Biomedical subjects

I van Alen-Boerrigter

Publications and source records attributed to I van Alen-Boerrigter.

4 recordsLinked to original sources

PubMed1996

Food-grade cloning and expression system for Lactococcus lactis.

A versatile set of cloning and expression vectors has been developed for application in self-cloning and other genetic modifications of Lactococcus lactis. The expression vectors were equipped with the controlled and strong lacA promoter of the lactococcal lactose operon. In addition, the transcriptional terminator of the aminopeptidase N gene, pepN, was inserted, which in some cases increased the genetic stabilities of the vectors and the cloned DNA. The small, 0.3-kb lacF gene encoding the soluble carrier enzyme IIALac was used as a dominant selection marker in the plasmid-free L. lactis strain NZ3000 carrying an in-frame deletion of the chromosomal lacF gene. Lactose-utilizing transformants were easily selected on lactose indicator plates at high frequencies and showed a copy number of approximately 50 plasmids per cell. All vectors were stably maintained in the lacF strain NZ3000 when grown on lactose, and only the high-level expression vectors showed some instability when their host was grown on glucose-containing medium. The application potentials of the expression vectors carrying the lacF marker were determined by cloning of the promoterless Escherichia coli gusA reporter gene under control of the lacA promoter followed by analysis of its expression. While in one of the vectors this resulted in a promoter-down mutation in the -10 region of the lacA promoter, in other vectors high-level and controlled expression of the gusA gene was observed.

Base Sequence↗

PubMed1996

Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis.

The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG 1363, which is a nisin-transposon- and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.

Base Sequence↗

PubMed1995

Metabolic engineering of Lactococcus lactis: influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions.

The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1363 was cloned on a multicopy plasmid under the control of the inducible L. lactis lacA promoter. More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L. lactis under inducing conditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene. The effect of alpha-acetolactate synthase overproduction on the formation of end products in various L. lactis strains was studied under different fermentation conditions. Under aerobic conditions and with an initial pH of 6.0, overexpression of the als gene resulted in significant acetoin production that amounted to more than one-third of the pyruvate converted. However, the effect of the alpha-acetolactate synthase overproduction was even more pronounced in the lactate dehydrogenase-deficient strain L. lactis NZ2700. Anaerobic cultivation of this strain resulted in a doubling of the butanediol formation of up to 40% of the converted pyruvate. When cultivated aerobically at an initial pH of 6.8, overexpression of the als gene in L. lactis NZ2700 resulted in the conversion of more than 60% of the pyruvate into acetoin, while no butanediol was formed. Moreover, at an initial pH of 6.0, similar amounts of acetoin were obtained, but in addition approximately 20% of the pyruvate was converted into butanediol. These metabolic engineering studies indicate that more than 80% of the lactose can be converted via the activity of the overproduced alpha-acetolactate synthase in L. lactis.

Acetolactate Synthase↗

PubMed1993

Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis.

The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase. Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23). Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged. The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site. Several mutant SK11 proteinases have novel properties not previously encountered in natural variants. Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity. Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase. Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.

Amino Acid Sequence↗