Genetics of actinorhodin biosynthesis by Streptomyces coelicolor A3(2)

A series of 76 mutants of Streptomyces coelicolor A3(2) specifically blocked in the synthesis of the binaphthoquinone antibiotic actinorhodin were classified into seven phenotypic classes on the basis of antibiotic activity, accumulation of pigmented precursors or shunt products of actinorhodin biosynthesis, and cosynthesis of actinorhodin in pairwise combinations of mutants. The polarity of cosynthetic reactions, and other phenotypic properties, allowed six of the mutant classes to be arranged in the most probable linear sequence of biosynthetic blocks. One member of each mutant class was mapped unambigiguously to the chromosomal linkage map in the short segment between the hisD and guaA loci, suggesting that structural genes for actinorhodin biosynthesis may form an uninterrupted cluster of chromosomal genes.     

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.     

Genetic analysis of absB, a Streptomyces coelicolor locus involved in global antibiotic regulation.

The filamentous soil bacterium Streptomyces coelicolor is known to produce four antibiotics which are genetically and structurally distinct. An extensive search for antibiotic regulatory mutants led to the discovery of absB mutants, which are antibiotic deficient but sporulation proficient. Genetic analysis of the absB mutants has resulted in definition of the absB locus at 5 o'clock on the genetic map. Multiple cloned copies of the actII-ORF4 gene, an activator of synthesis of the antibiotic actinorhodin, restore actinorhodin biosynthetic capability to the absB mutants. These results are interpreted to mean that the failure of absB mutants to produce antibiotics results from decreased expression of the antibiotic genes. The absB gene is proposed to be involved in global regulation of antibiotic synthesis.     

abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.     

Initiation of actinorhodin export in Streptomyces coelicolor

Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.     

Nutritional control of actinorhodin production byStreptomyces coelicolor A3(2): suppressive effects of nitrogen and phosphate

Summary Actinorhodin production inStreptomyces coelicolor A3(2) was relatively insensitive to the carbon source concentration but was elicited by nitrogen or phosphate depletion, or by a decline in the growth rate. In starch-glutamate media with nitrogen limitation, increasing the nitrogen supply delayed the onset of antibiotic synthesis and, at concentrations above 30 mM, decreased its rate. In a similar medium with phosphate limitation, increasing the initial phosphate concentration delayed actinorhodin formation and, above 2.5 mM, reduced the rate of synthesis. Experiments in which actinorhodin synthesis was elicited by phosphate depletion at various nitrogen concentrations demonstrated strong suppression by residual glutamate. Cultures in which actinorhodin biosynthesis was initiated by nitrogen depletion were not similarly suppressed by increasing amounts of residual phosphate. The results suggest that actinorhodin production inS. coelicolor A3(2) responds to interacting physiological controls, notable among which is nitrogen catabolite regulation.     

Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str(r)), gentamicin (Gen(r)), or rifampin (Rif(r)), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the beta-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.     

Selection of objective function in genome scale flux balance analysis for process feed development in antibiotic production

Using flux variability analysis of a genome scale metabolic network of Streptomyces coelicolor, a series of reactions were identified, from disparate pathways that could be combined into an actinorhodin-generating mini-network. Candidate process feed nutrients that might be expected to influence this network were used in process simulations and in silico predictions compared to experimental findings. Ranking potential process feeds by flux balance analysis optimisation, using either growth or antibiotic production as objective function, did not correlate with experimental actinorhodin yields in fed processes. However, the effect of the feeds on glucose assimilation rate (using glucose uptake as objective function) ranked them in the same order as in vivo antibiotic production efficiency, consistent with results of a robustness analysis of the effect of glucose assimilation on actinorhodin production.