Biosynthetic methods have the potential to deliver value-added chemicals from renewable feedstocks. However, despite major advances in metabolic engineering and synthetic biology, the rapid engineering of microbes to deliver high yields and titers of target compounds remains as a challenge. Here, we disclose a new chemical catalysis-based strategy for expanding the types of products available from unmodified microorganisms. By combining Gluconobacter oxidans as a whole cell biocatalyst in a single pot with a lysine organocatalyst, we demonstrate that aqueous solutions of Cn n-aliphatic alcohols are converted to C2n α,β-unsaturated aldehydes in a single pot in mild conditions. This carbon-doubling reaction works with a range of C2–C6 alcohol substrates. In the absence of the lysine organocatalyst, only n-aliphatic carboxylic acids are observed, indicating that the organocatalyst intercepts metabolic intermediates and redirects flux toward target chemicals. Taken together, our work reveals a new strategy of flux redirection to expand the scope of products from biosynthetic processes.

In the past two years, the COVID‐19 pandemic has caused over 5 million deaths and 250 million infections worldwide. Despite successful vaccination efforts and emergency approval of small molecule therapies, diverse antivirals are still needed to combat the inevitable viral resistance that will arise. The main protease of SARS‐CoV‐2 (Mpro) is an attractive drug target due to the clinical success of protease inhibitors against other viruses, such as HIV and HCV. However, in order to combat resistance, diverse chemical scaffolds need to be identified that have the potential to be developed into potent inhibitors. To this end, we screened a high‐content protease inhibitor library against Mpro in vitro, in order to identify structurally diverse compounds that may be further developed into antiviral leads. Our high‐content screening efforts retrieved 27 hits each with >50% inhibition in our Mpro FRET assay. Of these, three natural product compounds, anacardic acid, aloesin, and aloeresin D, were chosen for follow up IC50 screening due to their strong druglike properties (high bioavailability, low toxicity). Further analysis via dose response curves revealed IC50 values of 6.8 μM, 38.9 μM, and 125.3 μM respectively. Molecular docking studies demonstrated that the three inhibitors bound at the catalytic active site of Mpro with varying binding energies (‐7.8 to ‐5.6 kcal/mol). Furthermore, we determined that our kinetic data from the Mpro FRET assay fit better to a sigmoidal Hill model than the standard Michaelis‐Menten hyperbola, indicating substantial cooperativity between enzyme monomers, suggesting that the dimerization interface could be an attractive target for allosteric inhibitors. In conclusion, we identified two natural product compounds from the Aloe plant (aloesin and aloeresin D) as novel scaffolds for Mpro inhibitor design and confirmed the cooperative kinetics of Mpro. These results advance our knowledge of structure‐function relationships in Mpro and offer new molecular scaffolds for inhibitor design.

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Nevirapine (NVP), a non‐nucleoside HIV reverse transcriptase (RT) inhibitor, is generally a well‐tolerated drug. However, certain toxicological issues have been reported, including idiosyncratic drug reactions (IDRs) such as skin rash and hepatotoxicity. Additionally, NVP is a known mechanism‐based inactivator (MBI) and inducer of CYP3A cytochrome P450 enzymes (CYPs), with the potential to cause drug‐drug interactions. It has been demonstrated previously that the 12‐hydroxynevirapine metabolite and a reactive quinone methide species, produced by CYP3A4 and CYP2B6, are responsible for the observed IDRs. The reactive quinone methide species also leads to mechanism‐based inactivation of CYP3A4, while NVP itself is an inducer of both CYP3A4 and CYP2B6. The goal of our study was to characterize a series of NVP analogs with respect to their safety and efficacy to treat HIV by examining target engagement efficiency and metabolic liability through in vitro characterization. Our hypothesis was that a NVP analog will be able to inhibit RT with similar efficacy, but with limited production of the toxic metabolites. Initial studies were conducted to determine the IC50 values of NVP and each analog in the RT inhibition assay, followed by determining the CYP induction/inhibition potential of NVP and its associated analogs in cryopreserved primary human hepatocytes. mRNA fold induction, CYP3A4 activity fold change, and % lactate dehydrogenase (LDH) release were all studied via qPCR, LC‐MS/MS, and LDH assays respectively. In total, NVP and eight analogs were tested. The RT assay IC50 of NVP was 0.5 uM and the three analogs with the lowest IC50 values were: nevirapine‐d3 (NVP‐d3, 0.8 uM), 2‐nitro nevirapine (2‐N‐NVP, 1.1 uM), and 3‐bromo nevirapine (3‐B‐NVP, 3.0 uM). The induction experiments demonstrated that CYP3A4 was induced ~2 fold at 25 uM NVP, and ~6 fold at 100 uM NVP. CYP2B6 showed higher induction, with ~5 fold at 25 uM NVP and ~13 fold at 100 uM NVP. For CYP3A4 activity, 25 uM NVP showed ~1.4 fold increase in activity, while 100 uM NVP resulted in no fold change. This is likely due to the combined induction/inhibition effect of NVP; i.e., at higher concentrations more NVP is present to inactivate CYP3A4 and thus inhibition wins out over induction, while at lower concentrations of NVP induction dominates. %LDH release along with observation of the monolayer revealed low levels of hepatocyte mortality. Overall, three analogs were identified as potential candidates to replace NVP, due to their low IC50 values in the RT inhibition assay and reduced potential for CYP induction. Further characterization of these analogs is ongoing with the goal to identify an efficacious and safer NVP analog that can be used to treat HIV in pregnant women and children in developing countries, where NVP is still often used.