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 across the take a look at colony (halo) was taken to be a semiquantitative measure of the cysteine secretion by the take a look at pressure. The zone of progress of the indicator strain around the particular 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 reworked into a host strain utilizing customary methods. FIG. 5 reveals the amino acid sequence of E. coli serine acetyltransferase. By the use of utilizing the previously mentioned mutagenesis strategies, a number of nucleotides within the DNA sequence are changed such that the amino acid sequence which is now encoded by the gene exhibits no less than one mutation in the sequence area from place ninety seven up to and including the amino acid in position 273, or at least one deletion is current within the carboxyterminal sequence area beginning with the amino acid in place 227, the place position 1 is the initiating methionine in FIG. 5 (SEQ ID NO: 1) and where 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 strain, thereby guaranteeing that it is only the cysteine-insensitive serine acetyltransferase, which has been introduced into the particular strain by transformation, which is synthesized. For the determination, use was manufactured from S30 extracts (cell homogenates centrifuged at 30,000 g and 4° C. for 20 minutes) of the selected revertants, the beginning strains and the comparability strain, N-Acetyl-L-Cysteine 98% custom sourcing E. coli W3110 (ATTC 27325). Plenty of revertants had been found whose serine acetyltransferase exercise nonetheless exhibited significant residual activity (Ki worth of between 5 and 50 μM) in 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 many revertants of cysteine-auxotrophic cysE E. coli strains. Preferably, the protein sequence of a novel serine acetyltransferase accommodates the amino acid substitution of a minimum of one of the cysE mutants specified in Tab. FIG. 10 reveals a nucleotide sequence mentioned on page 21 of the specification. Determination of the enzymic exercise 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 analysis was used to verify 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 identical time allowing satisfactory enzymic activity to be retained. An enzyme activity test is carried out on the chosen strains so as 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 a minimum of one suggestions-resistant cysE allele. In order to find out the extent of the cysteine insensitivity of the altered enzyme, the secretion of cysteine by the pressure is first of all measured in a semiquantitative, so-called cross-feeding take a look at. Bacteria are ideally suitable for preparing a novel strain. L-cysteine-related products are all products that 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 doable to abolish the management at an vital biosynthetic management level, thereby amplifying the production of numerous compounds which are situated downstream of this management point. So as to find out the ability to secrete cysteine in a liquid medium by quantitatively determining cysteine, 50 selected cysE revertants have been incubated in 20 ml of customary manufacturing medium at 30° C. and at 170 rpm for 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 factor solution (cf. Subsequent to the mutagenesis, which has been carried out, for instance, as described, the mutants having the specified phenotype are chosen, for example 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 take a look at 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 each case eliminated after 24 and 48 hours and diluted, the place acceptable, and the cysteine concentration within the cell-free supernatant was decided calorimetrically using the strategy of Gaitonde, M. K. (1967), Biochem.
