The amino acid acetyl-l-carnitine, or ALCAR, outcomes from the synthesis of carnitine, whereas the amino acid cysteine results in NAC. The radius of the feeding auriole around the check colony (halo) was taken to be a semiquantitative measure of the cysteine secretion by the check strain. The zone of growth of the indicator strain round the actual inoculation streak (halo) serves as a semiquantitative measure of the cysteine secretion. For the purpose of expressing the altered serine acetyltransferase enzyme, the suggestions-resistant cys-E alleles are remodeled into a host pressure using customary strategies. FIG. 5 exhibits the amino acid sequence of E. coli serine acetyltransferase. By the use of using the previously mentioned mutagenesis strategies, one or more nucleotides within the DNA sequence are changed such that the amino acid sequence which is now encoded by the gene exhibits at the least one mutation in the sequence area from place 97 up to and including the amino acid in position 273, or at the least one deletion is present within the carboxyterminal sequence area starting with the amino acid in position 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 in the host pressure, 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 willpower, 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). Plenty of revertants had been discovered whose serine acetyltransferase exercise still exhibited significant residual exercise (Ki value of between 5 and 50 μM) within the presence of differing concentrations of the inhibitor, L-cysteine. Mutants possessing the specified 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 at least one of the cysE mutants specified in Tab. FIG. 10 exhibits a nucleotide sequence mentioned on page 21 of the specification. Determination of the enzymic activity and the inhibitor constant, Ki, and in addition the cross-feeding take a look at, had been carried out in analogy with the outline in Example 1. DNA sequence evaluation was used to affirm that the deletions had been appropriately 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 in the cysteine sensitivity whereas at the same time permitting sufficient enzymic exercise to be retained. An enzyme activity test is carried out on the chosen 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 truth that they possess a cysteine metabolism which is deregulated by no less than one suggestions-resistant cysE allele. In order to determine the extent of the cysteine insensitivity of the altered enzyme, the secretion of cysteine by the pressure is initially measured in a semiquantitative, so-called cross-feeding check. Bacteria are ideally appropriate for making ready a novel pressure. L-cysteine-related merchandise are all merchandise which are derived from L-cysteine, i.e. sulfur-containing compounds which require L-cysteine for their preparation. The suggestions-resistant cys-E alleles render it attainable to abolish the management at an vital biosynthetic management point, thereby amplifying the manufacturing of numerous compounds which are situated downstream of this management level. So as to find out the ability to secrete cysteine in a liquid medium by quantitatively figuring out cysteine, 50 chosen cysE revertants have been incubated in 20 ml of normal production medium at 30° C. and at 170 rpm N-Acetyl-L-Cysteine 98% for supplements a period of forty eight hours.

The usual production medium consisted of 15 g/L glucose, 0.08 g/L bactotryptone, 0.04 g/L yeast extract, 5 mg/L vitamin B1, 3 g/L KH2 PO4, 12 g/L K2 HPO4, 0.Three 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 element solution (cf. Subsequent to the mutagenesis, which has been carried out, for instance, as described, the mutants having the desired phenotype are selected, for instance by the use 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 suitable for determining the cysteine sensitivity of any serine acetyltransferase enzyme, together with the enzymes which have a modified carboxyl terminus. A sample (10 μl) was in every case eliminated after 24 and forty eight hours and diluted, the place acceptable, and the cysteine focus in the cell-free supernatant was determined calorimetrically utilizing the method of Gaitonde, M. K. (1967), Biochem.