Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli

O-methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power. In this study, we developed an in vitro prototyping workflow that allowed us to screen ~30 enzymes against five substrates in three days with high reproducibility. We combined in vitro transcription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus-far uncharacterized O-methyltransferases for future use as biocatalysts

Band and momentum dependent electron dynamics in superconducting Ba(Fe1−xCox)2As2{\rm Ba(Fe_{1-x}Co_{x})_2As_2} as seen via electronic Raman scattering

We present details of carrier properties in high quality ${\rm Ba(Fe_{1-x}Co_{x})_2As_2}$ single crystals obtained from electronic Raman scattering. The experiments indicate a strong band and momentum anisotropy of the electron dynamics above and below the superconducting transition highlighting the importance of complex band-dependent interactions. The presence of low energy spectral weight deep in the superconducting state suggests a gap with accidental nodes which may be lifted by doping and/or impurity scattering. When combined with other measurements, our observation of band and momentum dependent carrier dynamics indicate that the iron arsenides may have several competing superconducting ground states.Comment: 5 pages, 4 figure

Pinpointing Gap Minima in Ba(Fe0.94_{0.94}Co0.06)2_{0.06})_{2}As2_2 \textit{via} Band Structure Calculations and Electronic Raman Scattering

A detailed knowledge of the gap structure for the Fe-pnictide superconductors is still rather rudimentary, with several conflicting reports of either nodes, deep gap minima, or fully isotropic gaps on the Fermi surface sheets, both in the $k_{x}-k_{y}$ plane and along the c-axis. In this paper we present considerations for electronic Raman scattering which can help clarify the gap structure and topology using different light scattering geometries. Using density functional calculations for the Raman vertices, it is shown that the location of the gap minima may occur on loops stretching over a portion of the c-axis in Ba(Fe$_{0.94}$Co$_{0.06})_{2}$As$_2$.Comment: 4+ pages, three figure

Genome sequencing and molecular networking analysis of the wild fungusAnthostomella pineareveal its ability to produce a diverse range of secondary metabolites

Background Filamentous fungi are prolific producers of bioactive molecules and enzymes with important applications in industry. Yet, the vast majority of fungal species remain undiscovered or uncharacterized. Here we focus our attention to a wild fungal isolate that we identified as Anthostomella pinea. The fungus belongs to a complex polyphyletic genus in the family of Xylariaceae, which is known to comprise endophytic and pathogenic fungi that produce a plethora of interesting secondary metabolites. Despite that, Anthostomella is largely understudied and only two species have been fully sequenced and characterized at a genomic level.Results In this work, we used long-read sequencing to obtain the complete 53.7 Mb genome sequence including the full mitochondrial DNA. We performed extensive structural and functional annotation of coding sequences, including genes encoding enzymes with potential applications in biotechnology. Among others, we found that the genome of A. pinea encodes 91 biosynthetic gene clusters, more than 600 CAZymes, and 164 P450s. Furthermore, untargeted metabolomics and molecular networking analysis of the cultivation extracts revealed a rich secondary metabolism, and in particular an abundance of sesquiterpenoids and sesquiterpene lactones. We also identified the polyketide antibiotic xanthoepocin, to which we attribute the anti–Gram-positive effect of the extracts that we observed in antibacterial plate assays.Conclusions Taken together, our results provide a first glimpse into the potential of Anthstomella pinea to provide new bioactive molecules and biocatalysts and will facilitate future research into these valuable metabolites

A balancing act: Evidence for a strong subdominant d-wave pairing channel in Ba0.6K0.4Fe2As2{\rm Ba_{0.6}K_{0.4}Fe_2As_2}

We present an analysis of the Raman spectra of optimally doped ${\rm Ba_{0.6}K_{0.4}Fe_2As_2}$ based on LDA band structure calculations and the subsequent estimation of effective Raman vertices. Experimentally a narrow, emergent mode appears in the $B_{1g}$ ($d_{x^2-y^2}$) Raman spectra only below $T_c$, well into the superconducting state and at an energy below twice the energy gap on the electron Fermi surface sheets. The Raman spectra can be reproduced quantitatively with estimates for the magnitude and momentum space structure of the s$_{+-}$ pairing gap on different Fermi surface sheets, as well as the identification of the emergent sharp feature as a Bardasis-Schrieffer exciton, formed as a Cooper pair bound state in a subdominant $d_{x^2-y^2}$ channel. The binding energy of the exciton relative to the gap edge shows that the coupling strength in this subdominant $d_{x^2-y^2}$ channel is as strong as 60% of that in the dominant $s_{+-}$ channel. This result suggests that $d_{x^2-y^2}$ may be the dominant pairing symmetry in Fe-based sperconductors which lack central hole bands.Comment: 10 pages, 6 Figure

The Pairing Mechanism in HTSC investigated by Electronic Raman Scattering

By means of electronic Raman scattering we investigated the symmetry of the energy gap Delta(k), its temperature dependence and its variation with doping of well characterized Bi2Sr2CaCu2O8+delta single crystals. The oxygen content delta was varied between the under- and the overdoped regime by subsequently annealing the same single crystal in Ar and O2, respectively. The symmetry analysis of the polarized electronic Raman scattering is consistent with a d_x^2-y^2-wave symmetry of the energy gap in both regimes. The gap ratio 2Delta_max/k_BT_c and its temperature dependence changes with doping similar to predictions of theories based on paramagnon coupling.Comment: 3 pages, LaTeX, 2 ps figures available on request to [email protected]