Effect of inoculum type on actinorhodin production by Streptomyces coelicolor A3(2)

Summary The production of actinorhodin by Streptomyces coelicolor in a defined medium was examined using spore and vegetative inocula. The spore inoculum yielded higher concentrations of biomass and actinorhodin as well as a higher maximum specific growth rate compared with the vegetative inoculum. Nevertheless, the productivity of the batch culture for actinorhodin formation with vegetative inoculum was higher than that with spore inoculum.     

Culture conditions promoting dispersed growth and biphasic production of actinorhodin in shaken cultures of Streptomyces coelicolor A3(2)

Media and culture conditions were developed for experiments on the physiology of secondary metabolism in Streptomyces coelicolor A3(2). Well dispersed mycelial growth was obtained in a buffered starch-glutamate-salts medium; a high (5%) starch concentration and addition of glass beads aided dispersal. Under the conditions developed, production of actinorhodin was suppressed during trophophase growth and began abruptly near the growth maximum.     

Metabolic flux analysis in Streptomyces coelicolor under various nutrient limitations

Metabolic flux analysis was applied to Streptomyces coelicolor continuous culture data obtained under nitrogen, phosphate, sulfate, and potassium limitations. The metabolic reaction network involved more than 200 reactions describing the major pathways as well as the secondary metabolism for the production of actinorhodin and excretion of certain metabolites. Linear programming was used for the optimization of specific growth rates and energy requirements. Two types of specific growth rates, stoichiometric and theoretical, were defined, maximized, and compared in order to investigate the microbial potential. Potassium limitation led to the largest and nitrogen limitation to the smallest difference between the stoichiometric and theoretical specific growth rates. Although the value of the maximum theoretical specific growth rate was close to that of the experimental specific growth rate with potassium limitation, this difference was the largest in the case of nitrogen limitation. Energy requirements during different nutrient limitations were also investigated. The model indicated that although the highest actinorhodin production rate was with nitrogen limitation, this was accompanied with the undesired excretion of certain metabolites.     

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.     

High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain overexpressing the pathway-specific activator gene actll-ORF4

Streptomyces lividans 1326 usually does not produce the red/blue colored polyketide actinorhodin in liquid culture even though it carries the entire actinorhodin biosynthesis gene cluster. The bacterium can be forced to produce this secondary metabolite by introducing actII-ORF4, the actinorhodin pathway-specific activator gene from Streptomyces coelicolor, on a multicopy plasmid. The production of actinorhodin by such a strain has been optimized by medium and process manipulations in fed-batch cultures. With high-yield cultivation conditions, 5 g actinorhodin/l are produced during 7 days of cultivation; or approximately 0.1 g actinorhodin/g dry weight (DW)/day in the production phase. The yield in this phase is 0.15 Cmol actinorhodin/Cmol glucose, which is in the range of 25% to 40% of the maximum theoretical yield. This high-level production mineral medium is phosphate limited. In contrast, nitrogen limitation resulted in low-level production of actinorhodin and high production of α-ketoglutaric acid. Ammonium as nitrogen source was superior to nitrate supporting an almost three times higher actinorhodin yield as well as a two times higher specific production rate. The wild-type strain lacking the multicopy plasmid did not produce actinorhodin when cultivated under any of these conditions. This work examines the actinorhodin-producing potential of the strain, as well as the necessity to improve the culture conditions to fully utilize this potential. The overexpression of biosynthetic pathway-specific activator genes seems to be a rational first step in the design of secondary metabolite overproducing strains prior to alteration of primary metabolic pathways for redirection of metabolic fluxes. Journal of Industrial Microbiology & Biotechnology (2002) 28, 103–111 DOI: 10.1038/sj/jim/7000219 Received 04 April 2001/ Accepted in revised form 30 October 2001     

Production of actinorhodin by Streptomyces coelicolor A3(2) grown in chemostat culture

Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (q(actinorhodin)) was measured. The highest q(actinorhodin) values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that q(actinorhodin) was highest at D = 0.06h(-1), which was well below the maximal D value tested (0.14 h(-1)). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577-582, 1997.     

Cell physiology and antibiotic production of Streptomyces coelicolor grown on solid medium

Summary In order to examine the physiology ofStreptomyces coelicolor when growing on solid media, we have employed a membrane overlay technique and used a new approach to extract substrate and product compounds from the agar. Comparisons made with liquid grown cultures indicate a change from non-growth associated productivity of actinorhodin in liquid culture, to growth associated production on agar plates. In contrast, the temporal control of methylenomycin production was virtually identical under both culture conditions. Considerable extracellular protein production was observed during growth on agar.     

EshA accentuates ppGpp accumulation and is conditionally required for antibiotic production in Streptomyces coelicolor A3(2)

Disruption of eshA, which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB, a close homologue of eshA, had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated. The loss of actinorhodin production in the eshA disruptant was restored by expression of a truncated relA gene, which increased the ppGpp level to the level in the wild-type strain, indicating that the reduced ppGpp accumulation in the eshA mutant was solely responsible for the loss of antibiotic production. Antibiotic production was also restored in the eshA mutant by introducing mutations into rpoB (encoding the RNA polymerase β subunit) that bypassed the requirement for ppGpp, which is consistent with a role for EshA in modulating ppGpp levels. EshA contains a cyclic nucleotide-binding domain that is essential for its role in triggering actinorhodin production. EshA may provide new insights and opportunities to unravel the molecular signaling events that occur during physiological differentiation in streptomycetes.     

Carbon flux distribution in antibiotic-producing chemostat cultures of Streptomyces lividans

The carbon metabolism of derivatives of Streptomyces lividans growing under phosphate limitation in chemostat cultures and producing the antibiotics actinorhodin and undecylprodigiosin was investigated. By applying metabolic flux analysis to a stoichiometric model, the relationship between antibiotic production, biomass accumulation, and carbon flux through the major carbon metabolic pathways (the Embden Meyerhoff Parnas and pentose-phosphate pathways) was analyzed. Distribution of carbon flux through the catabolic pathways was shown to be dependent on growth rate, as well as on the carbon and energy source (glucose or gluconate) used. Increasing growth rates promoted an increase in the flux of carbon through glycolysis and the pentose-phosphate pathway. The synthesis of both actinorhodin and undecylprodigiosin was found to be inversely related to flux through the pentose-phosphate pathway.     

Comparison of the activity of immobilised and freely suspended Streptomyces coelicolor A3(2)

Streptomyces coelicolor was immobilised naturally in porous support materials and its growth, glucose uptake and actinorhodin production were compared with freely suspended culture using defined and complex media. When the defined medium was used, the most pronounced difference between the two cultures was the accumulation of actinorhodin extracellulary in freely suspended and intracellularly in immobilised cultures. In the complex medium, however, actinorhodin was excreted by both cultures. In addition, the complex medium yielded 50 times as much actinorhodin compared to the defined medium. Further increases in product concentration were obtained by repeated batches of immobilised culture, which showed stability for at least 3 months.     

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.     

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.     

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.     

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3′,5′ monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 μM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co-ordinate antibiotic biosynthesis.