Characterization of Enzymes Involved in Lipid Biosynthesis from the Photosynthetic Bacteria <em>Synechocystis PCC6803</em> and <em>Blastochloris viridis</em>

Abstract

Cyanobacteria are unicellular prokaryotic algae that perform oxidative photosynthesis similar to plants. The cells harbour thylakoid membranes composed of lipids related to those of chloroplasts in plants, to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including TAG or wax esters, which are found in plants, animals and some bacteria, nevertheless remained unclear in cyanobacteria. This thesis shows that the cyanobacterium Synechocystis sp. PCC6803 accumulates both TAG and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to the acyltransferase domain of plant esterase/lipase/thioesterase (ELT) proteins, is essential for TAG and phytyl ester synthesis in Synechocystis. The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in TAG and wax ester synthesis.<br /> Synechocystis harbours a second ORF (slr1807) with sequence similarity to the hydrolase domain of plant ELT proteins. Analysis of the Synechocystis slr1807 mutant reveals that slr1807 is involved in the accumulation of phytyl esters. However, the recombinant slr1807 enzyme did not enhance the triacylglycerol or phytyl ester synthesis activity when it was combined with the recombinant slr2103 enzyme in in vitro enzyme assays.<br /> The galactolipids digalactosyldiacylglycerol (αβDGDG) and monogalactosyldiacylglycerol (βMGDG) are the most abundant membrane lipids in photosynthetic organisms including plants, green algae and cyanobacteria. Different organisms employ different pathways with different galactosyltransferases to synthesize βMGDG and αβDGDG. In plants and green algae, βMGDG is produced by MGD1, and βMGDG is the substrate for αβDGDG synthesis by the DGDG synthase DGD1. The photosynthetic purple bacterium Blastochloris viridis contains a plant MGD1-type processive galactosyltransferase (BviMgdP) which converts diacylglycerol directly to ββDGDG, without accumulating βMGDG as intermediate, when expressed in E. coli. In this thesis, BviMgdP, was introduced into the Arabidopsis galactolipid-deficient mgd1 and dgd1 mutants to study its activity in planta and its capacity to complement lipid and photosynthetic deficiency of the plant mutants. The introduction of BviMgdP in dgd1 plants resulted in the accumulation of ββDGDG instead of the plant-type αβDGDG. While growth, chlorophyll content and DGDG amount were similar to WT, photosynthesis was only partially complemented presumably due to different anomeric configurations of the outermost galactose in DGDG (β vs. α). Transfer of BviMgdP into the Arabidopsis mgd1 null mutant resulted in partial complementation with dwarfed, but green, transgenic plants. Interestingly, most mgd1-BviMgdP lines contained higher amounts of ββDGDG than MGDG in the leaves, in contrast to Arabidopsis and other plants, where MGDG is ~2fold more abundant than DGDG. The accumulation of small amounts of MGDG in mgd1-BviMgdP plants might be due to low MGDG synthase activity of BviMgdP in the plant environment, or to MGD2/MGD3 from Arabidopsis which could contribute to MGDG synthesis in the transgenic plants.<br /> In addition to MGDG and DGDG, B. viridis accumulates an anionic glycolipid, i.e glucuronosyldiacylglycerol (GlcADG). The GlcADG synthase (BviGlcADS) from B. viridis shows high similarity to the agrobacterial Agt. Therefore, B. viridis contains three pathways for the synthesis of MGDG (BviMgdN), DGDG (BviMgdP) and GlcADG (BviGlcADS).<br /> The bacterial enzymes involved in lipid synthesis reported in this thesis show sequence similarity to plant lipid enzymes found in chloroplasts. These results emphasize the importance of the cyanobacterial progenitor (slr2103, slr1807) or lateral gene transfer from bacteria, i.e. the presence of MGD1-related (BviMgdP, BviMgdN), and SQD2-related sequences (BviGlcADS) indicate, that bacteria represent the origin of chloroplast enzymes.