High-level heterologous production of propionate in engineered Escherichia coli

A propanologenic (i.e., 1-propanol-producing) bacterium Escherichia coli strain was previously derived by activating the genomic sleeping beauty mutase (Sbm) operon. The activated Sbm pathway branches out of the tricarboxylic acid (TCA) cycle at the succinyl-CoA node to form propionyl-CoA and its derived metabolites of 1-propanol and propionate. In this study, we targeted several TCA cycle genes encoding enzymes near the succinyl-CoA node for genetic manipulation to identify the individual contribution of the carbon flux into the Sbm pathway from the three TCA metabolic routes, that is, oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt. For the control strain CPC-Sbm, in which propionate biosynthesis occurred under relatively anaerobic conditions, the carbon flux into the Sbm pathway was primarily derived from the reductive TCA branch, and both succinate availability and the SucCD-mediated interconversion of succinate/succinyl-CoA were critical for such carbon flux redirection. Although the oxidative TCA cycle normally had a minimal contribution to the carbon flux redirection, the glyoxylate shunt could be an alternative and effective carbon flux contributor under aerobic conditions. With mechanistic understanding of such carbon flux redirection, metabolic strategies based on blocking the oxidative TCA cycle (via ∆sdhA mutation) and deregulating the glyoxylate shunt (via ∆iclR mutation) were developed to enhance the carbon flux redirection and therefore propionate biosynthesis, achieving a high propionate titer of 30.9 g/L with an overall propionate yield of 49.7% upon fed-batch cultivation of the double mutant strain CPC-Sbm∆sdhA∆iclR under aerobic conditions. The results also suggest that the Sbm pathway could be metabolically active under both aerobic and anaerobic conditions.     

2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii

The acetogenic bacterium Acetobacterium woodii is able to reduce CO₂ to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD⁺-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO₂ to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.     

Highly efficient somatic-mutation identification using Escherichia coli mismatch-repair detection

The discovery of somatic mutations in cancer tissue is extremely laborious, time-consuming and costly. In an evaluation comparing mismatch repair detection (MRD) against Sanger sequencing for somatic-mutation detection, we found that MRD had a specificity of 96% and a sensitivity of 92%. Our results showed that MRD is a robust and cost-effective alternative to Sanger sequencing for identifying somatic mutations in human tumors.     

Evaluation of NMR spectral data of urine in conjunction with measured clinical chemistry and histopathology parameters to assess the effects of liver and kidney toxicants

Single low and high doses of several compounds with known renal toxic effects (para-aminophenol, puromycin aminonucleoside, sodium chromate, and hexachlorobutadiene,) or known liver toxic effects (galactosamine, allyl alcohol, and thioacetamide) were administered to male Wistar rats in groups of 4 or 8 for each compound. Predose urine samples (Day 0) and samples from post-dosing (Days 1–4) were collected for each rat and monitored by 1D ¹H NMR. Principal component analysis (PCA) of the NMR spectra was used to investigate differences between dose levels for each compound individually. The findings from PCA at both dose levels for each compound were examined in the context of the corresponding clinical chemistry and pathology data collected during the study. The PCA clustering of NMR spectra from rats dosed with each individual compound were shown to be associated with the measured levels of creatinine, BUN, AST, ALT and histopathology findings. Finally, scaled-to-maximum, aligned, and reduced trajectories (SMART) analysis was applied to compare the temporal metabolic trajectories obtained for each animal at each dose level of the administered compounds. By day 4, the SMART trajectories for allyl alcohol and hexachlorobutadiene had returned to predose levels indicating a recovery response, however, the high dose SMART trajectories for para-aminophenol, puromycin aminonucleoside, sodium chromate, and galactosamine did not appear to return to predose levels indicating a prolonged toxic effect.     

An integrated approach for the comparative analysis of a multigene family: The nicotianamine synthase genes of barley

Recent genomic projects reveal that about half of the gene repertoire in plant genomes is made up by multigene families. In this paper, a set of structural and phylogenetic analyses have been applied to compare the differently sized nicotianamine synthase (NAS) gene families in barley and rice. Nicotianamine acts as a chelator of iron and other heavy metals and plays a key role in uptake, phloem transport and cytoplasmic distribution of iron, challenging efforts for the breeding of iron-efficient crop plants. Nine barley NAS genes have been mapped, and co-linearity of flanking genes in barley and rice was determined. The combined analyses reveal that the NAS multigene family members in barley originated through at least one duplication event that occurred before the divergence of rice and barley. Additional duplications appear to have occurred within each of the species. Although we detected no evidence for positive selection of recently duplicated genes within species, codon-based tests revealed evidence for positive selection having contributed to the divergence of some amino acids. The integrated comparative and phylogenetic analysis improved our current view of NAS gene family evolution, might facilitate the functional characterization of individual members and is applicable to other multigene families. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Comparison of blood pro/antioxidant levels before and after acute exercise in athletes and non-athletes

The aims of our study were to assess the redox state of adolescent athletes and non-athletes both at rest and after acute exposure to physical load and to find relations between parameters of redox state and morphofunctional characteristics of subjects. 58 young handball players and 37 non-athletes were subjected to body composition analysis, measuring of maximal oxygen consumption and blood sampling immediately before and after a maximal progressive exercise test. At rest, athletes had significantly higher superoxide dismutase (SOD) and catalase (CAT) activity, higher levels of glutathione (GSH) and nitric oxide (NO) and lower levels of lipid peroxidation (TBARS) compared with non-athletes. A maximal exercise test induced statistically significant rise of superoxide anion radical (O2-), hydrogen peroxide (H2O2) and NO levels in non-athletes, while TBARS levels decreased. Athletes experienced the fall in NO levels and the fall in CAT activity. After exercise, athletes had significantly lower levels of O2- compared with non-athletes. Two way repeated measures ANOVA showed that the response of O2-, NO and TBARS to the exercise test was dependent on the sports engagement (training experience) of subjects. Significant correlations between morphofunctional and redox parameters were found. These results suggest that physical fitness affects redox homeostasis.     

The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinson's disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.     

Prostaglandin E2 stimulates p53 transactivational activity through specific serine 15 phosphorylation in human synovial fibroblasts. Role in suppression of c/EBP/NF-kappaB-mediated MEKK1-induced MMP-1 expression

Cyclooxygenase-2 (COX-2) overexpression has been linked to cell survival, transformation, and hyperproliferation. We examined the regulation of the tumor suppressor gene p53 and p53 target genes by prostaglandin E(2) (PGE(2)) in human synovial fibroblasts (HSF). PGE(2) induced a time-dependent increase in p53 Ser(15) phosphorylation, with no discernible change in overall p53 levels. PGE(2)-dependent Ser(15) phosphorylation was apparently mediated by activated p38 MAP kinase as SB202190, a p38 kinase inhibitor, blocked the response. Overexpression of a MKK3 construct, but not MKK1, stimulated SB202190-sensitive p53 Ser(15) phosphorylation. PGE(2)-stimulated [phospho-Ser(15)]p53 transactivated a p53 response element (GADD45)-luciferase reporter in transiently transfected HSF (SN7); the effect was compromised by overexpression of a dominant-negative mutant (dnm) of p53 or excess p53S15A expression plasmid but mimicked by a constitutively active p53S15E expression construct. PGE(2), wtp53 expression in the presence of PGE(2), and p53S15E suppressed steady-state levels of MEKK1-induced MMP-1 mRNA, effects nullified with co-transfection of p53 dnm or p53S15A. MEKK1-induced MMP-1 promoter-driven luciferase activity was largely dependent on a c/EBPbeta-NF-kappaB-like enhancer site at -2008 to -1972 bp, as judged by deletion and point mutation analyses. PGE(2), overexpression of p53wt with PGE(2), or p53S15E abolished the MEKK1-induced MMP-1 promoter luciferase activity. Gel-shift/super gel-shift analyses identified c/EBPbeta dimers and c/EBPbeta/NF-kappaB p65 heterodimers as binding species at the apparent site of MEKK1-dependent transactivation. PGE(2)-stimulated [phospho-Ser(15)]p53 abrogated the DNA binding of c/EBPbeta dimers and c/EBPbeta/NF-kappaB p65 heterodimers. Our data suggest that COX-2 prostaglandins may be implicated in p53 function and p53 target gene expression.     

Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump which converts atmospheric oxygen to water and couples the oxygen reduction reaction to the creation of a membrane proton gradient. The structure of the enzyme has been solved; however, the mechanism of proton pumping is still poorly understood. Recent calculations from this group indicate that one of the histidine ligands of enzyme's CuB center, His291, may play the role of the pumping element. In this paper, we report on the results of calculations that combined first principles DFT and continuum electrostatics to evaluate the energetics of the key energy generating step of the model-the transfer of the chemical proton to the binuclear center of the enzyme, where the hydroxyl group is converted to water, and the concerted expulsion of the proton from delta-nitrogen of His291 ligand of CuB center. We show that the energy generated in this step is sufficient to push a proton against an electrochemical membrane gradient of about 200 mV. We have also re-calculated the pKa of His291 for an extended model in which the whole Fe(a3)-CuB center with their ligands is treated by DFT. Two different DFT functionals (B3LYP and PBE0), and various dielectric models of the protein have been used in an attempt to estimate potential errors of the calculations. Although current methods of calculations do not allow unambiguous predictions of energetics in proteins within few pKa units, as required in this case, the present calculation provides further support for the proposed His291 model of CcO pump and makes a specific prediction that could be targeted in the experimental test.