Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Virginia A
Somayeh Gharaie Fathabad - Morgan State University
Co-Author(s): AnithaChristy S. Arumanayagam, Methodist Hospital Research Institute, TX ; Behnam Tabatabai, Morgan State University; Huan Chen, National High Magnetic Field Laboratory, FL; Jie Lu, National High Magnetic Field Laboratory, FL and Florida State University, FL; Viji Sitther, Morgan State University, MD
Over the last few years, increasing environmental consequences of fossil fuel have driven scientists to produce biofuel as a sustainable and environmentally friendly alternative to fossil fuels. The freshwater cyanobacterium, Fremyella diplosiphon, is an ideal biofuel feedstock due to its ability to produce lipids in its thylakoid membrane. The objective of the present study was to overexpress the lipid synthesis gene, sterol desaturase (SD), in this model organism and evaluate its effect on total lipid content and fatty acid composition. F. diplosiphon (strain B481) total RNA was extracted, reverse transcribed to cDNA, and homologs of the SD gene amplified by PCR. The pGEM-7Zf (+) expression plasmid with native promoters was constructed, cloned, and transformed into wild type (WT) F. diplosiphon via electroporation. Overexpression of the target gene in the transformant was detected using quantitative reverse transcriptase-PCR. In the next phase, total lipid content and fatty acid methyl ester (FAME) composition in WT and transformant were compared using gravimetric analysis and gas chromatography mass spectrometry (GC-MS). Successful overexpression of the SD gene resulted in a transformant designated as B481-SD, which exhibited a 64-fold increase in mRNA transcript level. Comparison of total lipid content revealed a 27.3% increase in B481-SD relative to the WT. GC-MS analysis indicated a 23% increase of unsaturated FAMEs in B481-SD transesterified lipids, with methyl octadecadienoate (C18:2) identified as the dominant desaturated component. We calculated the theoretical chemical and physical properties of resultant FAME confirming biodiesel with a cetane number and oxidative stability above minimum fuel standards. Using two-dimensional gas chromatography with time-of-flight mass spectrometry, we identified several FAME components that were not previously detected in 1D GC-MS such as C12:0, C15:0, C18:3, and C18:4. Our findings indicated that SD gene overexpression enhanced total lipid content and essential unsaturated fatty acid abundance, thus augmenting F. diplosiphon capacity as a commercial source of biofuel. Future efforts will be aimed towards scale-up studies maximizing biomass accumulation, collection, and extraction/conversion of lipid to verify F. diplosiphon B481-SD potential for large-scale biodiesel production.
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Funder Acknowledgement(s): This study was supported in part by the National Institutes of Health Award UL1GM118973.
Faculty Advisor/Mentor: Dr. Viji Sitther, firstname.lastname@example.org
Funder Acknowledgement(s): Funder Acknowledgement(s): This study was supported in part by the National Institutes of Health Award UL1GM118973.
Faculty Advisor: Dr. Viji Sitther, email@example.com
Role: I performed all the molecular and lipid work. We sent our sample for detecting FAME abundance using Two dimensional comprehensive gas chromatography- time of flight mass spectrometry to another lab that has the required instrumentation.