Increased production of zeaxanthin and other pigments by application of genetic engineering techniques to Synechocystis sp. strain PCC 6803.

The psbAII locus was used as an integration platform to overexpress genes involved in carotenoid biosynthesis in Synechocystis sp. strain PCC 6803 under the control of the strong psbAII promoter. The sequences of the genes encoding the yeast isopentenyl diphosphate isomerase (ipi) and the Synechocystis beta-carotene hydroxylase (crtR) and the linked Synechocystis genes coding for phytoene desaturase and phytoene synthase (crtP and crtB, respectively) were introduced into Synechocystis, replacing the psbAII coding sequence. Expression of ipi, crtR, and crtP and crtB led to a large increase in the corresponding transcript levels in the mutant strains, showing that the psbAII promoter can be used to drive transcription and to overexpress various genes in Synechocystis. Overexpression of crtP and crtB led to a 50% increase in the myxoxanthophyll and zeaxanthin contents in the mutant strain, whereas the beta-carotene and echinenone contents remained unchanged. Overexpression of crtR induced a 2.5-fold increase in zeaxanthin accumulation in the corresponding overexpressing mutant compared to that in the wild-type strain. In this mutant strain, zeaxanthin becomes the major pigment (more than half the total amount of carotenoid) and the beta-carotene and echinenone amounts are reduced by a factor of 2. However, overexpression of ipi did not result in a change in the carotenoid content of the mutant. To further alter the carotenoid content of Synechocystis, the crtO gene, encoding beta-carotene ketolase, which converts beta-carotene to echinenone, was disrupted in the wild type and in the overexpressing strains so that they no longer produced echinenone. In this way, by a combination of overexpression and deletion of particular genes, the carotenoid content of cyanobacteria can be altered significantly.

Rubisco activase transcript (rca) abundance increases when the marine unicellular green alga Chlorococcum littorale is grown under high-CO2 stress.

cDNA and the corresponding genomic DNA region encoding Rubisco activase were isolated from the unicellular green alga Chlorococcum littorale. The deduced amino acid sequence encoded by the cDNA was 403 amino acids long and exhibited important homology with those of other known Rubisco activases. Its N-terminal sequence was similar to the chloroplastic transit peptides in Chlamydomonas reinhardtii. The mature protein had a predicted molecular mass of 42 kDa. Five introns were located inside the genomic gene encoding Rubisco activase (rca). Genomic Southern blots indicated that two copies of the rca gene were present in the genome of C. littorale. The level of rca messenger RNA increased when cells of C. littorale were subjected to high-CO2 stress (i.e. grown under at least 20% CO2). Hsp70 heat-shock protein was also induced under high-CO2 conditions and, as expected, was also induced at 35 degrees C. The rca gene, in contrast, was not induced at 35 degrees C, indicating that this gene was induced in response to the high CO2 concentration and not to general stress. A search of the promoter-binding proteins by a gel retardation assay showed that, under the high-CO2 conditions, a protein(s) which was probably an activator of the rca transcription was synthesized.

A protein is involved in accessibility of the inhibitor acetazolamide to the carbonic anhydrase(s) in the cyanobacterium Synechocystis PCC 6803.

A gene, zam (for resistance to acetazolamide), controlling resistance to the carbonic anhydrase inhibitor acetazolamide, is described. It has been cloned from a spontaneous mutant, AZAr-5b, isolated from the cyanobacterium Synechocystis PCC 6803, for its resistance to this drug (Bédu et al., Plant Physiol 93: 1312-1315, 1990). This mutant, besides its resistance to acetazolamide, displayed an absence of catalysed oxygen exchange activity on whole cells, suggestive of a deficiency in carbonic anhydrase activity. The gene was isolated by screening a genomic library of AZAr-5b, and selecting for the capacity to transfer the AZAr phenotype to wild-type cells. A system leading to forced homologous recombination in the host chromosome, using a platform vector, was devised in order to bypass direct selection difficulties. The putative encoded protein, 782 amino acids long, showed some homology with four eukaryotic and prokaryotic proteins involved in different cellular processes, one of them suppressing a phosphatase deficiency. The mutated allele of AZAr-5b showed an in-frame 12 nucleotide duplication, which should not interfere with translation, and might result from transposition of a mobile element. Integration into a wild-type genome of either the spontaneous mutated allele or one inactivated by insertional mutagenesis conferred the character of resistance, but not the deficiency in oxygen exchange, indicating that the two phenotypic aspects of AZAr-5b corresponded to two independent mutations. A working hypothesis explaining the phenotypes of the mutants is that the presence of the Zam protein would be necessary for the inhibitor to reach (one of) the two carbonic anhydrases present in this strain. This, however, would be a secondary action, the physiological role of the protein still being cryptic.

A protein involved in co-ordinated regulation of inorganic carbon and glucose metabolism in the facultative photoautotrophic cyanobacterium Synechocystis PCC6803.

The involvement of a gene of Synechocystis PCC6803, icfG, in the co-ordinated regulation of inorganic carbon and glucose metabolism, was established. The icfG gene codes for a 72 kDa protein, which shows no homology with those registered in data libraries. Expression of icfG required glucose, the actual inducer probably being glucose-6-phosphate, and was independent of light and of the external inorganic carbon concentration. Mutants carrying an inactivated copy of icfG were constructed. Their growth characteristics were identical to those of the wild type under all regimes except in limiting inorganic carbon with glucose being present either before or after the transfer to the limiting conditions. These conditions completely prevented growth, both in the light and in the dark. The inhibition could be relieved by several intermediates of the tricarboxylic acid cycle. Assays of various enzymic activities related to inorganic carbon uptake and to its assimilation via either the Calvin cycle or phosphoenolpyruvate carboxylase did not reveal the level of action of IcfG. Possible models include a blockage of the assimilation of both carbon sources in the absence of IcfG, or the inhibition of Ci incorporation route(s) essential under limiting inorganic carbon conditions, even when glucose is present, and even in the dark.