Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762
Performing Department
Food and Animal Sciences
Non Technical Summary
Sustainable production of energy in the form of biofuels is a major global challenge as mankind faces an increasing demand for renewable energy, while there is an ongoing struggle to decrease dependency on fossil fuels. Lipid-based biofuels could become a sustainable source of bioenergy that will satisfy part of the demand for renewable energy. Second generation lipid-based biofuels (i.e., biodiesel) are produced from microbes with a high propensity for lipid production (i.e., oleaginous cells), but to date, there is no large-scale production process for microbial lipid-based fuels despite the intense research efforts. The major reason for this discrepancy is that oleaginous microbes are not cost-competitive in the production of biofuels. However, new tools for the genetic modification of oleaginous microbes and high-throughput methods that have been developed recently have facilitated the generation microbial strains that produce lipids more efficiently. In this project, we will make use of the recently developed tools and techniques to improve sustainable bioenergy production.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Sustainable production of energy in the form of biofuels is a major global challenge as mankind faces an increasing demand for renewable energy, while there is an ongoing struggle to decrease dependency on fossil fuels. Lipid-based biofuels could become a sustainable source of bioenergy that will satisfy part of the demand for renewable energy. Second generation lipid-based biofuels (i.e., biodiesel) are produced from microbes with high propensity for lipid production (i.e., oleaginous cells), but to date there is no large-scale production process for microbial lipid-based fuels despite the intense research efforts. The major reason for this discrepancy is that wild type oleaginous microbes are not cost-competitive in production of biofuels. However, new tools for the genetic manipulation of oleaginous microbes and high-throughput methods that have been developed recently have facilitated the generation microbial strains that produce lipids more efficiently. These developments have created opportunities to improve the existing bioenergy production processes. This proposal aims at this knowledge gap.In this project, we will make use of the recently developed tools and techniques to improve sustainable bioenergy production. To achieve this goal, we will build expertise in molecular food biotechnology by creating a critical mass of expertise across food science and biology to advance our understanding of lipid metabolism at the molecular level and use this knowledge to develop sustainable production processes for lipid-based biofuels. Molecular lipid biotechnology can be broadly defined as the integration of food biotechnology with lipid biology and omics sciences. This exciting research area of food biotechnology has benefited from the rapid advances in computational science and biotechnology and the increased availability of omics tools that have led to a better understanding of the lipid biosynthetic pathways and the regulatory mechanisms that govern lipid biosynthesis. These discoveries have fostered a rapid growth in areas, such as lipidomics, transcriptomics, nutrigenomics, etc. However, there is an apparent lack of multidisciplinary research programs in most 1890 institutions and especially at AAMU thatintegrate the above-mentioned disciplines to address the sustainability of lipid-based biofuels.This proposal seeks to fill this gap by establishing a research and training program that integrates expertise from food science (Dr. Fakas-AAMU), plant science (Dr. Sripathi-AAMU), biology (Dr. Paton-Vogt-Duquesne University), and microbial biotechnology (Dr. Papanikolaou-Agricultural University of Greece) to tackle challenges in sustainable bioenergy production. The new research program aligns well with the strategic plan of Alabama A&M University, where developing new undergraduate and graduate programs in new areas relevant to the 21th century and providing students with global and interdisciplinary perspectives, is a top priority. The department of Food and Animal Sciences at AAMU has already taken the first steps towards this direction by establishing a research program in food biotechnology. The next phase of this building process will be the establishment of this international multidisciplinary research program which will open underexplored avenues for collaboration and provide AAMU students with much needed international exposure.Specific objective 1: Identify novel genes that mediate triacylglycerol (TAG) biosynthesis in Y. lipolytica by molecular genetics and lipidomicsSpecific objective 2: Comparative transcriptomics of Y. lipolyticaSpecific objective 3: Train the new generation of food biotechnologists by experiential learning and by formal training in Molecular Food Biotechnology
Project Methods
Specific objective 1: Identify novel genes that mediate triacylglycerol (TAG) biosynthesis in Y. lipolytica by molecular genetics and lipidomicsWe propose to identify the enzyme(s) involved in TAG biosynthesis in Y. lipolytica by an integrated approach that will combine lipid biochemistry and molecular genetics with lipidomics. The proposed approach will make use of the expertise developed in the previous funding period and build on the current infrastructure to develop capacity in Y. lipolytica genetic manipulation and lipidomics. The use of Y. lipolytica as a model for oleaginous cells has facilitated the characterization of the biosynthetic pathways that mediate and regulate lipid accumulation. Using yeast as a model for oleaginous cells (e.g., adipocytes) allows for the generation of "knockout" and other mutant strains in a fraction of the time needed to do analogous work in higher eukaryotes (e.g., mice). Knockouts of lipid biosynthetic genes may alter the lipid metabolic fluxes which result in changes in the amounts of lipid molecular species that reflect the function of the gene(s) that has been disrupted. In contrast, overexpression of key lipid biosynthetic genes (e.g., PAH1) is expected to drive the lipid pathway towards the biosynthesis of TAG. Monitoring these changes has been a challenge for lipid biotechnologists because of the lack of proper instrumentation which would allow the quantification of the subtle fluctuations in lipid amounts. The use of the powerful tool of lipidomics has revolutionized the field of lipid biotechnology, because it provides a snapshot of most lipid molecular species which allows for monitoring changes in specific lipid pools. The use of lipidomics is increasing because of the decreasing costs and increased availability of the instrumentation (i.e., mass spectrometer). Especially, lipidomics is expected to transform the research in food science because it can elucidate the interactions between nutrition, food components, and human metabolism. However, expertise in lipidomics is lacking at AAMU and other HBCUs.Specific objective 2: Undertake comparative transcriptomics of Y. lipolyticaWe propose to analyze and compare the transcriptomes of the knockouts that lack one or more genes involved in lipid metabolism and gain insights into the regulation of lipid biosynthesis. The transcriptome is the total mRNA in a cell that reflects the genes that are actively expressed. Comparative transcriptomics is the analysis and comparison of transcriptomes which reveals changes in the expression of genes under certain conditions. For example, knockout of genes that are thought to be involved in lipid metabolism may affect the transcription of key lipid metabolic genes that regulate the flux of metabolites in the lipid biosynthetic pathway. In Y. lipolytica, the very few transcriptomic studies mostly focused on the comparative analysis of the transcriptome during the lipid production phase to identify genes that are differentially expressed in cells that have shifted their metabolism to lipid accumulation. These studies have revealed a complex regulatory network of genes that are differentially expressed during biomass production and during lipid accumulation. These genes are involved in amino acid and sugar metabolism, protein and nucleic acid metabolism and the regulation of transcription and translation among many metabolic processes. The complexity of these analyses can be simplified by looking for specific changes in the mRNA levels of lipid biosynthetic genes caused by a certain mutation. In this research, we will analyze and compare the transcriptomes of wild type and knockout Y. lipolytica strains that lack one or more phosphatidate phosphatase genes. We will then look for genes whose expression is changed in the knockouts and more specifically for genes that may be involved in lipid biosynthesis. These analyses may reveal genes that complement the knockouts, for example by supplying biosynthetic precursors (e.g., DAG) for lipid biosynthesis.Specific objective 3: Train the new generation of food biotechnologists by experiential learning and by formal training in Molecular Food BiotechnologyEarly exposure to research is "one of the most powerful instructional tools" (NSF, 1989) that has been linked to increased student retention, especially in STEM fields. Experiential learning through meritorious research is one of the cornerstones of the Food Biotechnology research program at AAMU. Ten undergraduate students have trained in the PD's lab in the past four years and two juniors have stated that their experience has sparked their interest for graduate school. We will build on this tradition by training two undergraduate students in molecular food biotechnology. Also, we will encourage the students to participate to the "Interdisciplinary Biology Research & Community-Engaged Learning" program at Duquesne University where Dr. Paton-Vogt serves as a student advisor. This formal collaboration between AAMU and Duquesne is in its first year and one undergraduate underrepresented student will participate in the program this year. The exposure to research is expected to increase student retention in the Food Science program and build the basis for ensuring a steady supply of students that will join the Food Biotechnology research team as undergraduate and then as graduate students.Two graduate (master's) underrepresented students will be recruited to the new molecular food biotechnology concentration and will be trained in molecular biotechnology, lipidomics and transcriptomics by Dr. Fakas and Dr. Sripathi. One graduate student will work on the construction of the mutant (knockout) strains and the lipidomics analysis with Dr. Fakas and Dr. Paton-Vogt, and the other on the transcriptomics research and the construction of gene networks with Dr. Sripathi. During their studies, students will be encouraged to pursue internships with biotechnology companies and the governmental agencies that will expose them to the challenges that biotechnology faces today and to some of the latest advances in the field. Part of their training will include an international component that will expose students to other cultures and make them aware of the common challenges that the global food supply chain faces. Our collaboration with the Food Science Department at the Agricultural University of Greece offers a unique opportunity for cultural exchange because it hosts students from many European countries (e.g., Spain, Portugal, Slovakia). Dr. Papanikolaou who has developed an extensive network of collaborations in the EU will host our students. We anticipate that after the completion of their studies, the students will enter the PhD in Food Science program.