Microbial preparation of substances from aromatic metabolism/I

Details Microbial preparation of substances from aromatic metabolism/I Microbial preparation of substances from aromatic metabolism/I

1999-04-26

2001-11-13

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S106000, C435S155000, C435S183000, C435S108000

Reexamination Certificate

active

06316232

ABSTRACT:

The invention relates to a process for the microbial preparation of substances, in particular aromatic amino acids.
Microbially prepared substances, such as fine chemicals, in particular aromatic amino acids, are of great economic interest, with the requirement for amino acids, for example, continuing to increase. Thus, L-phenylalanine, for example, is used for preparing medicaments and, in particular, also in the preparation of the sweetener aspartame (&agr;-L-aspartyl-L-phenylalanine methyl ester). L-tryptophan is required as a medicament and as an additive to animal feeds; there is likewise a need for L-tyrosine as a medicament and also as a raw material in the pharmaceutical industry. In addition to isolation from natural materials, biotechnological preparation is a very important method for obtaining amino acids in the desired optically active form under economically justifiable conditions. Biotechnological preparation is effected either using enzymes or using microorganisms. The latter, microbial, preparation enjoys the advantage that simple and inexpensive raw materials can be employed. Since the biosynthesis of amino acids in the cell is controlled in a wide variety of ways, however, a large number of attempts have already been made to increase product formation. Thus, amino acid analogues, for example, have been employed in order to switch off the regulation of biosynthesis. For example, mutants of
Escherichia coli
permitting an increased production of L-phenylalanine were obtained by selecting for resistance to phenylalanine analogues (GB-2,053,906). A similar strategy also led to overproducing strains of Corynebacterium (JP-19037/1976 and JP-39517/1978) and Bacillus (EP-0,138,526). Furthermore, microorganisms which have been constructed using recombinant DNA techniques are known in which the regulation of biosynthesis is likewise abolished, with the genes which encode key enzymes which are no longer subject to feedback inhibition being cloned and expressed. As a prototype, EP-0,077,196 describes a process for producing aromatic amino acids in which a 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase) which is no longer subject to feedback inhibition is overexpressed in
E. coli
. EP-0,145,156 describes an
E. coli
strain in which chorismate mutase/prephenate dehydratase are additionally overexpressed for the purpose of producing L-phenylalanine.
A feature common to the abovementioned strategies is that the intervention for improving production is restricted to the biosynthesis pathway which is specific for the aromatic amino acids. However, in order to increase production still further, efforts must be made to improve the provision of the primary metabolites, phosphoenolpyruvate (PEP) and erythrose 4-phosphate (Ery4P), which are required for producing aromatic amino acids.
PEP is an activated precursor of the glycolysis product pyruvate; Ery4P is an intermediate in the pentose phosphate pathway.
Various attempts have been made to achieve a specific improvement in the provision of the said primary metabolite Ery4P. An increased flow of carbon through the pentose phosphate pathway was achieved in
E. coli
by switching off the enzyme phosphoglucose isomerase; this resulted in tryptophan being formed (Mascarenhas D. et al., Appl. Environ. Microbiol. 57 (1991) 2995-99). U.S. Pat. No. 5,168,056 disclosed that overexpression of a transketolase, achieved by means of recombinant DNA techniques, makes it possible to obtain an increased provision of Ery4P and, as a consequence, an improvement in the formation of L-tryptophan, L-tyrosine or L-phenylalanine as products. Both Draths K. M. et al. (J. Am. Chem. Soc. 114 (1992) 3956-62) and Flores N. et al. (Nat. Biotechnol. 14 (1996) 620-3) demonstrated the same effect. However, as Feldmann has shown, simply increasing the activity of transketolase in
Zymomonas mobilis
strains lacking transaldolase activity leads to the enrichment of metabolites of the pentose phosphate pathway in the cells and, as a consequence, to negative effects on cell growth (Feldmann S. D. et al., Appl. Microbiol. Biotechnol. 38 (1992) 354-61). This physiological effect may possibly be attributable to an excessive intracellular concentration of sedoheptulose-7-phosphate. The inventors have also found a corresponding inhibition of biomass growth as a consequence of transketolase overexpression in the case of
E. coli
tal
+
strains.
The object of the invention is, therefore, to make available an alternative process for producing substances, in particular aromatic amino acids, which process is distinguished by an increased provision of intracellular metabolic intermediates for the synthesis of these substances without suffering from the above-cited disadvantages of the processes, which disadvantages are the consequence of only increasing the activity of the transketolase.
Surprisingly, this object is achieved, according to the invention, by making available a process for the microbial preparation of substances, in which process the activity of a transaldolase is increased in a microorganism which is producing these substances, with the activity of a phosphoenolpyruvate (PEP)-dependent sugar-uptake system in this microorganism being present, reduced or absent.
This result is particularly surprising in that it is in no way self-evident that transaldolases play an important role in the growth of microorganisms and in their production of substances. This particularly applies in the case of growth on hexoses. Thus, yeast strains are known, for example, which are still able to grow on glucose despite having a mutation in the transaldolase gene (Schaaff L. et al., Eur. J. Biochem. 188 (1990) 597-603). Accordingly, a transaldolase should not exert any essential influence on cell growth. This is supported by the fact that bacterial species are also known which are able to grow in the absence of a transaldolase (Feldmann S. D. et al., Appl. Microbiol. Biotechnol. 38 (1992) 354-62).
In addition, hexoses such as glucose, in contrast to xylose and other pentoses, can also be metabolized through degradation pathways other than the pentose phosphate pathway. It is therefore in no way self-evident that transaldolase has an essential function in glucose degradation.
It may also be noted that an increased flow by way of the enzymes of the pentose phosphate pathway was measured in vitro in extracts of
Saccharomyces cerevisiae
cells in which the activity of a transaldolase was increased (Senac T. et al., Appl. Environ. Microbiol. 57 (1991) 1701-6). However, the experimental conditions which were selected in this study do not permit any transfer of the results to the natural phenomena of metabolic physiology which occur in living microorganisms, in particular bacteria. In addition, it may be noted that Liao J. C. et al. (Biotechn. Bioeng. 52 (1996) 129-140 have shown that when the activities of a transaldolase and AroG are simultaneously increased, DAHP formation increases. This effect is to be ascribed mainly to the increase in AroG activity. An increase in the production of substances as a result of an increase in transaldolase activity as such is not disclosed or indicated. It is therefore in no way to be expected that there is a causal relationship between an increase in transaldolase activity in microorganisms and the provision of Ery4P for the improved production of substances. Increasing the activity (overexpression) of a transaldolase in accordance with the present invention makes available an alternative process for implementing an increased flow of carbon through the pentose phosphate pathway and consequently effecting an improved provision of the primary metabolite Ery4P. An increased quantity of Ery4P is consequently available for the microbial synthesis of substances in whose synthesis at least one intermediate of the pentose phosphate pathway, and in particular Ery4P, is involved. The inventors have shown that the improved availability of Ery4P occurs both in microorganisms in which the activity of a PEP-dependent sugar-uptake system i

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