Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the -35 and -10 consensus promoter sequences were completely randomized and some variability was introduced in the -35 (position 6) and -10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB.
View Article and Find Full Text PDFJ Mol Microbiol Biotechnol
September 2010
The glycolytic enzyme phosphoglycerate mutase (PGM), which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, was examined in Lactococcus lactis with respect to its function, kinetics and glycolytic flux control. A library of strains with PGM activities ranging between 15-465% of the wild-type level was constructed by replacing the native promoter of pgm with synthetic promoters of varying strengths. The specific growth rate and glucose flux were found to be maximal at the wild-type level at which PGM had no flux control.
View Article and Find Full Text PDFSystem-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-nothing approach has been the use of a synthetic promoter library (SPL) that is based on randomized sequences flanking the consensus -10 and -35 promoter regions and allows for fine-tuning of bacterial gene expression.
View Article and Find Full Text PDFLactococcus lactis is known to be capable of respiration under aerobic conditions in the presence of haemin. In the present study the effect of respiration on ATP production during growth on different sugars was examined. With glucose as the sole carbon source, respiratory conditions in L.
View Article and Find Full Text PDFThe lactose transporter and beta-galactosidase from Streptococcus thermophilus, encoded by the lacSZ operon, were introduced into the lactose-negative strain Lactococcus lactis MG1363 and the expression of the lacSZ operon was modulated by substitution of the native promoter with randomized synthetic promoters. A series of strains with various expression levels of lacSZ were examined for their fermentation of lactose. Strains with a high expression level were found to metabolize lactose in a similar manner to S.
View Article and Find Full Text PDFThe fermentation pattern of Lactococcus lactis with altered activities of the las enzymes was examined on maltose. The wild type converted 65% of the maltose to mixed acids. An increase in phosphofructokinase or lactate dehydrogenase expression shifted the fermentation towards homolactic fermentation, and with a high level of expression of the las operon the fermentation was homolactic.
View Article and Find Full Text PDFIn Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the las operon. We used metabolic control analysis to study the role of this organization. Earlier studies have shown that, at wild-type levels, LDH has no control over glycolysis and growth rate, but high negative control over formate production (C(Jformate)LDH=-1.
View Article and Find Full Text PDFGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in Lactococcus lactis (B. Poolman, B. Bosman, J.
View Article and Find Full Text PDFLactococcus lactis MBP71 deltathyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B.
View Article and Find Full Text PDFAntonie Van Leeuwenhoek
August 2002
The understanding of control of metabolic processes requires quantitative studies of the importance of the different enzymatic steps for the magnitude of metabolic fluxes and metabolite concentrations. An important element in such studies is the modulation of enzyme activities in small steps above and below the wild-type level. We review a genetic approach that is well suited for both Metabolic Optimization and Metabolic Control Analysis and studies on the importance of a number of glycolytic enzymes for metabolic fluxes in Lactococcus lactis.
View Article and Find Full Text PDFUsing molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.
View Article and Find Full Text PDFMost genomes are much more complex than required for the minimum chemistry of life. Evolution has selected sophistication more than life itself. Could this also apply to bioenergetics? We first examine mechanisms through which bioenergetics could deliver sophistication.
View Article and Find Full Text PDFWe studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F(1) domain of the membrane-bound (F(1)F(0)) H(+)-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F(1)-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate.
View Article and Find Full Text PDFThe nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F(1) part of the membrane-bound (F(1)F(0)) H(+)-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio.
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