The amino acid acetyl-l-carnitine, or ALCAR, outcomes from the synthesis of carnitine, while the amino acid cysteine leads to NAC. The radius of the feeding auriole around the test colony (halo) was taken to be a semiquantitative measure of the cysteine secretion by the test strain. The zone of development of the indicator pressure round the actual inoculation streak (halo) serves as a semiquantitative measure of the cysteine secretion. For the aim of expressing the altered serine acetyltransferase enzyme, the suggestions-resistant cys-E alleles are reworked into a host strain utilizing customary strategies. FIG. 5 exhibits the amino acid sequence of E. coli serine acetyltransferase. By the use of utilizing the previously talked about mutagenesis strategies, one or more nucleotides in the DNA sequence are changed such that the amino acid sequence which is now encoded by the gene exhibits at the least one mutation within the sequence area from place 97 up to and together with the amino acid in position 273, or no less than one deletion is present in the carboxyterminal sequence area beginning with the amino acid in place 227, the place place 1 is the initiating methionine in FIG. 5 (SEQ ID NO: 1) and the place the mutation of Met to Ile in position 256 is excluded.

Preferably, the gene of the native, cysteine-delicate serine acetyltransferase is inactivated within the host strain, thereby making certain that it's only the cysteine-insensitive serine acetyltransferase, which has been launched into the actual pressure by transformation, which is synthesized. For the dedication, use was manufactured from S30 extracts (cell homogenates centrifuged at 30,000 g and 4° C. for 20 minutes) of the selected revertants, the starting strains and the comparison strain, E. coli W3110 (ATTC 27325). Numerous revertants had been found whose serine acetyltransferase activity still exhibited vital residual activity (Ki value of between 5 and 50 μM) within the presence of differing concentrations of the inhibitor, L-cysteine. Mutants possessing the desired properties (insensitivity of the serine acetyltransferase to cysteine) are sought among the revertants of cysteine-auxotrophic cysE E. coli strains. Preferably, the protein sequence of a novel serine acetyltransferase comprises the amino acid substitution of a minimum of one of the cysE mutants specified in Tab. FIG. 10 shows a nucleotide sequence mentioned on web page 21 of the specification. Determination of the enzymic activity and the inhibitor fixed, Ki, and likewise the cross-feeding take a look at, had been carried out in analogy with the description in Example 1. DNA sequence analysis was used to affirm that the deletions had been accurately introduced.

It has been discovered, 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 while at the same time allowing ample enzymic exercise to be retained. An enzyme activity take a look at is carried out on the selected strains in order to find out the extent of the cysteine tolerance of the altered serine acetyltransferase. Such strains of microorganisms are characterized by the fact that they possess a cysteine metabolism which is deregulated by at the very least one feedback-resistant cysE allele. So as to determine the extent of the cysteine insensitivity of the altered enzyme, the secretion of cysteine by the pressure is to begin with measured in a semiquantitative, so-referred to as cross-feeding check. Bacteria are preferably suitable for getting ready a novel strain. L-cysteine-associated merchandise are all products that are derived from L-cysteine, i.e. sulfur-containing compounds which require L-cysteine for their preparation. The feedback-resistant cys-E alleles render it doable to abolish the control at an vital biosynthetic control point, thereby amplifying the N-Acetyl-L-Cysteine 98% production of a lot of compounds that are situated downstream of this control level. In order to determine the ability to secrete cysteine in a liquid medium by quantitatively determining cysteine, 50 selected cysE revertants were incubated in 20 ml of commonplace manufacturing medium at 30° C. and at 170 rpm for a period of forty eight hours.

The usual manufacturing medium consisted of 15 g/L glucose, 0.08 g/L bactotryptone, 0.04 g/L yeast extract, 5 mg/L vitamin B1, three g/L KH2 PO4, 12 g/L K2 HPO4, 0.3 g/L MgSO4×7 H2 O, 0.1 g/L NaCl, 5 g/L (NH4)2 SO4, 14.7 mg/L CaCl2×2 H2 O, 2 mg/L FeSO4×2 H2 O, 1 g/L Na3 citrate×2 H2 O, 5 g/L Na2 S2 O3×5 H2 O and 1 ml/L trace component answer (cf. Subsequent to the mutagenesis, which has been carried out, for example, as described, the mutants having the specified phenotype are chosen, for instance by means of plating on to cysteine-free medium and subsequently figuring out the extent to which the mutated serine acetyltransferase is sensitive to cysteine. The above-described enzyme check is appropriate for figuring out the cysteine sensitivity of any serine acetyltransferase enzyme, together with the enzymes which have a modified carboxyl terminus. A pattern (10 μl) was in each case removed after 24 and forty eight hours and diluted, the place applicable, and the cysteine focus within the cell-free supernatant was determined calorimetrically using the tactic of Gaitonde, M. K. (1967), Biochem.