Towards the bioproduction of methacrylic acid: a case study on the use of decarboxylases

In this study, the biocatalytic production of methacrylic acid (MAA), a commodity chemical with a large global market, was investigated. The target was to develop MAA formation via enzymatic decarboxylation of itaconic, mesaconic, citraconic and citramalic acid. Several candidate enzymes were tested, but none catalysed the target reactions. Therefore, a novel high throughput screening method for decarboxylases was developed to enable protein engineering. The screening assay detected gaseous CO2 with a pH-dependent colorimetric reaction, in a 96-well format. Focused mutagenesis was then performed on the mevalonate diphosphate decarboxylases (MVD) from Saccharomyces cerevisiae and from Picrophilus torridus. However, MAA formation was not observed, even after screening over 3800 variants. Further investigation was then performed on some of the candidates to understand why MAA was not formed and to develop alternative strategies for enzyme selection. P. torridus MVD was discovered to belong to a new class of enzymes, mevalonate-3-kinase, and to be part of a novel archaeal mevalonate pathway. The enzyme also catalysed the formation of isobutene from 3-hydroxyisovalerate with unprecedented production rates. S. cerevisiae and Aspergillus niger phenylacrylic acid decarboxylases, previously believed to be cofactor-free enzymes, were proposed to require divalent metals and a novel organic cofactor. The enzymatic mechanism of Pseudomonas fluorescens α-amino-β-carboxymuconate-ε-semialdehyde (ACMS) decarboxylase was investigated. Through inhibition studies, it was demonstrated that the aldehyde and amino moieties of ACMS could be directly involved in catalysis, in contrast to the previously reported mechanism. This information partially explains why some of these candidates could not catalyse the target reaction, and provides insights into their potential future use for the bioproduction of MAA. Although this route does not seem feasible at present, this study gives foundation and guidance for future investigations

Proteome changes in the skin of the grape cultivar Barbera among different stages of ripening

<p>Abstract</p> <p>Background</p> <p>Grape ripening represents the third phase of the double sigmoidal curve of berry development and is characterized by deep changes in the organoleptic characteristics. In this process, the skin plays a central role in the synthesis of many compounds of interest (<it>e.g</it>. anthocyanins and aroma volatiles) and represents a fundamental protective barrier against damage by physical injuries and pathogen attacks. In order to improve the knowledge on the role of this tissue during ripening, changes in the protein expression in the skin of the red cultivar Barbera at five different stages from <it>véraison </it>to full maturation were studied by performing a comparative 2-DE analysis.</p> <p>Results</p> <p>The proteomic analysis revealed that 80 spots were differentially expressed throughout berry ripening. Applying a two-way hierarchical clustering analysis to these variations, a clear difference between the first two samplings (up to 14 days after <it>véraison</it>) and the following three (from 28 to 49 days after <it>véraison</it>) emerged, thus suggesting that the most relevant changes in protein expression occurred in the first weeks of ripening. By means of LC-ESI-MS/MS analysis, 69 proteins were characterized. Many of these variations were related to proteins involved in responses to stress (38%), glycolysis and gluconeogenesis (13%), C-compounds and carbohydrate metabolism (13%) and amino acid metabolism (10%).</p> <p>Conclusion</p> <p>These results give new insights to the skin proteome evolution during ripening, thus underlining some interesting traits of this tissue. In this view, we observed the ripening-related induction of many enzymes involved in primary metabolism, including those of the last five steps of the glycolytic pathway, which had been described as down-regulated in previous studies performed on whole fruit. Moreover, these data emphasize the relevance of this tissue as a physical barrier exerting an important part in berry protection. In fact, the level of many proteins involved in (a)biotic stress responses remarkably changed through the five stages taken into consideration, thus suggesting that their expression may be developmentally regulated.</p

Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway

Bio-production of fuels and chemicals from lignocellulosic C5 sugars usually requires the use of the pentose phosphate pathway (PPP) to produce pyruvate. Unfortunately, the oxidation of pyruvate to acetyl-coenzyme A results in the loss of 33 % of the carbon as CO2, to the detriment of sustainability and process economics. To improve atom efficiency, we engineered Escherichia coli to utilize d-xylose constitutively using the Weimberg pathway, to allow direct production of 2-oxoglutarate without CO2 loss. After confirming enzyme expression in vitro, the pathway expression was optimized in vivo using a combinatorial approach, by screening a range of constitutive promoters whilst systematically varying the gene order. A PPP-deficient (ΔxylAB), 2-oxoglutarate auxotroph (Δicd) was used as the host strain, so that growth on d -xylose depended on the expression of the Weimberg pathway, and variants expressing Caulobacter crescentus xylXAB could be selected on minimal agar plates. The strains were isolated and high-throughput measurement of the growth rates on d-xylose was used to identify the fastest growing variant. This strain contained the pL promoter, with C. crescentus xylA at the first position in the synthetic operon, and grew at 42 % of the rate on d-xylose compared to wild-type E. coli using the PPP. Remarkably, the biomass yield was improved by 53.5 % compared with the wild-type upon restoration of icd activity. Therefore, the strain grows efficiently and constitutively on d-xylose, and offers great potential for use as a new host strain to engineer carbon-efficient production of fuels and chemicals via the Weimberg pathway

The putative mevalonate diphosphate decarboxylase from Picrophilus torridus is in reality a mevalonate-3-kinase with high potential for bioproduction of isobutene

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway.MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme’s potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min-1 g cells-1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min-1 mg protein-1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway

17β-estradiol effects on human coronaries and grafts employed in myocardial revascularization: a preliminary study

Background: This study was undertaken to compare the in vitro effects of 17β-estradiol on human epicardial coronary arteries, resistance coronary arteries and on arterial vessels usually employed as grafts in surgical myocardial revascularization. Methods: Coronary artery rings (descending coronary artery, right coronary artery, circumflex coronary artery, first septal branch) and arterial graft rings (internal thoracic artery, gastro-epiploic artery) obtained from human heart donors with heart not suitable to cardiac transplantation were connected to force transducer for isometric force recording. Precontracted specimens with and without endothelium were exposed to increasing concentration of 17β-estradiol (3–30–300–3000 nmol/l) and to vehicle (0.1% v/v ethanol). We also evaluated the effects of 17β-estradiol on vessels before and 20 minutes after exposure to L-monomethyl-arginine and indomethacin. Results: 17β-estradiol induced a significant relaxation in all precontracted vessels (mean maximum effect: 78,6% ± 8,5). This effect was not different among the different rings and was not related to the presence of endothelium. N-monomethyl-L-arginine and indomethacin did not modify 17β-estradiol relaxant effect. Conclusion: The vasodilator action of the 17β-estradiol is similar on coronary arteries, resistance coronary arteries and arterial vessels usually employed as grafts in myocardial revascularization

Assessment of 29 candidate genes for milk traits in Italian dairy cattle

Several investigations have recently searched for significant association between gene polymorphisms and milk traits in livestock and model species. In several cases, it remains rather difficult to assess if the observed effects are caused by the mutation tested, by a nearby mutation in the same gene or by a mutation in a different gene or DNA region in linkage disequilibrium with the former. As a consequence, only in a few cases (e.g., κ-casein, SCD, DGAT1) the causative mutation seems to have been identified and, even when evidence is rather clear, genetic heterogeneity and genetic background may influence the size of allele substitution effects. Therefore, the significance of gene-trait associations and the estimate of their effect have to be verified in any new population in which this information is planned to be used, to estimate its actual utility in gene assisted breeding. In the SelMol project, we selected 29 candidate genes on the basis of known relationships between physiological or biochemical processes and evidence of significant association with milk traits in cattle, in related (e.g., sheep and goats) and model (e.g., mouse) species. A total of 106 SNPs were selected, using either information available in literature, or in silico, searching the NCBI dbSNP database. SNPs found significantly associated in other investigations were preferentially targeted. Otherwise non-synonymous SNPs and those in putative control regions (e.g., in promoter binding sites) were selected from dbSNP. If within a gene no SNP having one of these characteristics was available in dbSNP, synonymous SNPs, occurring in introns and untranslated non-control regions were chosen. DNA was extracted from semen of elite sires. SNPs polymorphism was confirmed by screening a panel of 32 individuals each of Pezzata Rossa (PR), Bruna Italiana (BI), and Frisona Italiana (FI) dairy cattle breeds. A total of 73 SNPs were confirmed as polymorphic in at least one breed: 63 in PR, 61 in BI, and 68 in FI. Polymorphic SNPs were genotyped on 400 individuals of PR and 600 of BI. Statistical tests were applied to detect selection sweeps, significant association to EBVs and phenotypic traits related to milk production and quality (milk yield, protein and fat yield and percentage), together with a number of functional traits (fertility, SCS as indicator of mastitis resistance, conformational traits, and milkability)

Combined fit to the spectrum and composition data measured by the Pierre Auger Observatory including magnetic horizon effects

The measurements by the Pierre Auger Observatory of the energy spectrum and mass composition of cosmic rays can be interpreted assuming the presence of two extragalactic source populations, one dominating the flux at energies above a few EeV and the other below. To fit the data ignoring magnetic field effects, the high-energy population needs to accelerate a mixture of nuclei with very hard spectra, at odds with the approximate E$^{-2}$ shape expected from diffusive shock acceleration. The presence of turbulent extragalactic magnetic fields in the region between the closest sources and the Earth can significantly modify the observed CR spectrum with respect to that emitted by the sources, reducing the flux of low-rigidity particles that reach the Earth. We here take into account this magnetic horizon effect in the combined fit of the spectrum and shower depth distributions, exploring the possibility that a spectrum for the high-energy population sources with a shape closer to E$^{-2}$ be able to explain the observations