Genetic Engineering and Nanotechnological Approaches to Enhance Lipid Production in Fremyella Diplosiphon, a Model Cyanobacterium
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Date
2019-03-25
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Department
Biology
Program
Doctor of Philosophy
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Abstract
In recent years, cyanobacteria have gained great importance as a potential source of biofuel due to their fast generation time and ability to directly convert carbon dioxide into free fatty acids. In this study, the sterol desaturase (SD) gene was overexpressed on the model cyanobacterium Fremyella diplosiphon B481 to enhance total lipid content and fatty acids. The effort resulted in a transformant designated as B481-SD with a 64-fold increase in mRNA transcript level. Results of gravimetric analysis and gas chromatography-mass spectrometry (GC-MS) revealed a 27.3% increase in total lipid content and a 23% increase in unsaturated fatty acid methyl esters (FAMEs) from B481-SD transesterified lipids relative to wild type (WT).
In the second phase, the effect of salinity stress on B481-SD growth, total lipid content, and fatty acid composition was examined. The halotolerant strain designated as B481-SDH exhibited a 38-fold increase in mRNA transcript level when compared to the WT. In addition, proteomic analysis revealed a band near the expected size of sterol desaturase in all strains. Four spots that potentially represent SD up-regulated in B481-SD and B481-SDH were identified using 2D-PAGE. In the third phase, the effect of various concentrations (0.05-3.2 mg L-1) of two iron nanoparticles (Nanofer 25 and 25s) on growth, photosynthetic pigmentation, total lipid content, and fatty acid composition of F. diplosiphon was investigated. Results of the study revealed a significant increase (P≤0.05) in growth of F. diplosiphon treated with Nanofer 25s ranging from 0.2-1.6 mg L-1 compared to the control; however, no significant difference was observed in growth of F. diplosiphon treated with Nanofer 25 relative to the control. Photosynthetic pigment quantification revealed no significant increases in chla and carotenoid accumulations in F. diplosiphon exposed to Nanofer 25s ranging from 0.4-1.6 mg L-1. Results of gravimetric analyses and GC-MS revealed significant increases in total lipid content and unsaturated fatty acid methyl esters in F. diplosiphon nano-treated transesterified lipids. The presence of iron nanoparticles inside F. diplosiphon was visualized using optical microscopy and transmission electron microscopy. Our findings address a key challenge in lipid productivity of cyanobacterial cultivation systems by increasing total lipid content and essential unsaturated fatty acids to maximize the potential of F. diplosiphon as a large-scale biofuel agent.