Isoprenoid biosynthesis in Synechocystis sp. strain PCC6803 is stimulated by compounds of the pentose phosphate cycle but not by pyruvate or deoxyxylulose-5-phosphate

Abstract

The photosynthetic cyanobacterium Synechocystis sp. strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-D-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E. coli. However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds. Fosmidomycin (at 1 micro M and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [(14)C]IPP incorporation stimulated by DHAP plus GA3P. To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium. The combined results suggest that the MEP pathway, as described for E. coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp. strain PCC6803. Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.

FIG. 1.

Pathway of isoprenoid synthesis in E. coli. The initial substrates GA3P and PYR lead to DXP and then to MEP in a reaction catalyzed by a reductoisomerase, which can be inhibited (notched arrow) by fosmidomycin. This is followed by formation of 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) and, subsequently, 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate (CDP-ME2P), 2-C-methyl-d-erythritol-2,4-cyclodiphosphate (ME-2,4cPP), and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate (HM-2B4PP). The LytB enzyme is considered to be the branch point leading to the eventual formation of IPP and DMAPP. IPP isomerase is present, but not essential. (The figure was modified from references , , and 33).

FIG. 2.

GA3P and dihydroxyacetone phosphate stimulate [¹⁴C]IPP incorporation in a cell extract of Synechocystis sp. strain PCC6803 grown photoautotrophically, but 1-deoxy-d-xylulose 5-phosphate and pyruvate do not. The following compounds were added individually or in combination: GA3P (1 mM) (▵), DHAP (500 μM) (▴), DXP (500 μM) (•), PYR (500 μM) (□), GA3P plus DHAP (500 μM) (▪), and GA3P plus PYR (500 μM) (○). COFS, cofactors alone (⋄). The cell extract (60,000 × g supernatant) (pH 7.7) (100 mM HEPES-KOH), cofactor mixture (500 μM ATP, 250 μM CTP, 100 μM thiamine diphosphate, 1 mM NADPH, 500 μM NADP, 1 mM FAD, 5 mM glutathione, 5 mM MgCl₂, 2.5 mM MnCl₂, 10 μM coenzyme B₁₂), and/or metabolites were incubated at 37°C. Incorporation of [¹⁴C]IPP into isoprenoids was verified by analyzing the petroleum ether extracts of the acid-hydrolyzed fraction (9).

FIG. 3.

Stimulation of [¹⁴C]IPP incorporation by phosphorylated sugars in cell extract of Synechocystis sp. strain PCC6803. Column numbers specify the supernatant (60,000 × g), with the cofactors and conditions given in the legend to Fig. 2, with individual incubation of the following: 1, ER4P (500 μM); 2, RU5P (500 μM); 3, GL6P (500 μM); 4, 6PG (500 μM); 5, FR6P (500 μM); 6, MEP (500 μM); 7, GA3P (1 mM); 8, erythrose (500 μM); 9, glucose (500 μM); 10, xylulose (500 μM); 11, sucrose (500 μM); and 12, glyceraldehyde (500 μM). Incubation (37°C) was for 30 min (gray bars) and 60 min (white bars). Incorporation of [¹⁴C]IPP into isoprenoids was verified by analyzing the petroleum ether extracts of the acid-hydrolyzed fraction (9). Each value is the mean + standard deviation of two (columns 1, 3, 5, 6, and 8 to 12) or three (columns 2, 4, and 7) independent experiments.

FIG. 4.

Stimulation of [¹⁴C]IPP incorporation by pentose phosphate cycle sugars in the cell-free supernatant fraction (60,000 × g) of Synechocystis PCC6803. Columns: 1, RU5P (500 μM); 2, RU5P (500 μM) plus RU5P-3-epimerase; 3, RU5P (500 μM) plus GA3P (1 mM); 4, RU5P (500 μM) plus GA3P plus RU5P-3-epimerase; 5, GA3P (1 mM); 6, 6PG (500 μM); 7, 6PG (500 μM) plus GA3P (1 mM); 8, 6PG (500 μM) plus GA3P plus 6PG-dehydrogenase plus RU5P-3-epimerase. Incubation was at 37°C for 30 min (gray bars) and 60 min (white bars) with buffer and cofactors, and analysis was performed as described in the legend to Fig. 2. Each value is the mean + standard deviation of three (columns 1, 5, and 6) or two (columns 2, 7, and 8) independent experiments, with columns 3 and 4 each representing a single experiment.

FIG. 5.

Growth of Synechocystis sp. strain PCC6803 in the presence of the MEP synthesis inhibitor fosmidomycin. Results are shown for growth in BG-11 growth medium alone (♦), BG-11 medium plus 1 μM fosmidomycin (▪), and BG-11 medium plus 1 mM fosmidomycin (▴). Cultures were grown at 20°C with 20 μM/m² s light in air and monitored at A₇₃₀. The data represent the average of three separate experiments.

FIG. 6.

Hypothetical pathway for the biosynthesis of the isoprenoid precursors IPP and DMAPP in Synechocystis sp. strain PCC6803 under photosynthetic conditions. The carbon atoms originate from photosynthetically produced phosphorylated sugars (e.g., GA3P, DHAP, GL6P, FR6P, 6PG, ER4P, and RU5P). These are shown in the study to stimulate isoprenoid synthesis in a cell-free system and could enter primarily via RU5P and/or XY5P, and after a reductoisomerase reaction, they could be converted to diphosphocytidyl hydroxymethyl d-erythritol (CDP-HME), followed by phosphorylation and cyclization to give 2,4 hydroxymethyl cyclodiphosphate (HME-2,4 cPP). LytB is considered to be near the branch point leading to formation of IPP and DMAPP (5). Interconversion between IPP and DMAPP appears to be lacking under these conditions.