It has been found, surprisingly, that the novel amino acid substitutions and/or amino acid deletions of the carboxyl terminus of the serine acetyltransferase lead to a diminution within the cysteine sensitivity whereas at the same time allowing ample enzymic exercise to be retained. It is possible to cut back the cysteine sensitivity of the serine acetyltransferase in vivo by producing, by means of expression vectors, antisense RNAs which are complementary to an outlined area of the 3′ coding strand of the native or remodeled cysE gene. The gene for serine acetyltransferase has already been cloned and the amino acid sequence which is deduced from the DNA sequence is understood (Denk, D. and Bock, A. 1987, J. Gen. Microbiol. As well as, O-acetylserine sulfhydrylase B (cysM) is ready to make the most of thiosulfate as a sulfur supply (Sirko, A. et al., 1987, J. Gen. Microbiol. Methods for introducing mutations at particular positions within a DNA fragment are recognized and are described, for instance, in the following publications: Sarkar, G., Sommer, S. S., 1990, BioTechniques 8: 404-407 describe site-particular mutagenesis using PCR; Ausubel, F. M. et al., 1987, pp. Another methodology of producing feedback-resistant cysE alleles consists in combining different level mutations which result in feedback resistance, thereby giving rise to multiple mutants possessing new properties.
Because of this, the suggestions-resistant cysE alleles are preferably built-in into the genome as single copies using customary methods. Strains which contain cysteine-sensitive proteins, for instance prokaryotes or yeasts, are used as host strains. Since, in principle, cysteine metabolism proceeds by the use of the identical metabolic route, which is known per se, in all microorganisms, and the techniques for use for making ready the novel strains are well-known, for instance from standard textbooks, and relevant to all microorganisms, novel strains might be ready from any microorganisms whatsoever. The invention additionally relates to the preparation of L-cysteine, or of products which are derived from L-cysteine, by means of cultivating novel microorganisms. The invention additionally pertains to microorganisms which comprise the suggestions-resistant cysE alleles. The current invention furthermore relates to DNA sequences which encode novel serine acetyltransferases. The coding sequences that are current on the vector are advantageously linked to regulatory elements that are required for expressing the coding sequences to the desired extent. Sequences which encode selective markers and/or reporter genes are also preferably present on the expression vector along with the regulatory components.
An additional improve in the cysteine yield will be achieved by additionally overexpressing the sulfate-lowering enzymes (encoded by the genes cysD, C, H, G, I and J) and the sulfhydrating enzymes (encoded by the genes cysK and cysM). The formation of L-cysteine itself is catalyzed by two O-acetylserine sulfhydrylase isoenzymes (EC 4.2.99.8), encoded by the genes cysK (O-acetylserine sulfhydrylase A) and cysM (O-acetylserine sulfhydrylase B), a reaction by which O-acetylserine capabilities as a β-alanyl donor and H2 S as a β-alanyl acceptor (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. The catalytic exercise of the totally different serine acetyltransferase enzymes is determined in the presence and absence of L-cysteine, and the inhibitor constant, Ki, is ascertained from this (Kredich and Tomkins, J. Biol. 1.1×10-6 M was decided within the presence of 0.1 mM acetyl-coenzyme A and 1 mM L-serine (Kredich, N-Acetyl-L-Cysteine 98% API suppliers N. M. 1971 and Tomkins G. M. 1966, J. Biol.
The novel serine acetyltransferases ideally have an inhibitor fixed, Ki, of from 0.005 to 2.3 mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, where serine acetyltransferases having at the least one mutation ideally possess an inhibitor constant, Ki, of from 0.015 to 2.3 mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, while serine acetyltransferases having not less than one carboxyterminal deletion preferably exhibit an inhibitor fixed, Ki, of from 0.005 to 0.03 mM within the presence of 1 mM L-serine and 0.1 mM acetyl-CoA. Strategies for integrating genes into the chromosome using vectors whose origins of replication have been removed are cutting-edge (Winans et al., 1985; J. Bacteriol. It is a part of the state of the art to dam or modify gene activity in a selected method via so-called reverse genetics using antisense RNA (Inouye, 1988, Gene 72: 25-34). Antisense RNA is the transcription product of the DNA strand which is complementary to the strand encoding the protein. The starting DNA fragment, encompassing, for instance, the wild-kind cysE gene, is recombined on a vector utilizing identified standard techniques for preparing recombinant DNA. The DNA of the wild-type cysE gene, or a cysE gene which has been inactivated by mutation, or a cysE gene which has been mutated and which already encodes a feedback-resistant serine acetyltransferase, is ideally used because the starting material for the mutagenesis.
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