Entropy balances of microbial product formation.

Considering the approach of Bermudez and Wagensberg (1986) devoted to the entropy balance of growing microorganisms some equations were developed which describe particularly the entropy balance of microbial product formation. The formula allows to determine the coefficients of resistance R(mn) and of coupling L(mn) according to rates of growth, product formation, maintenance metabolism and heat evolution assuming a linear relationship between thermodynamic fluxes and forces. In order to check the usefulness of the derived model appropriate experimental data of two microbial batch processes concerning production of L-lysine and the antibiotic nourseothricine were taken into account. The results showed similar courses of entropy balances despite different pathways of product formation which were characterized by an overshoot of entropy production at the beginning of biosynthesis of the primary and secondary metabolite. This fact was interpreted as a more general phenomenon for microorganisms under inbalanced nutritional conditions.

Use of chemostat for selection ofStreptomyces chrysomallus mutants altered in the induction of D-glucose isomerase.

D-glucose isomerase ofStreptomyces chrysomallus PL45 is inducible by D-xylose only. In mutants obtained by means of a selection procedure in a chemostat the isomerase was induced in xylose-free medium containing glucose as carbon source.

Xylose catabolism by a mutant ofStreptomyces chrysomallus altered in the regulation of D-glucose isomerase.

A mutant ofS. chrysomallus with a D-glucose isomerase inducible by D-glucose or its catabolites was characterized. In contrast to the wild-type strain it showed a decreased catabolite repression by D-glucose of D-xylose consumption and a constitutive pentose phosphate pathway as well. A hypothesis concerning altered induction pattern of its D-glucose isomerase is discussed.

Theoretically possible yields of microbial secondary products.

Balance equations for some representatives of the main biogenetic groups of secondary metabolites were formulated which provide the known and probable reactions within the biosynthetic routes. The calculated yields Yp/s max with respect to the usual substrate glucose range between 0.37 and 0.9 and, obviously, they agree well with the theoretical yields of biochemically related parts of cell dry mass. The sugar derivatives correspond to carbohydrates, the beta-lactams and shikimate derivatives to proteins, and finally, the polyketides and mevalonate derivatives to lipids. From this stoichiometric point of view the secondary metabolism thus seems to possess the same biosynthetic efficiency as adequate parts of primary metabolism under the conditions of retarded growth. The introduction of the Yp/s max values into an equation for substrate consumption regarding the growth, maintenance and product formation (van der Beek and Roels 1984) delivered yields which demonstrated that more than 70% of the glucose flowed into byproducts or waste metabolites, respectively.

Influence of inorganic phosphate on the lipid synthesis of a phosphate-deregulated mutant of Streptomyces noursei.

Phosphate-dependent changes of the mycelial lipid composition were studied in the streptothricin-producing parental strain Streptomyces noursei JA 3890 b/2 and its mutant RG 2. In contrast to its ancestor, the mutant was capable of producing the antibiotic nourseothricin even when large quantities of inorganic phosphate were present in the medium. The apparent insensitivity of the secondary metabolism to phosphate inhibition corresponds to a decreased level of phospholipids in the presence of excessive inorganic phosphate and, during phosphate limitation, to a much higher production of the alkaline phosphatases. A model is discussed which proposed the control by a common genetic element of both the phospholipid and antibiotic production.

Influence of nourseothricin on growth and secondary metabolism of Streptomyces noursei JA 3890b.

The nourseothricin-producing S. noursei strain JA 3890b possessed a high degree of resistance to its own antibiotic when grown in submerged cultures started from mycelium samples as inocula. In contrast, both the outgrowth of spores and the development of surface colonies from mycelium samples were severely inhibited in the presence of relatively low concentrations of nourseothricin, suggesting that the producer organism is susceptible to the autotoxic metabolite in particular stages of its development. Nourseothricin production by submerged cultures has been found to be independent of negative feedback regulation by the antibiotic.

[A statistical method for the determination of the yield correlation of antibiotic-producing microorganisms]. / Ein statistisches Verfahren zur Ermittlung der Leistungs-Korrelation antibioticabildender Mikroorganismen im Labor- und Technikumsmassstab.

The antibiotic yields of industrial selectants must be checked at several steps of cultivation. Here the question arises whether the activities at the different levels of cultivation are correlated. The common coefficient of correlation cannot be used because repeated determinations of the yield at one selectant result in different values. In order to have only one fixed value per selectant we define the mean value around which the observed values are varying. But these mean values cannot be observed. Thus, an adequate method of correlation similar to that in Guiard and Herrendörfer (1977) was used. The method is demonstrated in two examples: With selectants of Streptomyces noursei for streptothricin titers in 20 ml- and 20 l-cultures as well as with selectants of Penicillium chrysogenum for potency indices in surface cultures and penicillin titers in 50 ml submerged cultures, respectively. In both cases the coefficients of correlation were above 0.7.

Isolation and biological properties of arsenate-resistant strains of Streptomyces noursei.

Arsenate-resistant (AsR) clones were obtained with high frequency from colony populations of streptothricin-producing strains of Streptomyces noursei by the paper strip method. Whereas in the AsR-strains obtained from both wild type and mutant NG 13 the antibiotic production was reduced to approx. 60% of the parental level, the AsR clones isolated from colony populations of mutant UV 12 displayed increased productivity (less than or equal to 150%). However, their improved capacity to produce streptothricins was lost rapidly after repeated cell propagation in submerged cultures, suggesting that unstable genetic elements were involved in enabling S. noursei to grow in the presence of toxic concentrations of arsenate.

ATP levels in phosphate-deregulated and arsenate-resistant mutants of Streptomyces noursei.

Mycelial levels of ATP and glucose-6-phosphate were investigated in mutants of streptothricin-producing S. noursei JA 3880b differing from the wild-type strain in antibiotic formation, in the control by inorganic phosphate of the secondary metabolism, and in the resistance to growth inhibition by toxic arsenate ions. As compared with the ancestral strain, mutants exhibited a lower content of ATP in the mycelium while addition of 0.1 M arsenate to growing cultures provoked only moderate changes in the level of this high-energy metabolite. The results suggest that there exists a correlation between growth resistance to arsenate and insensitivity to phosphate inhibition of the secondary metabolism, on the one hand, and the capacity to produce streptothricin-type antibiotics, on the other.

Relation of anabolic-catabolic glucose utilization in growth-limited cultures of Streptomyces griseus.

Phenotypically different submerged mycelium conserves had been produced from a spore conserve of the HP-strain Streptomyces griseus and proofed in a product formation culture as a test system. The phenotypical characters induced on the base of the genotype proved in a cultivation cycle during 30-34 reduplications of the biomass constant. Employing the HP phenotype we investigated the possibility of economizing the substrate turnover by utilizing the anabolic potential for the synthesis of secondary substances and/or reducing the conservation catabolism during the stationary growth stage. As criteria for that served the stoichiometric turnover equation of the streptomycin biosynthesis and the quotient qO2/qGluc taking at full substrate oxidation the numerical value 6. During the stationary growth stage the relation of maintenance anabolism to maintenance catabolism in addition to the formation as secondary substances is not fixed in the tested HP phenotypes, but in a striking manner variable. The relation of by-product synthesis to secondary metabolism synthesis, too, is variable in the stationary growth stage with constant maintenance catabolism. Due to those response reactions on phenotypical manipulations an economization of the substrate turnover during the product formation stage with stationary growth is not possible in the streptomycin producer Streptomyces griseus.

Biochemical characteristics of non-streptomycin-producing mutants of Streptomyces griseus. II. Lipids and fatty acid composition of vegetative mycelia.

Five non-streptomycin-producing non-aerial-mycelium-forming mutants (Str-Amy-) of Streptomyces griseus obtained either by spontaneous degeneration or during continuous cultivation of the high-producing aerial-mycelium-forming parent strain HP (Str+Amy+) were checked with regard to the composition of mycelial lipid material. All the Str-Amy- derivatives differed from their ancestor strain HP by an increased ration of 12-methyltetradecanoic acid (aC15:0) to isopalmitic acid (iC16:0) during growth on a chemically defined medium lacking branched-chain amino acids. This finding attests alterations in the availability of precursors for the biosynthesis of methyl-branched fatty acids. The qualitative composition of phospholipids and other polar lipids in one mutant group was found to be similar to the progenitor strain but, additionally, both a yellow pigment and a neutral lipid component were produced in excess. A second type of mutant differed by its incapability to form ornithinolipids even under phosphate limitation. Changes of phospholipid composition were demonstrated in the course of fermentation. Formation of ornithinolipid was suppressed by an excess of inorganic phosphate in the medium, while the portions of phosphatidylethanolamine and cardiolipin increased strongly. Furthermore, the formation of ornithinolipids was influenced by nitrogen sources. these results suggest that the composition of membrane of S. griseus varies in dependence upon the composition of the medium and the age of the mycelium.

Biochemical characteristics of non-streptomycin-producing mutants of Streptomyces griseus. I. Role of NAD (P)-glycohydrolase in cell differentiation.

Five non-streptomycin-producing mutants of an industrial strain of Streptomyces griseus lacking aerial mycelium formation were compared with their genetic ancestor and another producing mutant with regard to the NAD(P)-glycohydrolase activity during cultivation on different media. By contrast to producing strains, all the Str- Amy- mutants displayed much lower mycelial and extracellular levels of enzyme, thus confirming earlier contentions concerning its involvement in control by phospho-adenosinediphospho-ribose of the intermediary metabolism at the sites of the citric acid cycle and the catabolism of the carbohydrates during distinct stages of cell differentiation. On the other hand, in the producing strain the biosynthesis of NAD(P)-glycohydrolase was demonstrated to depend on the regime of fermentation. Repeated stages of submerged cultivation in stirred fermentors resulted in suppression of enzyme formation without concomitant change of the mycelial capacity to produce streptomycin. This suggests that there is no direct involvement of NAD(P)-glycohydrolase in the control of antibiotic biosynthesis.

Control by phospho-adenosinediphospho-ribose of NADP-dependent isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase in Streptomyces griseus.

The metabolic function of NAD(P)-glycohydrolase in the streptomycin-producing Streptomyces griseus was investigated. Phospho-adenosinediphospho-ribose, the product of NAD(P)-glycohydrolase reaction was shown to interfere as a competitive inhibitor not only with the glucose-6-phosphate dehydrogenase (VORONINA et al. 1978) but also with the NADP-dependent isocitrate and 6-phospho-gluconate dehydrogenases. Inhibition kinetics were studied with isocitrate dehydrogenase from pig heart and 6-phosphogluconate dehydrogenase from yeast as well as with mycelial extracts of a mutant of S. griseus lacking NAD(P)-glycohydrolase.

Anabolic-catabolic glucose utilization and product formation of Streptomyces griseus.

If in stationary growth phases appearing in the submerged cultivation of Streptomyces griseus several times the consumption rates of glucose and oxygen and the rates of streptomycin formation are put in relation, the following results are obtained: The yield coefficient Ysp of glucose was below 0.1. Since from the stoichiometric equation of turnover for the biosynthesis of the streptomycin follows that the substrate and the product are in the same weight relation, it was possible to check whether the quantity of glucose that does not appear in the streptomycin is used for the energy supplying synthesis or conservation reactions. As a characteristic value was built up the quotient qo3/qgluc that in total substrate oxidations takes the numerical value 6. This quotient varied between 2.0 and 6.0 so that anabolic side reactions during the production of secondary substances were concluded from. As possibilities are discussed syntheses of analytically disregarded primary metabolites or preliminary steps of the biosynthesis of streptomycin. Due to the decrease of the enthalpy production by anabolic reaction steps in stationary growth phases follows a physiological-energetical importance of microbial product syntheses with likely evolutive action.