IMPROVING INDUSTRIAL PERFOMANCE OF Chlamydomonas reinhardtii THROUGH GENETIC ENGINEERING: A FOCUS ON STRESS TOLERANCE AND IRON TRANSPORT

IRT2;Biofuels;Metabolic Engineering;BCL-XL;Algae;Genetic Engineering;Chemical & Biomolecular Engineering

Garcia, Luis F.Betenbaugh, Michael J. ;

Johns Hopkins University

关键词: IRT2;    Biofuels;    Metabolic Engineering;    BCL-XL;    Algae;    Genetic Engineering;    Chemical & Biomolecular Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/44659/GARCIA-THESIS-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Algal cultures exhibit the potential as robust systems for pharmaceutical production, as sustainable sources of nutrition both for humans and livestock, and as renewable biomass feedstock for biofuel production.Algal biomass grows quickly, requires minimal nutrient supplementation and sunlight, and has the ability to sequester environmental CO2. Although these properties seem attractive, algal industrial cultivation is still far from optimal; leading to non-ideal culture conditions which induce cellular stress that cause a loss in productivity. Furthermore, bioprocessing of algal biomass for biofuel production is still in its infancy and encounters many obstacles and bottlenecks after mass cultivation, mainly cell separation from culture.In this study we aim to address these industrial issues by employing the power of genetic and metabolic engineering. Specifically, we employ a two-fold plan to increase the stress tolerance and intracellular iron concentration of model organism, Chlamydomonas reinhardtii, by overexpressing mammalian anti-apoptotic BCL-XL protein and the native membrane coupled iron transporter IRT2. In order to achieve our goal a gene construct driven the native hsp70/rbcS2 tandem promoter was constructed containing the appropriate gene of interest and carrying a Hygromycin B resistance marker for downstream selection. After transformation, resulting colonies were screened via colony PCR; positive colonies were subcultured and exposed to a variety apoptosis inducing agents tailored to mimic stresses involved with industrial cultivation; mainly photooxidative damage, exposure to reactive oxygen species, osmotic pressure change, and intracellular damage caused by high irradiance. While the transgenic cell line experienced a reduced growth rate, it reached a higher cell density and featured higher cell longevity in comparison to the wild type after prolonged culture. Furthermore, in all stress related experiments the transgenic cell line outperformed the wild type, often with stark phenotypic changes. Moreover, RT-PCR analysis confirmed RNA level expression of this peptide allowing us to conclude that the transgenic cell line was producing a functional form of this recombinant anti-apoptotic protein leading to more robust stress tolerance.