Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase.

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme's global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme's access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

Enabling high-power, broadband THz generation with 800-nm pump wavelength.

The organic terahertz (THz) generation crystal BNA has recently gained traction as a source for producing broadband THz pulses. When pumped with 100 fs pulses, the thin BNA crystals can produce relatively high electric fields with frequency components out to 5 THz. However, the THz output with 800-nm pump wavelength is limited by the damage threshold of the material, particularly when using a 1 kHz or higher repetition rate laser. Here, we report that the damage threshold of BNA THz generation crystals can be significantly improved by bonding BNA to a high-thermal conductivity sapphire window. When pumped with 800-nm light from an amplified Ti:sapphire laser system, this higher damage threshold enables generation of 2.5× higher electric field strengths compared to bare BNA crystals. We characterize the average damage threshold for bare BNA and BNA-sapphire, measure peak-to-peak electric field strengths and THz waveforms, and determine the nonlinear transmission in BNA. Pumping BNA bonded to sapphire with 3 mJ 800-nm pulses results in peak-to-peak electric fields exceeding 1 MV/cm, with broadband frequency components >3 THz. This high-field, broadband THz source is a promising alternative to tilted pulse front LiNbO3 THz sources, enabling many research groups without optical parametric amplifiers to perform high-field, broadband THz spectroscopy.

Boron-Templated Dimerization of Allylic Alcohols To Form Protected 1,3-Diols via Acid Catalysis.

We report an unprecedented boron-templated dimerization of allylic alcohols that generates a 1,3-diol product with two stereogenic centers in high yield and diastereoselectivity. This acid-catalyzed reaction is achieved via in situ formation of a boronic ester intermediate that facilitates selective cyclization and formation of a cyclic boronic ester product. High yields are observed with a variety of allylic alcohols, and mechanistic studies confirm the role of boron as a template for the reaction.