Progress 09/01/15 to 11/30/18
Outputs
Target Audience:Postdoctoral associate, graduate student, undergraduate students and high school students. One postdoctoral associate, two African-American graduatestudents were trained in laborary techniques in Food Biotechnology. Twenty African-American undergraduate students received formal classroom training in biochemistry and biotechnology.Thirty African-American high school students (juniors and seniors) attended a two-week summer school in Food Biotechnology. The students attended lectures on Food Science and Food Biotechnology and were trained on molecular biology techniques. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities: One postdoctoral associate, two graduate and two undergraduate students were trained by the PD in Food Biotechnology. The training included: laboratory techniques, preparation of research presentations and preparation of manuscripts for publication. The graduate students took the Introduction to Biotechnology and Food Enzymes courses that were developed as part of the new curriculum supported by this grant. The undergraduate students took the Agricultural Biochemistry course which introduced them to the Food Biotechnology curriculum. Professional development: The PD was invited to give a lecture at Duquesne University where he presented the results of this research. The PD also attended the CBG project director's meeting. The PD and the Ph.D. student attended the annual Experimental Biology conference twice where the student presented his research. The graduate student received a travel award from the American Society of Biochemistry and Molecular Biology to present his work. There he had the opportunity to attend networking events and meet with scientists and potential employers. The graduate student also spent two months at Rutgers University where he was trained by our collaborator Dr. George Carman, a renowned lipid biochemist. Additionally, the graduate studentpresented his work at the annual Institute of Food Technologists conference where he won 1st place in the Biotechnology section; as well as the master's studentpresented her work at this same annual Institute of Food Technologists conference. How have the results been disseminated to communities of interest?The results of our research have been disseminated to the scientific community by presentations in conferences and publications in scientific journals. Also, the results of the summer programs were presented to an audience that included family members of the high school students, who were mostly African-Americans. The presentations enhanced their understanding of Food Science as a discipline and the importance of this research for the well-being of the community. They were also made aware of Food Science as a field and the different careers available to graduates with Food Science degrees. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
It is common knowledge that there is an underused talent pool among minorities which can contribute to the economic growth of the US. A good example is the life sciences sector, where African-Americans hold roughly 2.5 percent of the jobs when their share in their general population is 12 percent. This is a sector that contributes greatly to innovation and the economy, and has largely relied on foreign talent who immigrates to the US. However, recent studies clearly show that this flow of talent is likely to decline in the next years because of increasing competition by developing economies. The solution to this problem is obvious: recruitment of minorities into science to replenish the pool of talent. The recruitment efforts of this project targeted African-American students, starting from high-school all the way to graduate school. As a result, 30 high school students, two undergraduate and two graduate students were trained in Food Biotechnology. One of the high-school students has entered our Food Science program and two more are expected to enter in the Fall '19. One graduate student will be graduating with a Ph.D. degree, one with an MS degree and two undergraduate students with BS in Food Science. These future graduates are expected to fill a critical gap in the food industry. The biodiesel industry is growing in the US, but the production of biodiesel generates vast amounts of raw glycerol, a byproduct with very limited usage. The accumulation of raw glycerol poses a threat to the viability of the biodiesel industry and needs to be addressed. In this project, we explored the feasibility of converting raw glycerol to food-grade functional oils that can partially substitute the dwindling fish oils. Although we were able to convert raw glycerol to microbial oil, the produced oil was toxic against human cells which indicated that raw glycerol would require some pretreatment before it can be used to produce food-grade oils. This drawback can be addressed through collaborative efforts that will explore sustainable methods to remove the toxic contaminants from raw glycerol. a) Design novel bioprocesses for the bioconversion of by-products to microbial lipids 1) Major activities completed / experiments conducted: Several Y. lipolytica strains were evaluated in their ability to convert raw glycerol to microbial lipids. The Po1d strain showed good conversion yields and is amenable to genetic manipulation so it was chosen for further experiments. 2) Data collected: We collected data on the growth kinetics of the Y. lipolytica strains on raw glycerol and on the amount of microbial lipids produced by these strains. We then analyzed the fatty acid composition and profile the produced lipids. 3) Summary statistics and discussion of results: Selection of proper strains is key to the outcome of a bioprocess. By evaluating the strains, we selected the best candidate for the conversion of raw glycerol to lipids. 4) Key outcomes: Selection of parent strain for the conversion of raw glycerol to microbial lipids. b) Studies on the lipid biosynthetic pathway in model microorganisms 1) Major activities completed / experiments conducted: A key lipid biosynthetic gene that has not been studied in detail in Y. lipolytica is the PAH1 gene which encodes for phosphatidate phosphatase (PAP). PAP regulates the flow of biosynthetic precursors to the lipid pathway and thus it was selected for further study. 2) Data collected: We developed methods for analyzing the PAP activity in Y. lipolytica. Then, we analyzed the levels of PAP activity, its subcellular localization and the effects of its cofactor. We further characterized the PAP activity by analyzing its sensitivity to the alkylating reagent N-ethylmaleimide. 3) Summary statistics and discussion of results: Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbors only a small amount of activity. PAP activity was regulated in a growth-dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. 4) Key outcomes: Our studies provide the first insights into the role of PAP activity in lipid biosynthesis by Y. lipolytica. c) Evaluate the performance of selected strains on complex substrates 1) Major activities completed / experiments conducted: To examine whether Y. lipolytica Po1d can grow on raw glycerol, the biomass yield was first measured on increasing concentrations of raw glycerol while pure glycerol was used as the control. 2) Data collected: Kinetics of growth and lipid production on pure and raw glycerol, lipid profiles, and fatty acid composition of the produced lipids. 3) Summary statistics and discussion of results: High concentrations of raw glycerol inhibited cell growth which was probably due to the various impurities in raw glycerol that can have a negative effect on microbial growth. 4) Key outcomes: Optimized culture conditions to produce microbial lipids from raw glycerol. d)Determine the bioactivity of microbial lipids produced on by-products 1) Major activities completed / experiments conducted: Lipid extracts from the raw and pure glycerol cultures were added to human liver cells and their effect on cell growth and viability was examined. 2) Data collected: Results from cell viability and proliferation assays. 3) Summary statistics and discussion of results: In-vitro cytotoxicity tests showed that the lipids produced from raw glycerol caused a significant decrease in cell viability and cell proliferation. These results indicated that the lipids produced from raw glycerol are not suitable for human nutrition. 4) Key outcomes: The lipids produced from raw glycerol are toxic to human liver cells and therefore not suitable for human consumption. e) Redesign the Food Biotechnology curriculum to include the latest developments in microbial and lipid biotechnology and to recruit and retain minority students 1) Major activities completed / experiments conducted: A new specialization in Food Biotechnology was developed by redesigning the Food Science curriculum to include topics in molecular biology, enzyme biotechnology and biochemistry. 2) Data collected: Number of students that specialized in Food Biotechnology. 3) Summary statistics and discussion of results: The implementation of the new curriculum has created a pipeline for students to pursue advanced degrees in Food Science in the area of Food Biotechnology. 4) Key outcomes: One graduate student will be graduating with a Ph.D. degree in Food Biotechnology, one graduate student with an MS in Food Biotechnology, and two undergraduate students with BS degrees in Food Science. f) Develop summer programs in Food Biotechnology to recruit minority school students 1) Major activities completed / experiments conducted: Two-week summer programs targeting African-American high school students were developed in collaboration with the North Alabama Center for Educational Excellence. The students attended lectures on topics in Food Science and in career opportunities for food scientists. 2) Data collected: The number of high school students who attended the summer school and then applied and were accepted into the Food Science program was monitored. Since many of the students who attended the summer school are still going through high school the monitoring period will be extended to track applications for the next two academic years. 3) Summary statistics and discussion of results and: Thirty minority high school students attended the summer school and one has entered the Food Science program. Two more minority high school students are expected to enroll in Food Science in Fall 2019. 4) Key outcomes: Minority students were introduced to Food Science and recruited to the Food Science curriculum.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose. Rahul Ukey; Taylor Carmon; Derell Hardman; Na'Taja Hill; Stylianos Fakas
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Progress 09/01/17 to 08/31/18
Outputs
Target Audience:Postdoctoral associate, graduate student, high school students. One postdoctoral associate and one graduate (Ph.D.) student are currently working on the project. Nine high school students (juniors and seniors) attended a two-week summer school in Food Biotechnology. The students attended lectures on Food Science and Food Biotechnology and were trained on molecular biology techniques Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral associate and two graduate students were trained in the Food Biotechnology lab. One graduate student received a travel award from the Federation of American Societies for Experimental Biology to present his work at the annual Experimental Biology conference. There he had the opportunity to attend networking events and meet with scientists and potential employers. How have the results been disseminated to communities of interest?Results of our work were presented at the annual Experimental Biology conference, and the annual Institute of Food Technologists conference where our graduate student won 1st place in the Biotechnology section. What do you plan to do during the next reporting period to accomplish the goals?In the next grant period, we will complete the work on the toxicity of the lipids produced on raw glycerol.
Impacts
What was accomplished under these goals?
a) Design novel bioprocesses for the bioconversion of by-products to microbial lipids We used wild type Y. lipolytica strains (i.e. Po1g and Po1d) to convert raw glycerol to lipids. In these bioprocesses, we studied the effect of the initial raw glycerol concentration on lipid accumulation to identify the optimum glycerol concentration for lipid production. We then studied the kinetics of the bioconversion of raw glycerol to lipids in flask cultures to establish the lipid accumulation phase. We also analyzed the lipid composition of cells that we collected in the peak of the lipid accumulation phase. b) Studies on the lipid biosynthetic pathway in model microorganisms and use of recombinant methods to increase lipid yields We deleted the PAH1 gene that encodes for phosphatidic acid phosphatase in Y. lipolytica and examined the effect of deletion on lipid biosynthesis during the growth cycle. Specifically, we looked at triglyceride production and phospholipid content in cells. We then extended our studies to cells growing under conditions that favor lipid accumulation to investigate the effect of PAH1 deletion on lipid biosynthesis. c) Evaluate the performance of selected mutant strains on complex substrates We selected the Y. lipolytica Po1d strain for further research and developed a bioprocess for lipid production from raw glycerol. We then studied the kinetics of lipid biosynthesis during growth on raw glycerol and compared them to the kinetics on pure glycerol. The results showed that raw glycerol is a good substrate for lipid production. The lipids that were produced on raw and pure glycerol were used in the bioactivity assays (goal d). d) Determine the bioactivity of microbial lipids produced on by-products We examined the effect of the lipids extracted from the raw glycerol cultures on the viability of THLE-3 cells and found that the lipid extracts reduced cell viability. We are currently investigating by which the lipids reduce cell viability. e) Redesign the Food Biotechnology curriculum to include the latest developments in microbial and lipid biotechnology and to recruit and retain minority students The Introduction to Biotechnology (FAS 671) course was redesigned to include the latest developments in the field of Biotechnology. A new textbook (Introduction to Biotechnology (2013) W.J. Thieman; M.A. Palladino) was introduced along with a lab component to introduce students to molecular biology techniques. The Advanced Food Biotechnology (FAS 771) course was developed as a lab course to teach students the basic techniques of microbial biotechnology including microbial culture techniques, DNA extraction, gel electrophoresis, PCR reactions etc. The Food Enzymes (FAS 615) course was redesigned to include a section in Enzyme Biotechnology where students are taught the methods and techniques for enzyme production using recombinant DNA technology. f) Develop summer programs in Food Biotechnology to recruit minority school students Ten high school students were trained in Food Biotechnology for two weeks. The students were introduced in the Food Science curriculum and learned basic techniques of molecular biology (i.e. DNA extraction and gel electrophoresis).
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Hardman D, Ukey R, Fakas S. 2018. The PAH1-encoded phosphatidate phosphatase plays a role in lipogenesis in the oleaginous yeast Yarrowia lipolytica. The FASEB Journal 32:539.20-539.20.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Hardman D, Ukey R, Fakas S. 2018. Phosphatidate phosphatase activity is induced during lipogenesis in the oleaginous yeast Yarrowia lipolytica. Yeast doi:10.1002/yea.3353:in press.
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:Postdoctoral associate, graduate student, high school students. One postdoctoral associate and one graduate (Ph.D.) student are currently working on the project. Nine high school students (juniors and seniors) attended a two-week summer school in Food Biotechnology. The students attended lectures on Food Science and Food Biotechnology and were trained on molecular biology techniques. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral associate and one graduate student were trained in the Food Biotechnology lab. The graduate student received a travel award from the American Society of Biochemistry and Molecular Biology to present his work at the annual Experimental Biology conference. There he had the opportunity to attend networking events and meet with scientists and potential employers. The graduate student also spent two months at Rutgers University where he was trained by Dr. George Carman, a renowned lipid biochemist. How have the results been disseminated to communities of interest?Results of our work were presented at the annual Experimental Biology conference and the annual Institute of Food Technologists conference. The PD was invited to give a seminar on our research at Duquesne University. What do you plan to do during the next reporting period to accomplish the goals?In the next grant period, we will make use of the engineered strains that we are currently constructing to convert raw glycerol to microbial lipids (goal a). Initial experiments will be done in flasks and the best producers will be selected. These strains will then be grow in a bioreactor to further optimize growth and lipid production.
Impacts
What was accomplished under these goals?
a) Design novel bioprocesses for the bioconversion of by-products to microbial lipids Work was initiated to examine the efficacy of bioconversion of raw glycerol, a by-product of the biodiesel industry, to lipids by Y. lipolytica strains belonging to the Food Biotechnology lab collection. Wild type and engineered strains (e.g. PAH1 overexpressors) will be used to convert raw glycerol to microbial lipids. We used wild type Y. lipolytica strains (i.e. Po1g and Po1d) to convert raw glycerol to lipids. In these bioprocesses, we studied the effect of the initial raw glycerol concentration on lipid accumulation to identify the optimum glycerol concentration for lipid production. We then studied the kinetics of the bioconversion of raw glycerol to lipids in flask cultures to establish the lipid accumulation phase. We also analyzed the lipid composition of cells that we collected in the peak of the lipid accumulation phase. b) Studies on the lipid biosynthetic pathway in model microorganisms and use of recombinant methods to increase lipid yields We deleted the PAH1 gene that encodes for phosphatidic acid phosphatase in Y. lipolytica and examined the effect of deletion on lipid biosynthesis during the growth cycle. Specifically, we looked at triglyceride production and phospholipid content in cells. We then extended our studies to cells growing under conditions that favor lipid accumulation to investigate the effect of PAH1 deletion on lipid biosynthesis. We are also in the process of constructing strains that overexpress PAH1 to test the effect of overexpression on lipid biosynthesis. c) Evaluate the performance of selected mutant strains on complex substrates This work will start at a later point, when the construction of the mutant strains will be completed (goal b). d) Determine the bioactivity of microbial lipids produced on by-products Work was initiated to examine the toxicity of the lipids produced on raw glycerol. We prepared lipid extracts from cells growing on raw glycerol to test their toxicity against human liver cells (Hep2G). We are currently optimizing the conditions of these bioassays. e) Redesign the Food Biotechnology curriculum to include the latest developments in microbial and lipid biotechnology and to recruit and retain minority students The Introduction to Biotechnology (FAS 671) course was redesigned to include the latest developments in the field of Biotechnology. A new textbook (Introduction to Biotechnology (2013) W.J. Thieman; M.A. Palladino) was introduced along with a lab component to introduce students to molecular biology techniques. The Advanced Food Biotechnology (FAS 771) course was developed as a lab course to teach students the basic techniques of microbial biotechnology including microbial culture techniques, DNA extraction, gel electrophoresis, PCR reactions etc. The Food Enzymes (FAS 615) course was redesigned to include a section in Enzyme Biotechnology where students are taught the methods and techniques for enzyme production using recombinant DNA technology. f) Develop summer programs in Food Biotechnology to recruit minority school students Nine high school students were trained in Food Biotechnology for two weeks. The students were introduced in the Food Science curriculum and learned basic techniques of molecular biology (i.e. DNA extraction and gel electrophoresis).
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Hardman, D. and S. Fakas (2017). "Regulation of phosphatidic acid phosphatase by high glucose in the oleaginous yeast Yarrowia lipolytica." The FASEB Journal 31(1 Supplement): 782.784-782.784.
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:Postdoctoral associate, undergraduate and postgraduate students, high school students. One postdoctoral associate was hired to develop molecular biology tools for manipulation of the yeast Yarrowia lipolytica. One graduate (Ph.D.) student and two undergraduate (freshmen) students are currently working on the project. We anticipate that two moreundergraduate (freshmen) students will start working in the project on Spring 2017. Ten high school students (juniors and seniors) attended a two week summer school in Food Biotechnology. The students attended lectures on Food Science and Food Biotechnology and were trained on molecular biology techniques. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral associate, one graduate and two undergraduate students were trained in the Food Biotechnology lab. The PD attended the Capacity Building project director conference. How have the results been disseminated to communities of interest?Three papers were published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?In the next grant period, we will make use of the engineered strains that we are currently constructing to convert raw glycerol to microbial lipids (goal a). Initial experiments will be done in flasks and the best producers will be selected. These strains will then be grow in a bioreactor to further optimize growth and lipid production. The lipids that will be produced on raw glycerol will be subjected to toxicological studies to ensure their safety (goal d).
Impacts
What was accomplished under these goals?
a) Design novel bioprocesses for the bioconversion of by-products to microbial lipids Work was initiated to examine the efficacy of bioconversion of raw glycerol, a by-product of the biodiesel industry, to lipids by Y. lipolytica strains belonging to the Food Biotechnology lab collection. Wild type and engineered strains (e.g. PAH1 overexpressors) will be used to convert raw glycerol to microbial lipids. b) Studies on the lipid biosynthetic pathway in model microorganisms and use of recombinant methods to increase lipid yields Two genes that are involved in lipid metabolism in Y. lipolytica were cloned: PAH1 and APP1. Both of these genes encode for Mg2+-dependent phosphatidic acid phosphatase activity in S. cerevisiae, but their activity and role in the oleaginous Y. lipolytica has not been studied yet. Both genes will be overexpressed in Y. lipolytica and their contribution to phosphatidic acid phosphatase activity will be examined. Also, the effect of the overexpression on lipid metabolism will be examined. In a complementary approach, PAH1 and APP1 genes will be deleted alone or in combination and the effect of their deletion on phosphatidic acid phosphatase activity will be examined. c) Evaluate the performance of selected mutant strains on complex substrates This work will start at a later point, when the construction of mutant strains will be completed (goal b). d) Determine the bioactivity of microbial lipids produced on by-products This work will start at a later point, when the bioprocesses will be developed (goal a). e) Redesign the Food Biotechnology curriculum to include the latest developments in microbial and lipid biotechnology and to recruit and retain minority students The Introduction to Biotechnology (FAS 671) course was redesigned to include the latest developments in the field of Biotechnology. A new textbook (Introduction to Biotechnology (2013) W.J. Thieman; M.A. Palladino) was introduced along with a lab component to introduce students to molecular biology techniques. The Advanced Food Biotechnology (FAS 771) course was developed as a lab course to teach students the basic techniques of microbial biotechnology including microbial culture techniques, DNA extraction, gel electrophoresis, PCR reactions etc. The Food Enzymes (FAS 615) course was redesigned to include a section in Enzyme Biotechnology where students are taught the methods and techniques for enzyme production using recombinant DNA technology. f) Develop summer programs in Food Biotechnology to recruit minority school students Ten high school students were trained in Food Biotechnology for two weeks. The students were introduced in the Food Science curriculum and learned basic techniques of molecular biology (i.e. DNA extraction and gel electrophoresis).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Fakas, S. (2016) Lipid biosynthesis in yeasts: A comparison of the lipid biosynthetic pathways between the model non-oleaginous yeast Saccharomyces cerevisiae and the model oleaginous yeast Yarrowia lipolytica. Engineering in Life Sciences in press. DOI: 10.1002/elsc.201600040
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Hardman, D., McFalls, D., and Fakas, S. (2016) Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis. Yeast in press. 10.1002/yea.3216
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Hardman, D., Fakas, S. (2016) Polyunsaturated fatty acids as dietary supplements: biological activities and sources. International Journal of Clinical Nutrition & Dietetics accepted.
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