three of the project. Specifically, I intend to build the designed plasmids that will enable me to append the mRFP protein onto the genes of interest as a fluorescent marker and ultimately for protein analysis. I will leverage methods that have been recently published regarding the genetic manipulation of this unique microbe, which include employing a suicide plasmid from E. coli. Once the plasmids are built and transformed into E. coli, I can harvest the plasmids and have them sequence verified by Eurofins Genomics to ensure the correct sequence was obtained. I will then need to repeat this procedure in R. palustris. After the new R. palustris strains are developed that contain the mrfp gene, I will then be able to perform a number of tests including (i) growing all strains to assess any changes in growth, (ii) validating changes in fluorescence, (ii) performing SDS-PAGE to visualize the proteins, (iii) conducting immunoblot analysis, (iv) coordinating immunogold labeling via transmission electron microscopy, (v) validating that PHA production is not impacted by the addition of this mrfp gene, (vi) and potentially conducting fluorescent microscopy if resources allow.

Impacts
What was accomplished under these goals? During thisfirst reporting period I have accomplished the first objective of this project, which is to analyze changes in gene expression for the four predicted phasins over time and on different carbon sources. Since Ihad to migrate the project from the University of Nebraska-Lincoln (UNL) to Mississippi State University, I initially had to engineer a new system to mimic the growing conditions that R. palustris requires to produce polyhydroxyalkanoates (PHAs). I was able to modify a Percival lighted growth chamber to fit the lighting and temperature requirements by installing two benchtop shakers into the chamber and assessing the lighting conditions with a photosynthetically active radiation (PAR) meter. I conducted a growth scan of wild type R. palustris to assess deviations from the previous chamber we were using to the new one at Mississippi State, and there was no statistical difference in the logarithmic growth pattern. I then grew R. palustris on p-coumarate or acetate to mid-exponential phase, starved the cells of nitrogen to induce bioplastic production, and kept growing the samples for another five days. I harvested RNA at the mid exponential, Day 1, and Day 5 time points to compare gene expression as nitrogen starvation progresses. I also grew wild type R. palustris in aerobic conditions on p-coumarate as a control since it does not produce bioplastics under aerobic conditions. Having worked extensively on a transcriptomics project involving RNA from R. palustris previously, I knew that RNA extraction was challenging for this organism. I engineered a new method for more efficient and reliable RNA extraction compared to what we have done previously (https://star-protocols.cell.com/protocols/2645). The optimized method I developed employs the Qiagen RNA Plus extraction kit and saves about six hours of time while also yielding more consistent high-quality RNA. The specific details of this method will be shared in the publication for others to use. All RNA was assessed for degradation via bleach gels, and only samples with strong double bands were used for cDNA conversion. All cDNA was tested with the 16SrRNA housekeeping primer to assure quality cDNA was being used for further qPCR assessment. For gene expression analysis, all oligomers were designed using BioCyc and SnapGene, and ordered from Eurofins Genomics. Oligomers were first tested via RT-PCR to obtain optimum primer concentrations, which were thenused for further assessments in qRT-PCR. Primer efficiencies for each primer at the optimum concentrations were obtained using serial-dilutedcDNA from samples grown on p-coumarate at mid-exponential growth phase. Only primers with a primer efficiencybetween 90 - 110% were used to ensure reliable comparisonwith the 16SrRNA housekeeping gene. All oligomers, their concentrations, and their efficiencies will be included in a detailed table in the publication for others to access. Data was analyzed to obtain the fold change (2^-ΔΔCt) of the genes relative to the 16SrRNA housekeeping gene, using aerobic expression as the control. Although I specified only exploring the gene expressions for the predicted phasins for objective one of the project, I also incorporated the intracellular Poly(3-hydroxybutyrate) (PHB) depolymerase gene as a control and to compare its expression across conditions. The expression of PHB Depolymerase is controlled by the cell to hydrolyzed PHB when needed, essentially breaking it down to be used for other metabolic functions. Please find the graph below for a visual depiction of the average fold changes and standard deviations. Interestingly, the RPA0089 and RPA3770 genes yieldedhigher expressions under nitrogen starvation comparedto the other two predicted phasin genes (RPA4138 and RPA4137), suggesting RPA0089 and RPA3770 could code for the dominant phasins involved in PHA production by R. palustris. In general, the expression of all genes, phasins and depolymerase, are decreased when using acetate as the carbon source instead of p-coumarate. The expression of PHB Depolymerase is higher at the mid-exponential phase of both the p-coumarate and acetate carbon sources, indicating the cells are activating PHB depolymerase to prevent the buildup of PHB during mid-exponential phase. Once nitrogen starvation is introduced, the cells begin to generate more PHB as a carbon and redox sink, and the gene expression of PHB depolymerase is decreased for the nitrogen-starved samples. This data provides the first analysis of gene expression for the four bioinformatically predicted phasins employed by R. palustris for PHA production and provides a nice foundation for future research. ? Objective 2 of this project will enable a deeper assessment of the protein expressions associated with these predicted phasin genes under different conditions via SDS-PAGE. Objective 3 will then allow me to analyze where the proteins are localized in the cells (if at all) via the immunogold labeling with transmission electron microscopy.

Publications