碩士 / 國立成功大學 / 化學工程學系 / 105 / In this study, CRISPRi (clustered regularly interspaced short palindromic repeats interference) was used for the first time to regulate expression of exogenously supplied rfp (red fluorescene protein) gene as a proof-of-concept, and endogenous phosphoenolpyruvate carboxylase (PEPC1) gene as a proof-of-function in Chlamydomonas reinhardtii CC-400. Based on the design of sgRNA binding site, the repression efficiency is about 19%. The application rate of 94% and stability of 7 generations via CRISPRi mediated gene regulation in C. reinhardtii have been demonstrated by RFP. Gene PEPC1 encoding proteins are essential for controlling the carbon flux that enters the TCA cycle and plays a crucial role in carbon partitioning of substrates in competition thus to up-regulate of diglyceride acyltransferase (DGAT) with more lipids synthesis. All CrPEPC1 down-regulated strains have lower chlorophyll colour, but higher biomass concentration and lipid accumulation rate. The outstanding strain generated the highest lipid content and productivity of 28.5% on dry cell weight (DCW) and 34.9 mg/L/day, respectively. This is about 74.4% and 94.2% higher than that of the wild-type. Another gene e-cyclase (LCYe) involving in carotenoid biosynthetic pathway was also tested in the study. The CrLCYe disturbed strains showed significant changes in -carotene production upto 2244.3 ug/g, which yield is 41.3% higher than that of the wide-type. The present results revealed that CRISPRi based transcriptional silencing was applicable in C. reinhardtii and expanded the way to improve the yield, titer and productivity of microalgae-based high-value products.
| Identifer | oai:union.ndltd.org:TW/105NCKU5063060 |
| Date | January 2017 |
| Creators | Pei-HsunKao, 高培勛 |
| Contributors | I-Son Ng, 吳意珣 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 90 |
Page generated in 0.0017 seconds