Propuesta del POTNC del centro poblado de Tuquina

According to Doull & Vining (1990a), most secondary metabolic pathways affected by phosphate are also regulated by the carbon and nitrogen sources. In such interactively regulated systems, carbon, nitrogen and/or phosphate depletion cause a decrease in the growth rate, which may itself have a role in the switch between primary and secondary metabolism (Demain, 1992). Indeed, Piret & Demain (1988) speculate that since secondary metabolite synthesis seems to be dependent on low growth rates in most systems, then maybe carbon, nitrogen and phosphate-limitation merely act to lower the growth rate and that perhaps growth rate alone is the controlling factor.

There are several examples where secondary metabolite synthesis is dictated by a number of factors. As discussed in Sections 1.3.2, 1.3.3 and 1.3.4, cephalosporin production by S. clavuligerus is affected by carbon, nitrogen and phosphate sources (Aharonowitz & Demain, 1977, 1978, 1979). Maximum production required a growth- limiting carbon source such as starch; a poorly-used nitrogen source such as asparagine; and a reduced level of phosphate. Fang & Demain (1995) demonstrated that ‘regulation reversal’ could occur under environmental modification such as oxygen restriction, such that ammonium and phosphate stimulated p-lactam production by S. clavuligerus.

Chatterjee et al. (1983) showed that chloramphenicol production by S. venezuelae was affected by carbon and nitrogen sources as well as the growth rate. Chloramphenicol production was found to be a growth-associated process and occurred in the presence of rapidly-utilised glucose and ammonium ions in defined media. Poor production accompanied fast growth, hence yields were increased by the use of growth- limiting carbon and nitrogen sources. Growth on poorer carbon sources (lactose, galactose, starch, cellobiose) increased the yield 1.7 to 2.4-fold compared to glucose. Amino acid nitrogen sources (proline, isoleucine) improved the yield- 3 to 4.5-fold compared to ammonium and 2 to 3-fold compared to nitrate ions. Higher yields were obtained by restricting nitrogen availability than by restricting carbon availability.

In a single culture, different products may respond by differing degrees to carbon, nitrogen and phosphate sources and growth rate. This lends itself to the possibility of manipulating or directing product synthesis. In an example cited by Demain (1992), Lilley and co-workers found in 1981 that production of antibiotics by S. cattleya required both reduced growth rate and nutrient deficiency. Thienamycin

synthesis required a low growth rate specifically due to phosphate-limitation; while it appeared that cephamycin formation occurred at reduced growth rates caused by any of carbon, nitrogen or phosphate depletion. Further work by Bushell & Fryday (1983) clarified these observations. It was demonstrated that the synthesis of at least three secondary metabolites by S. cattleya was initiated by specific nutrient deficiency. In a culture grown in defined medium containing glucose, ammonium ions and phosphate, product formation occurred after the peak in biomass at 33h. Melanin production began at 55h, cephamycin production at 70h and thienamycin production at 115h, corresponding to the depletion of glucose, ammonium and phosphate respectively. It was thought possible to direct secondary product synthesis and obtain sequential product formation.

Actinorhodin production by S. coelicolor is also found to be initiated by a reduction in growth rate and is sensitive to the nitrogen and phosphate sources (Doull & Vining, 1990b). It is not affected by the carbon source, beyond the availability of acetate precursors (Hodgson, 1982). Doull & Vining (1990b) grew S. coelicolor in a defined medium containing starch, glutamate and phosphate. Increasing the nitrogen and phosphate concentrations above growth-limiting levels both delayed the initiation of and reduced the rate of actinorhodin production. The nitrogen source had a greater effect than the phosphate source. The specific role of each was unclear since actinorhodin was still produced when both sources were present in excess as long as the growth rate decreased (after maximal biomass accumulation).

Further work by Hobbs et al. (1990) showed that the production of both actinorhodin and undecylprodigiosin was affected by only the nitrogen and phosphate sources. In a defined medium containing glucose and nitrate ions, undecylprodigiosin production was growth-associated while actinorhodin production was delayed until the growth rate decreased. Proline also permitted actinorhodin formation while ammonium salts did not. Actinorhodin production was prevented by as little as ImM ammonium chloride while it required over 50mM ammonium chloride to inhibit undecylprodigiosin synthesis. The authors concluded that growth rate was not a controlling factor since glycine did not support actinorhodin formation although the growth rate on glycine varied by only 10% from that on proline. Phosphate had a less extreme effect on production. Actinorhodin synthesis was inhibited by 24mM phosphate while

affected antibiotic production even when grown in media containing nitrate ions as the nitrogen source, it was concluded that phosphate had a greater effect than nitrogen, but that regulation by these sources was interactive.

Subsequently, Hobbs et al. (1992) noted that secondary metabolite synthesis by

S. coelicolor could be manipulated by ‘physiological steering’. In 1990 these authors showed that undecylprodigiosin and actinorhodin production could be separated by increasing ammonium levels in the medium. In 1992, the authors demonstrated that exclusive methylenomycin synthesis could be obtained by the use of defined media containing glucose, alanine and elevated phosphate concentrations to prevent actinorhodin synthesis. Obanye et al. (1996) reported that methylenomycin production occurred when carbon flux through the pentose phosphate pathway increased relative to other carbon metabolic pathways. The authors noted the possibility that carbon metabolic pathways might be affected by the medium phosphate concentration.

More recently, Liao et al. (1995) analysed the relationship between actinorhodin production and growth on a large number of carbon and nitrogen sources. In defined media, sources supporting rapid growth {e.g. starch and glutamate) gave a biphasic production pattern; while sources supporting slower growth {e.g. maltose and nitrate) allowed production during rapid growth. The authors concluded that actinorhodin production by S. coelicolor was associated with sub-optimal growth rate.

These reports demonstrate that although high levels of carbon, nitrogen and phosphate permitting rapid growth may interfere with product biosynthesis, it is also possible to use these effects to manipulate Streptomyces metabolism and encourage the synthesis of a desired product rather than an unwanted one.