It has been found, surprisingly, that the novel amino acid substitutions and/or amino acid deletions of the carboxyl terminus of the serine acetyltransferase result in a diminution within the cysteine sensitivity whereas at the identical time permitting enough enzymic activity to be retained. It is possible to scale back the cysteine sensitivity of the serine acetyltransferase in vivo by producing, by means of expression vectors, antisense RNAs that 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 known (Denk, D. and Bock, A. 1987, J. Gen. Microbiol. In addition, O-acetylserine sulfhydrylase B (cysM) is ready to make the most of thiosulfate as a sulfur source (Sirko, A. et al., 1987, J. Gen. Microbiol. Methods for introducing mutations at specific positions inside a DNA fragment are identified and are described, for example, in the following publications: Sarkar, G., Sommer, S. S., 1990, BioTechniques 8: 404-407 describe site-specific mutagenesis using PCR; Ausubel, F. M. et al., 1987, pp. Another method of producing feedback-resistant cysE alleles consists in combining totally different point mutations which lead to suggestions resistance, thereby giving rise to a number of mutants possessing new properties.

For this reason, the suggestions-resistant cysE alleles are ideally built-in into the genome as single copies using customary methods. Strains which include cysteine-delicate proteins, for instance prokaryotes or yeasts, are used as host strains. Since, in principle, cysteine metabolism proceeds by the use of the same metabolic route, which is understood per se, in all microorganisms, and the techniques to be used for getting ready the novel strains are well-known, for instance from normal textbooks, and applicable to all microorganisms, novel strains can be ready from any microorganisms by any means. The invention additionally relates to the preparation of L-cysteine, or of merchandise that are derived from L-cysteine, by way of cultivating novel microorganisms. The invention additionally pertains to microorganisms which comprise the feedback-resistant cysE alleles. The current invention moreover pertains to DNA sequences which encode novel serine acetyltransferases. The coding sequences that are present on the vector are advantageously linked to regulatory parts which are required for expressing the coding sequences to the specified extent. Sequences which encode selective markers and/or reporter genes are also ideally present on the expression vector along with the regulatory components.

A further improve in the cysteine yield can be achieved by moreover overexpressing the sulfate-lowering enzymes (encoded by the genes cysD, C, N-Acetyl-L-Cysteine 98% contract manufacturing 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 during which O-acetylserine functions 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 activity of the totally different serine acetyltransferase enzymes is set 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 determined within the presence of 0.1 mM acetyl-coenzyme A and 1 mM L-serine (Kredich, N. M. 1971 and Tomkins G. M. 1966, J. Biol.

The novel serine acetyltransferases preferably have an inhibitor fixed, Ki, of from 0.005 to 2.Three mM within the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, where serine acetyltransferases having at the least one mutation preferably possess an inhibitor fixed, Ki, of from 0.015 to 2.Three mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, whereas serine acetyltransferases having a minimum of one carboxyterminal deletion ideally exhibit an inhibitor fixed, Ki, of from 0.005 to 0.03 mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA. Strategies for integrating genes into the chromosome utilizing vectors whose origins of replication have been removed are state of the art (Winans et al., 1985; J. Bacteriol. It's part of the state-of-the-art to block or modify gene exercise in a particular manner by way of so-known as reverse genetics utilizing 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 example, the wild-kind cysE gene, is recombined on a vector using recognized standard strategies for preparing recombinant DNA. The DNA of the wild-sort 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 suggestions-resistant serine acetyltransferase, is ideally used because the beginning material for the mutagenesis.