42 research outputs found
Fine Tuning of the Lactate and Diacetyl Production through Promoter Engineering in Lactococcus lactis
Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15±0.08 mM to 9.94±0.07 mM, and the corresponding diacetyl production increased from 1.07±0.03 mM to 4.16±0.06 mM with the intracellular NADH/NAD+ ratios varying from 0.711±0.005 to 0.383±0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H2O-forming NADH oxidase activity led to 76.95% lower H2O2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H2O2 accumulation and prolong cell survival
Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats
The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key
regulator of metabolism, stress resistance and longevity. Apart from its role as
an important redox carrier, NAD+ also serves as the sole substrate for
NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an
important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins
which play an important role in a wide variety of processes, including
senescence, apoptosis, differentiation, and aging. We examined the effect of
aging on intracellular NAD+ metabolism in the whole heart, lung, liver and
kidney of female wistar rats. Our results are the first to show a significant
decline in intracellular NAD+ levels and NAD∶NADH ratio in all organs
by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These
changes in [NAD(H)] occurred in parallel with an increase in lipid
peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in
total antioxidant capacity in these organs. An age dependent increase in DNA
damage (phosphorylated H2AX) was also observed in these same organs. Decreased
Sirt1 activity and increased acetylated p53 were observed in organ tissues in
parallel with the drop in NAD+ and moderate over-expression of Sirt1
protein. Reduced mitochondrial activity of complex I–IV was also observed
in aging animals, impacting both redox status and ATP production. The strong
positive correlation observed between DNA damage associated NAD+ depletion
and Sirt1 activity suggests that adequate NAD+ concentrations may be an
important longevity assurance factor
Extraction and Measurement of NAD(P)+ and NAD(P)H
Nicotinamide adenine dinucleotides are critical redox-active substrates for countless catabolic and anabolic reactions. Ratios of NAD+ to NADH and NADP+ to NADPH are therefore considered key indicators of the overall intracellular redox potential and metabolic state. These ratios can be measured in bulk conditions using a highly sensitive enzyme cycling-based colorimetric assay (detection limit at or below 0.05 μM or 1 pmol) following a simple extraction procedure involving solutions of acid and base. Special considerations are necessary to avoid measurement artifacts caused by the presence of endogenous redox-active metabolites, such as phenazines made by diverse Pseudomonas species (see Chapter 25)