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Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5’ 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

An amine transaminase was engineered for the efficient production of a chiral precursor to sacubitril, (2R,4S)-5-([1,1′-biphenyl]-4-yl)-4-amino-2-methylpentanoic acid, a key component in the blockb...

Maximal efficiency from enzyme cascades Enzymes are highly selective catalysts that can be useful for specific transformations in organic synthesis. Huffman et al. combined designer enzymes in a multistep cascade reaction (see the Perspective by O'Reilly and Ryan). The approach eliminates purification steps, recycles expensive cofactors, and couples favorable and unfavorable reactions. With the target molecule islatravir, an experimental HIV drug, they optimized five enzymes by directed evolution to be compatible with unnatural substrates and stable in the reaction conditions. Stereochemical purity was amplified at every enzymatic step, and the final synthesis was both atom economical and efficient. Science , this issue p. 1255 ; see also p. 1199

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence

One-pot reactions that combine non-enzymatic and biocatalytic transformations represent an emerging strategy in chemical synthesis. Some of the most powerful chemoenzymatic methodologies, although uncommon, are those that form a carbon–carbon (C–C) bond and a stereocenter at one of the reacting carbons, thereby streamlining traditional retrosynthetic disconnections. Here we report the one-pot, chemoenzymatic conversion of amides to enantioenriched alcohols. This transformation combines a nickel-catalyzed Suzuki–Miyaura coupling of amides in aqueous medium with an asymmetric, biocatalytic reduction to provide diarylmethanol derivatives in high yields and enantiomeric excesses. The synthetic utility of this platform is underscored by the formal syntheses of both antipodes of the pharmaceutical orphenadrine, which rely on ketoreductase enzymes that instill complementary stereoselectivities. We provide an explanation for the origins of stereoselectivity based on an analysis of the enzyme binding pockets.

A scalable and efficient synthesis of the GPR40 agonist MK-8666 was developed from a simple pyridine building block. The key step to set the stereochemistry at two centers relied on an enzymatic dynamic kinetic reduction of an unactivated ketone. Directed evolution was leveraged to generate an optimized ketoreductase that provided the desired trans alcohol in >30:1 dr and >99% ee. Further, it was demonstrated that all four diastereomers of this hydroxy-ester could be prepared in high yield and selectivity. Subsequently, a challenging intramolecular displacement was carried out to form the cyclopropane ring system with perfect control of endo/exo selectivity. The endgame coupling strategy relied on a Pd-catalyzed C–O coupling to join the headpiece chloropyridine with the benzylic alcohol tailpiece.

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT–RH over p2-NC and CA-p2. In kinetic assays, RT–RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT–RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

Carbonic anhydrase (CA) is one of nature’s fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme’s sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

An accurate force field is essential to computational protein design and protein fold prediction studies. Proper force field tuning is problematic, however, due in part to the incomplete modeling of the unfolded state. Here, we evaluate and optimize a protein design force field by constraining the amino acid composition of the designed sequences to that of a well behaved model protein. According to the random energy model, unfolded state energies are dependent only on amino acid composition and not the specific arrangement of amino acids. Therefore, energy discrepancies between computational predictions and experimental results, for sequences of identical composition, can be directly attributed to flaws in the force field's ability to properly account for folded state sequence energies. This aspect of fixed composition design allows for force field optimization by focusing solely on the interactions in the folded state. Several rounds of fixed composition optimization of the 56-residue β1 domain of protein G yielded force field parameters with significantly greater predictive power: Optimized sequences exhibited higher wild-type sequence identity in critical regions of the structure, and the wild-type sequence showed an improved Z-score. Experimental studies revealed a designed 24-fold mutant to be stably folded with a melting temperature similar to that of the wild-type protein. Sequence designs using engrailed homeodomain as a scaffold produced similar results, suggesting the tuned force field parameters were not specific to protein G.