| Popis: |
Microalgae have great potential for generating biofuels, pharmaceuticals, and other commercially valuable products. The goal of this thesis project was to improve this potential by investigating the expression and function of two key Calvin cycle enzymes in Chlamydomonas reinhardtii. Overexpression of sedoheptulose-1,7-bisphosphatase (SBPase) and fructose-1,6-bisphosphatase (FBPase) improves carbon fixation and growth rate in higher plants, but nothing has been reported regarding the role of these enzymes in photosynthetic output and biomass in C. reinhardtii, a model green alga. I first set out to determine how overexpression of C. reinhardtii SBPase affects Chlamydomonas growth by making nuclear transgenic lines expressing the SBPase-encoding gene SBP1 under control of the HSP70A/RBCS2 promoter and RBCS2 3'UTR. One transgenic line that overexpressed SBPase showed a promising result by reaching the exponential and stationary growth phases faster than the control strain, but expression of transgenic SBPase in this strain diminished significantly within 6 months, so I next set out to overexpress SBP1 and the FBPase encoding gene (FBP1) directly in the chloroplast, to avoid the possibility of gene silencing. To this end, I generated new chloroplast expression vectors with C. reinhardtii FBP1 and SBP1 coding sequences synthesized with C. reinhardtii chloroplast codon-bias and flanked with 5'psbD and 3'psbA regulatory sequences. The FBP1 vector integrated properly into the chloroplast genome and all FBP1 transformants tested accumulated ~4-fold increased levels of FBPase protein and 1.5-fold increased levels of FBPase activity; unfortunately, however, none of the SBP1 transformants expressed SBPase protein. Surprisingly, the FBPase-overexpressing transformants did not grow faster than the wild type, and in fact under mixotrophic conditions with 5% CO2 they grew more slowly and reached a maximum cell density that was 1.5-fold lower than the wild type did, and under photoautotrophic conditions they grew more slowly and reached maximum cell densities that were 1.4-fold lower (atmospheric CO2) and 1.7-fold lower (5% CO2) than the wild type. To learn more about regulation of FBP1 and SBP1 mRNA and FBPase and SBPase protein levels I cultured C. reinhardtii cells photoautotrophically under 12 hour light:12 hour dark conditions and analyzed mRNA levels by RT-qPCR and protein by western blot. I found FBP1 and SBP1 transcripts to be highly inducible by light, up to ~8 and ~9 fold, respectively, while FBPase and SBPase protein levels were only modestly elevated in the light ~2.5 and ~6 fold, respectively. So in conclusion, I determined that Chlamydomonas FBP1 and SBP1 appear to be light-regulated genes, that overexpressing SBPase might be a promising strategy for improving microalgal growth, but that overexpression of FBPase has a neutral or detrimental effect on growth and biomass productivity in C. reinhardtii, depending on growth conditions. |
