Galen P. Miley

A scalable platform to identify fungal secondary metabolites and their gene clusters

The genomes of filamentous fungi contain up to ~90 biosynthetic gene clusters (BGCs), encoding diverse secondary metabolites, an enormous reservoir of untapped chemical potential. However, recalcitrant genetics, cryptic expression, and unculturability prevent the systematic exploitation of these gene clusters and harvesting of their products. With heterologous expression of fungal BGCs largely limited to expression of single or partial clusters, we established a scalable process for expression of large numbers of full-length gene clusters, called FAC-MS. Using Fungal Artificial Chromosomes (FACs) with Metabolomic Scoring (MS) we screened 56 secondary metabolite BGCs from diverse fungal species for expression in A. nidulans. Fifteen new metabolites were discovered and confidently assigned to their BGCs. A new macrolactone, valactamide A, and its hybrid PKS-NRPS gene cluster were characterized extensively using this integrated platform. Regularizing access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

Total Synthesis of Tambromycin Enabled by Indole C–H Functionalization

The total synthesis of tambromycin (1), a recently isolated tetrapeptide, is reported. This unusual natural product possesses a highly modified tryptophan-derived indole fragment fused to an α-methylserine-derived oxazoline ring, and a unique noncanonical amino acid residue named tambroline (11). A convergent synthesis of tambromycin was achieved by a 13-step route that leveraged recent developments in the field of C-H functionalization to prepare the complex indole fragment, as well as an efficient synthesis of tambroline that featured a diastereoselective amination of homoproline.

Complex EUV imaging reflectometry: spatially resolved 3D composition determination and dopant profiling with a tabletop 13nm source

With increasingly 3D devices becoming the norm, there is a growing need in the semiconductor industry and in materials science for high spatial resolution, non-destructive metrology techniques capable of determining depth-dependent composition information on devices. We present a solution to this problem using ptychographic coherent diffractive imaging (CDI) implemented using a commercially available, tabletop 13 nm source. We present the design, simulations, and preliminary results from our new complex EUV imaging reflectometer, which uses coherent 13 nm light produced by tabletop high harmonic generation. This tool is capable of determining spatially-resolved composition vs. depth profiles for samples by recording ptychographic images at multiple incidence angles. By harnessing phase measurements, we can locally and nondestructively determine quantities such as device and thin film layer thicknesses, surface roughness, interface quality, and dopant concentration profiles. Using this advanced imaging reflectometer, we can quantitatively characterize materials-sciencerelevant and industry-relevant nanostructures for a wide variety of applications, spanning from defect and overlay metrology to the development and optimization of nano-enhanced thermoelectric or spintronic devices.

Sub-wavelength transmission and reflection mode tabletop imaging with 13nm illumination via ptychography CDI

EUV lithography is promising for addressing upcoming, <10nm nodes for the semiconductor industry, but with this promise comes the need for reliable metrology techniques. In particular, there is a need for actinic mask inspection in which the imaging wavelength matches that of the intended lithography process, so that the most relevant defects are detected. Here, we demonstrate tabletop, ptychographic, coherent diffraction imaging (CDI) in reflection- and transmission-modes of extended samples, using a 13 nm high harmonic generation (HHG) source. We achieve the first sub-wavelength resolution EUV image (0.9λ) in transmission, the highest spatial resolution using any 13.5 nm source to date. We also present the first reflection-mode image obtained on a tabletop using 12.7 nm light. This work represents the first 12.7 nm reflection-mode image using any source of a general sample.

Wide Field-of-View Reflection-Mode Ptychographic Imaging Microscope with Tabletop 12.7 nm High Harmonic Illumination

High-resolution imaging is an invaluable tool for understanding nanoscale systems. In particular, tabletop extreme ultraviolet (EUV) coherent diffractive imaging (CDI) techniques based on high harmonic generation (HHG) can combine femtosecond (fs) pulse durations with nanometer resolution and elemental-, spin-, electronicand magnetic-sensitivity, proving to be an ideal probe of complex nanostructured systems [1-2]. In this work, we demonstrate a novel technique for glancing-incidence, large field-of-view, reflection-mode ptychographic imaging using a tabletop 12.7nm HHG source. To our knowledge, this is the first demonstration of reflection-mode imaging at 12.7nm on a tabletop, as well as the first ~13nm reflection-mode image using any source of an extended sample of general composition (ie., not fabricated on a multilayer mirror).