It has been long known that algae have the potential to produce a diverse range of metabolic products including lipid and starch, which could be utilized as a fuel feedstock. Despite the capacity of algae to synthesize and store large amounts of lipids and starch, algae are not currently a commercially viable feedstock for biofuel. The metabolite storage in algae can depend on the availability of nutrients such that nutrient starvation can boost the storage of lipid and carbohydrate. These nutrient-status-induced changes in lipid and starch are underpinned by altered expression of several metabolite-related genes. However, many aspects of fatty acid and carbohydrate biosynthesis are not well understood. Furthermore, the genetic regulators of nutrient starvation-induced carbohydrate and lipid accumulation are unknown in microalgae. Therefore, this PhD focused on screening cultivation conditions, in particular Phosphorus (P) and Nitrogen (N) limited conditions that induce metabolic changes, evaluated a rapid microalgal screening method, which was used to identify putative metabolism regulators, and characterized in detail the role of one P-starvation regulator, called PSR1 (Phosphorus starvation response 1). For establishing suitable culture conditions, the microalga Chlamydomonas reinhardtii was cultured in five different P and N-limited conditions and screened for metabolic changes using Fourier transform infrared spectroscopy (FT-IR) at different phases of growth. The FT-IR spectral changes were visualized by multivariate statistical tools such as principal component analysis (PCA) and principal component-discriminant function analysis (PC-DFA). Clear clustering based on nutrient availability and metabolic changes demonstrates the potential and sensitivity of FT-IR in screening multiple culture conditions. The potential of FT-IR was further tested by screening mutant strains of C. reinhardtii that were defective in response to nutrient starvation. Nine lines with mutation in one or more of the PSR1, SNRK2.1 or SNRK2.2 genes and a wild type were screened by FT-IR for P and N starvation-induced metabolic changes. PCA, PC-DFA and predictive partial least squares discriminant analysis (PLS-DA) of FT-IR spectra, clearly distinguished wild type from mutant strains and clustered mutants with similar genetic backgrounds, demonstrating the potential of FT-IR to detect and differentiate specific genetic traits. The changes in lipid and carbohydrate profile under nutrient stress and in the different strains were validated by biochemical analysis and liquid chromatography-mass spectrometry (LC-MS).This thesis demonstrated that PSR1 is an important regulator of neutral lipid and starch biosynthesis. Transcriptomic analysis on wild type and psr1 mutant under P-starvation was performed to identify transcripts induced by P-starvation that were mis-regulated in psr1. Mainly transcripts encoding starch and triacylglycerol enzymes were affected. To further evaluate the role of PSR1 in regulating lipid and starch metabolism, complementation of psr1 and overexpression by PSR1 was performed. The P-starvation phenotype was clearly rescued in the complementation lines, and overexpression lines showed increased expression of P homeostasis genes and increased Pi accumulation in cells, with an increase in total starch content and number of starch granules. Clear increases in expression of key starch biosynthesis genes such as soluble starch synthase (SSS1, SSS5) and starch phosphorylase (SP1) was observed, which correlated with increased starch content in the overexpression lines. A carbon shift was observed as a decrease in neutral lipid was coupled with the increase in starch content. All together these findings suggest that PSR1 is a key transcriptional regulator of global metabolism, and demonstrated successful transcriptional engineering of microalgae.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:756799 |
| Date | January 2015 |
| Creators | Bajhaiya, Amit |
| Contributors | White, Keith ; Pittman, Jon |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/metabolite-analysis-of-chlamydomonas-reinhardtii-and-transcriptional-engineering-for-biofuel-production(185995ba-d1be-44ff-a87a-140c19655d31).html |
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