It has been discovered, surprisingly, that the novel amino acid substitutions and/or amino acid deletions of the carboxyl terminus of the serine acetyltransferase result in a diminution in the cysteine sensitivity while at the identical time permitting enough enzymic exercise to be retained. It is feasible to cut back the cysteine sensitivity of the serine acetyltransferase in vivo by producing, by the use of expression vectors, antisense RNAs which are complementary to an outlined region of the 3′ coding strand of the native or reworked 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 able to make the most of thiosulfate as a sulfur source (Sirko, A. et al., 1987, J. Gen. Microbiol. Methods for introducing mutations at particular positions within a DNA fragment are identified and are described, for instance, in the next publications: Sarkar, G., Sommer, S. S., 1990, BioTechniques 8: 404-407 describe site-particular mutagenesis utilizing PCR; Ausubel, F. M. et al., 1987, pp. Another method of producing feedback-resistant cysE alleles consists in combining totally different level mutations which result in suggestions resistance, thereby giving rise to a number of mutants possessing new properties.
For that reason, the feedback-resistant cysE alleles are preferably built-in into the genome as single copies utilizing customary methods. Strains which include cysteine-sensitive proteins, for instance prokaryotes or yeasts, are used as host strains. Since, in principle, cysteine metabolism proceeds by way of the identical metabolic route, which is known per se, in all microorganisms, and the methods to be used for preparing the novel strains are well known, for example from standard textbooks, and applicable to all microorganisms, N-Acetyl-L-Cysteine 98% manufacturers China novel strains can be prepared from any microorganisms in any respect. The invention also relates to the preparation of L-cysteine, or of products that are derived from L-cysteine, by means of cultivating novel microorganisms. The invention additionally relates to microorganisms which include the feedback-resistant cysE alleles. The present invention furthermore pertains to DNA sequences which encode novel serine acetyltransferases. The coding sequences that are present on the vector are advantageously linked to regulatory parts that are required for expressing the coding sequences to the desired extent. Sequences which encode selective markers and/or reporter genes are also preferably current on the expression vector along with the regulatory components.
An additional increase in the cysteine yield could be achieved by additionally overexpressing the sulfate-decreasing 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 during which O-acetylserine features as a ?-alanyl donor and H2 S as a ?-alanyl acceptor (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. The catalytic exercise of the totally different serine acetyltransferase enzymes is decided within 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. 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 within the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, where serine acetyltransferases having at least one mutation ideally possess an inhibitor fixed, Ki, of from 0.015 to 2.Three mM within the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, whereas serine acetyltransferases having not less than 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 eliminated are state-of-the-art (Winans et al., 1985; J. Bacteriol. It is part of the cutting-edge to dam or modify gene activity in a particular method by way of so-referred to 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-type cysE gene, is recombined on a vector using identified standard strategies for getting ready 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 suggestions-resistant serine acetyltransferase, is ideally used as the starting material for the mutagenesis.
