Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

The oligopeptide transport system (Opp) of Lactococcus lactis belongs to the class of binding protein-dependent ABC-transporters. This system has the unique capacity to mediate the uptake of peptides from 4 up to at least 18 residues. Kinetic analysis of peptide binding to the binding protein, OppA, revealed a relationship between the peptide dissociation constants and the length of the ligand. The dissociation constants varied from submicromolar for dodecapeptides to millimolar for pentapeptides. This implies that the residues 6-12 of the peptide contribute to the binding affinity, and, in contrast to the current views on peptide binding by homologous proteins, these residues must interact with OppA. Analysis of pre-steady-state kinetics of binding showed that the observed differences in the -values result primarily from variations in the dissociation rate constants. These results are discussed in relation to the affinity constant for transport of these substrates. Overall, the data suggest that the slow dissociation rate constants for the larger peptides are rate determining in the translocation of peptides across the membrane.

No abstract

The kinetic properties of wild-type and mutant oligopeptide binding proteins of Lactococcus lactis were determined. To observe the properties of the mutant proteins in vivo, the oppA gene was deleted from the chromosome of L. lactis to produce a strain that was totally defective in oligopeptide transport. Amplified expression of the oppA gene resulted in an 8-to 12-fold increase in OppA protein relative to the wild-type level. The amplified expression was paralleled by increased bradykinin binding activity, but had relatively little effect on the overall transport of bradykinin via Opp. Several site-directed mutants were constructed on the basis of a comparison of the primary sequences of OppA from Salmonella enterica serovar Typhimurium and L. lactis, taking into account the known structure of the serovar Typhimurium protein. Putative peptide binding-site residues were mutated. All the mutant OppA proteins exhibited a decreased binding affinity for the high-affinity peptide bradykinin. Except for OppA(D471R), the mutant OppA proteins displayed highly defective bradykinin uptake, whereas the transport of the low-affinity substrate KYGK was barely affected. Cells expressing OppA(D471R) had a similar K m for transport, whereas the V max was increased more than twofold as compared to the wild-type protein. The data are discussed in the light of a kinetic model and imply that the rate of transport is determined to a large extent by the donation of the peptide from the OppA protein to the translocator complex.

No abstract

Although glucose is an inexpensive substrate widely used as a carbon source in Escherichia coli recombinant fermentation technology, 10-30% of the carbon supply is wasted by excreting acetate. In addition to the loss of carbon source, the excretion of a weak acid may result in increased energetic demands and hence a decreased yield. Because glucose can enter the cell via several transport systems, isogenic strains defective in one or two of these transport systems were constructed. The effects of changes in the glucose uptake capacity on the in vivo flux distribution to a desired end product (beta-galactosidase) and to acetate were studied. The lack of one of the components (IICB(Glc) protein) of the glucose-phosphoenolpyruvate phosphotransferase system (Glc-PTS) reduced the growth rate significantly. The maintenance of a low-copy plasmid in this strain resulted in further arrest of the growth rate. However, beta-galactosidase production had no effect on growth rate. This strain directed more carbon into biomass and carbon dioxide, and less into acetate. Beta-galactosidase was produced in amounts not significantly different from the wild-type strain from half the amount of glucose. An explanation for the experimental results is given, making use of published results on metabolic regulation.

No abstract

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600 MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400 MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600 MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600 MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600 MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600 MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics.