Summary: The gene cluster of is normally expressed shortly before sporulation in the aerial mycelium, leading to production of the grey polyketide spore pigment. By placing the genes under the control of the thiostrepton-inducible promoter, they were artificially expressed on plasmids or in the chromosome during vegetative growth in a strain deleted for the genes, which control biosynthesis of the polyketide antibiotic actinorhodin. Certain combinations of -ORFI-VII led to production of mycelial pigments; these were exported into the medium when -ORFI was absent, but poorly in its presence. Combined with comparative sequence data, the results allowed deductions to be made, or confirmed, about the normal roles of the eight known genes, -ORFI-VIII, as follows: -ORFIII, IV, V encode the three components (ketosynthase, chain length factor and acyl carrier protein) of the ‘minimal’ polyketide synthase (PKS) needed for assembly of the carbon chain of the spore pigment precursor; -ORFII, VI, VII are likely to be involved in cyclizations of the nascent carbon chain; -ORFVIII controls a late step in the spore pigment biosynthetic pathway, probably a hydroxylation; and -ORFI may encode a protein needed for correct targeting or retention of spore pigment at an appropriate cellular location. In other experiments, genes encoding components of the -PKS and -PKS were artificially co-expressed. Each of the three minimal PKS subunit genes could complement lesions in the corresponding -PKS genes to produce actinorhodin or related mycelial pigments, and each of the three minimal PKS genes could complement lesions in the minimal PKS genes to cause spore pigmentation. Thus the two sets of PKS subunits, which are encoded by genes that have presumably diverged from a common ancestor, are still capable of biochemical ‘cross-talk’, but this is normally prevented because the gene sets are expressed in different ‘tissues’ of the differentiated colony. Ectopic expression of sets of -PKS genes presumed to be sufficient to assemble a carbon chain caused inhibition of early growth of the strains, perhaps by causing interference with fatty acid biosynthesis; this yielded circumstantial evidence that the -PKS gene products can also interact with those of the fatty acid synthase(s) of the organism.


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