Overproduction Of Metabolites Research Articles

Overproduction of microbial products—Facts and ideas

Overproduction of microbial metabolites is related to developmental phases of microorganisms. Inducers, effectors, inhibitors and various signal molecules play a role in different types of overproduction. Primary and secondary metabolism are interconnected. Biosynthesis of enzymes catalyzing metabolic reactions in microbial cells is controlled by well-known positive and negative mechanisms, e.g. induction, repression, catabolite repression, mechanisms controlling enzyme activity include isosteric and allosteric interactions, e.g. competitive and non-competitive inhibition, allosteric effects, molecular conversion etc. Biosynthesis of secondary metabolites is catalyzed by unaltered enzymes of primary metabolism, by altered enzymes of primary metabolism and by specific enzymes of secondary metabolism. In addition to classical mutagenesis and selection of suitable microbial cells, methods of molecular genetics are used in the overproduction of microbial products.

The mixed substrate concept, applied for microbial syntheses of metabolites

Microbial overproduction of metabolites is a response to suboptimal conditions for growth and multiplication. It is an energy-wasting process in terms of life insofar as a part of energy of the carbon source remains in the metabolite. From an energetic point of view microbial overproduction can be divided into two categories: i) energy-consuming, ii) energy-yielding. The amount of energy required or made available is considered to be responsible for discrepancies between carbon metabolism-determined possible and experimentally obtained yields. Since the expenditure of energy must be provided by oxidation of carbon source more substrate is consumed than required according to the metabolic pathway. In the case of energy-yielding synthesis energy must be discharged. Various possibilities exist. Since metabolic sequences not involved in the synthesis of the proper product are not switched off completely other synthetic processes and even growth can occur. The energy is thus discharged at the expense of substrate. To increase the experimental yield the energy produced or consumed has to be maintained low. This can be achieved by means of substrate mixtures. The synthesis of by-products and growth are difficult to prevent completely. However, growth can be quite desirable since the catalyst is renewed thus making the product synthesis possible.

The relationship between the energetic efficiency in different micro-organisms and the rate and type of metabolite overproduced

Data regarding the degree of energy conservation as determined by the $$Y_{O_2 }^{\max } $$ and the highest rates of metabolite production reported for various micro-organisms have been collated and analysed. The results have indicated that the highest rates of metabolite production occur in micro-organisms possessing low efficiencies of energy conservation. Moreover, in the case of exopolysaccharide production the oxidation state of the polymer is inversely related to the $$Y_{O_2 }^{\max } $$ value of the producing organism. In general, the rate of ATP turnover associated with exopolysaccharide production or the potential rate associated with over-production of other metabolites is inversely related to the $$Y_{O_2 }^{\max } $$ value of the producing organism. Analysis of current production rates for a range of metabolites suggests that there is scope for major improvements of existing processes by careful selection of appropriate micro-organisms.

Organic aciduria and butyryl CoA dehydrogenase deficiency in BALB/cByJ mice.

A metabolic screening program of inbred strains of mice has detected a marked organic aciduria in the BALB/cByJ strain. Gas chromatographic and mass spectrometric analysis identified large quantities of n-butyrylglycine plus lesser quantities of ethylmalonic acid. Crosses with the nonexcreting C57BL/6J strain indicate that this condition is inherited as an autosomal recessive trait. Independently from this screening a variant with no detectable enzyme activity of butyryl CoA dehydrogenase (BCD) in liver and kidney of the BALB/cByJ strain but not other BALB/c sublines was discovered. Data from a three-point cross indicated that the null variant maps to the structural locus for the enzyme, Bcd-1, on chromosome 5. The findings indicate that a mutation at or near Bcd-1 in the BALB/cByJ strain resulted in a biochemical abnormality manifest as the BCD deficiency. It is concluded that accumulation of butyryl CoA due to a block in the oxidation of short-chain fatty acids results in an overproduction of organic metabolites leading to the observed organic aciduria. The fact that other BALB/c substrains do not exhibit this abnormality further suggests that this disorder reflects subline divergence within the BALB/c family.

5-Fluorouracil stimulation of naphthoquinone synthesis in Nectria haematococca (Berk. & Br.) Wr.

A large number of growth inhibitors were tested for their effects on the pigmentation of Nectria haematococca. Some of them (5-fluorouracil, 5-fluorouridine, 6-azauracil, cycloheximide, nystatin, benomyl, some amino acid analogs) stimulated the synthesis of naphthoquinones. The others (fluoroacetate, dl-ethionine, puromycin, methothrexate, N-phosphonacetyl- l-aspartate, 5-fluorocytosine, and other pyrimidine analogs) were ineffective. The highest amounts of pigments were detected in cultures containing 5-fluorouracil (5FU). Maximal synthesis was obtained only on adding 5FU during early vegetative growth. The action of 5FU was neither as a mutagen nor as a naphthoquinone precursor. Uracil and thymine decreased the pigmentation-enhancing effect of 5FU. Incorporation of [2- 14C]5FU was determined and part of the radioactivity was found in nucleic acids. The significance of these observations is discussed in the light of knowledge about (1) the mechanisms of action of 5FU and (2) the relationships between growth rate and overproduction of secondary metabolites in micro-organisms.

Novel mechanisms controlling arginine metabolism in Neurospora.

Overproduction and compartmentalization of metabolites are recognized as elements of pathway control and pathway switching in the metabolism of arginine in Neurospora .

Mechanism of protection against “reperfusion injury” by aprotinin Roles of polymorphonuclear leucocytes and oxygen radicals

We have proposed that reperfusion injury results from the overproduction of reactive oxygen metabolites by PMNas a result of increased sensitivity to oxygen during the low oxygen phase. Aprotinin was demonstrated to inhibit overproduction and also radical production evoked by chemotactic peptide, 50% inhibitions occuring in the range 5–20 μM. Over this range, no effect on cell viability was observed. This inhibitory effect may provide a scientific basis for the protective effect of aprotinin in reperfusion induced injury.

Studies on the overproduction of indole-containing metabolites by a methanol-utilizing yeast

Production of indole-containing metabolites ("indoles") from methanol has been studied using a mutant ofHansenula polymorpha resistant to 5-fluorotryptophan. Whereas the wild-type culture produces only a small amount of indoles, the mutant is partially deregulated and overproduces indoles. Indoles production was studied in batch and continuous culture and in a washed-cell system. When the pH was above 4.0, indoles production was growth-associated, in both minimal and complex media, and batch or continuous culture. When the pH was below or equal to 4.0, a low phosphate concentration was found to improve production. In a phosphate-deficient washed-cell suspension system, the addition of an amino acid such as methionine at 5 mM increased specific productivity by more than 60%. Addition of cycloheximide at 50 mg/L decreased residual growth and increased maximum productivity of indoles by more than 60%. When the antibiotic was added at 1000 mg/L, growth was completely inhibited and indoles production continued for about 35 h.

ChemInform Abstract: CURRENT STRATEGIES IN THE SEARCH FOR BIOACTIVE MICROBIAL METABOLITES

New microbial metabolite screening currently requires an interdisciplinary team consisting of microbial ecologists, geneticists, molecular biologists, biochemists, microbial physiologists, fermentation technologists and chemists. The increasing difficulty in discovering new compounds of clinical potential can only be overcome by maximising the input of the above disciplines. Possible strategies for constructing a genetically diverse ‘bank’ of microorganisms, including naturally occurring and genetically manipulated strains, are reviewed. Also, the design of environmental conditions optimal for the over-production of metabolites and the types of screens that are selective for compounds of potential utility are discussed. The value of target-selective screen techniques is illustrated with experiences gained in the search for new β-lactams.