Glucose Metabolism in
            <i>Lactococcus lactis</i>
            MG1363 under Different Aeration Conditions: Requirement of Acetate To Sustain Growth under Microaerobic Conditions

Knowledge of how the activity of enzymes is affected under in vivo conditions is essential for analyzing their regulation and constructing models that yield an integrated understanding of cell behavior. Current kinetic parameters for Lactococcus lactis are scattered through different studies and performed under different assay conditions. Furthermore, assay conditions often diverge from conditions prevailing in the intracellular environment. To establish uniform assay conditions that resemble intracellular conditions, we analyzed the intracellular composition of anaerobic glucose-limited chemostat cultures of L. lactis subsp. cremoris MG 1363. Based on this, we designed a new assay medium for enzyme activity measurements of growing cells of L. lactis, mimicking as closely as practically possible its intracellular environment. Procedures were optimized to be carried out in 96-well plates, and the reproducibility and dynamic range were checked for all enzyme activity measurements. The effects of freezing and the carryover of ammonium sulfate from the addition of coupling enzymes were also established. Activities of all 10 glycolytic and 4 fermentative enzymes were measured. Remarkably, most in vivo-like activities were lower than previously published data. Yet, the ratios of V max over measured in vivo fluxes were above 1. With this work, we have developed and extensively validated standard protocols for enzyme activity measurements for L. lactis.

Section: Resultsmentioning

confidence: 99%

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

Goel

Santos

Vos

et al. 2012

“…These results show that acetyl-CoA levels were sufficient to sustain a base-level ester production, but the equal levels of productivity of L. lactis NZ9000 clones producing different SAAT levels indicates that acetyl-CoA levels may limit ester formation. Glucose, which was used as a carbon source in our experiments, is converted mainly to L-lactate, whereas approximately 2% is converted to acetyl-CoA; ultimately, ethanol, acetate, or biomass formation occurs (16,50). In our clones, at least three enzymes may compete for acetyl-CoA pools (phosphotransacetylase, acetaldehyde/alcohol dehydrogenase, and SAAT), and the relative amount of acetyl-CoA used for ester production depends on the amounts and kinetic parameters of these enzymes.…”

Section: Discussionmentioning

confidence: 99%

Expression of Plant Flavor Genes in Lactococcus lactis

Hernández

Molenaar

Beekwilder³

et al. 2007

Lactic acid bacteria, such as Lactococcus lactis, are attractive hosts for the production of plant-bioactive compounds because of their food grade status, efficient expression, and metabolic engineering tools. Two genes from strawberry (Fragaria x ananassa), encoding an alcohol acyltransferase (SAAT) and a linalool/nerolidol synthase (FaNES), were cloned in L. lactis and actively expressed using the nisin-induced expression system. The specific activity of SAAT could be improved threefold (up to 564 pmol octyl acetate h ؊1 mg protein ؊1 ) by increasing the concentration of tRNA 1 Arg , which is a rare tRNA molecule in L. lactis. Fermentation tests with GM17 medium and milk with recombinant L. lactis strains expressing SAAT or FaNES resulted in the production of octyl acetate (1.9 M) and linalool (85 nM) to levels above their odor thresholds in water. The results illustrate the potential of the application of L. lactis as a food grade expression platform for the recombinant production of proteins and bioactive compounds from plants.

“…The lower activity of PDH without riboflavin could limit the supply of acetyl-CoA, which is essential for fatty acid biosynthesis, and thereby limit growth. One way to overcome this limitation is to add acetate to the medium, which can be taken up and converted into acetylCoA (40). Indeed, by adding acetate (15 mM), it was possible to stimulate growth in the absence of riboflavin at 37°C (Fig.…”

Section: Effect Of Riboflavin On Growth and Fluxes At High Temperaturesmentioning

confidence: 99%

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Chen

Shen

Solem

et al. 2013

Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen. Lactococcus lactis is a mesophilic bacterium with an optimum growth temperature around 30°C (1) that is widely used in the dairy industry for production of cheese and buttermilk, where it plays a crucial role in flavor and texture formation. During cheese production, L. lactis is frequently exposed to a variety of stresses, such as heat stress (2). Superoptimal temperatures cause the denaturation of macromolecules and induce chaperone proteins, such as DnaK, GroEL, and GroES, which then help proteins to fold correctly (2). The chaperones are essential for growth at elevated temperatures, and mutants with defects in chaperone activity display a pronounced temperature-sensitive phenotype (3). The opposite effect is sometimes seen when overexpressing chaperones, and in L. lactis this results in improved fitness and lactic acid production at high temperatures (4, 5). The viability of some L. lactis strains at high temperatures is also improved by an incorporation of high concentrations of NaCl into the medium or a preadaptation prior to inoculation (6), and this phenomenon was demonstrated to be correlated with an induction of several chaperones (7). Meanwhile, metabolic responses are regarded as important factors in handling heat stress. It has been demonstrated that genes in several metabolic pathways are important for handling heat stress (8), and a recently recognized c-di-AMP-specific phosphodiesterase in L. lactis was also found to be important for heat tole...