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The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an “extein dependence,” whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting “promiscuous” inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins.

Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient protein trans-splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1α in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine 1 . As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus , which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale 2 , 3 . Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues. De novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast and in vitro chemical coupling to vinblastine is carried out, positioning yeast as a scalable platform to produce many monoterpene indole alkaloids.

Liquid–liquid phase separation (LLPS) of SynGAP and PSD-95, two abundant proteins that interact in the postsynaptic density (PSD) of neurons, has been implicated in modulating SynGAP PSD enrichment in excitatory synapses. However, the underlying regulatory mechanisms remain enigmatic. Here we report that O -GlcNAcylation of SynGAP acts as a suppressor of LLPS of the SynGAP/PSD-95 complex. We identified multiple O -GlcNAc modification sites for the endogenous SynGAP isolated from rat brain and the recombinantly expressed protein. Protein semisynthesis was used to generate site-specifically O -GlcNAcylated forms of SynGAP, and in vitro and cell-based LLPS assays demonstrated that T1306 O -GlcNAc of SynGAP blocks the interaction with PSD-95, thus inhibiting LLPS. Furthermore, O -GlcNAcylation suppresses SynGAP/PSD-95 LLPS in a dominant-negative manner, enabling sub-stoichiometric O -GlcNAcylation to exert effective regulation. We also showed that O -GlcNAc-dependent LLPS is reversibly regulated by O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA). These findings demonstrate that OGT- and OGA-catalysed O -GlcNAc cycling may serve as an LLPS-regulating post-translational modification. SynGAP and PSD-95 are two abundant proteins that form a complex and undergo liquid–liquid phase separation (LLPS) in the postsynaptic density of neurons. Now, O -GlcNAcylation of SynGAP has been found to suppress LLPS of the SynGAP/PSD-95 complex, and O -GlcNAc-dependent LLPS was also shown to be dynamically regulated by the enzymes O -GlcNAc transferase and O -GlcNAcase.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern 1 . For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings 2 . Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the m 2 6 A 2058 nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance. Structure-guided design and component-based synthesis are used to produce iboxamycin, a novel ribosome-binding antibiotic with potent activity against Gram-positive and Gram-negative bacteria.

α-Synuclein forms amyloid fibrils that are critical in the progression of Parkinson’s disease and serves as the pathological hallmark of this condition. Different posttranslational modifications have been identified at multiple sites of α-synuclein, influencing its conformation, aggregation and function. Here, we investigate how disease-related phosphorylation and O-GlcNAcylation at the same α-synuclein site (S87) affect fibril structure and neuropathology. Using semi-synthesis, we obtained homogenous α-synuclein monomer with site-specific phosphorylation (pS87) and O-GlcNAcylation (gS87) at S87, respectively. Cryo-EM revealed that pS87 and gS87 α-synuclein form two distinct fibril structures. The GlcNAc situated at S87 establishes interactions with K80 and E61, inducing a unique iron-like fold with the GlcNAc molecule on the iron handle. Phosphorylation at the same site prevents a lengthy C-terminal region including residues 73 to 140 from incorporating into the fibril core due to electrostatic repulsion. Instead, the N-terminal half of the fibril (1–72) takes on an arch-like fibril structure. We further show that both pS87 and gS87 α-synuclein fibrils display reduced neurotoxicity and propagation activity compared with unmodified α-synuclein fibrils. Our findings demonstrate that different posttranslational modifications at the same site can produce distinct fibril structures, which emphasizes link between posttranslational modifications and amyloid fibril formation and pathology. Here, the authors use cryo-EM to show that phosphorylating or O-GlcNAcylating α-synuclein on serine 87 leads to the formation of two distinct fibril structures. Both structures display reduced neurotoxicity and propagation activity.

Late-stage modification of drug molecules is a fast method to introduce diversity into the already biologically active scaffold. A notable number of analogs of mefloquine, chloroquine, and hydroxychloroquine have been synthesized, starting from the readily available active pharmaceutical ingredient (API). In the current review, all the modifications sites and reactivity types are summarized and provide insight into the chemistry of these molecules. The approaches include the introduction of simple groups and functionalities. Coupling to other drugs, polymers, or carriers afforded hybrid compounds or conjugates with either easily hydrolyzable or more chemically inert bonds. The utility of some of the compounds was tested in antiprotozoal, antibacterial, and antiproliferative assays, as well as in enantiodifferentiation experiments.

Tetrahydropapaverine (THP) and papaverine are plant natural products with clinically significant roles. THP is a precursor in the production of the drugs atracurium and cisatracurium, and papaverine is used as an antispasmodic during vascular surgery. In recent years, metabolic engineering advances have enabled the production of natural products through heterologous expression of pathway enzymes in yeast. Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products. Biosynthesis of THP and papaverine has not been achieved to date, in part because multiple pathway enzymes have not been elucidated. Here, we describe the development of an engineered yeast strain for de novo biosynthesis of THP. The production of THP is achieved through heterologous expression of two enzyme variants with activity on nonnative substrates. Through protein engineering, we developed a variant of N-methylcoclaurine hydroxylase with activity on coclaurine, enabling de novo norreticuline biosynthesis. Similarly, we developed a variant of scoulerine 9-O-methyltransferase capable of O-methylating 1-benzylisoquinoline alkaloids at the 3′ position, enabling de novo THP biosynthesis. Flux through the heterologous pathway was improved by knocking out yeast multidrug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 μg/L. Finally, we demonstrate a strategy for papaverine semisynthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce semisynthesized papaverine through oxidation of biosynthesized THP.

In this study, we describe the semisynthesis of cost-effective photosensitizers (PSs) derived from chlorophyll a containing different substituents and using previously described methods from the literature. We compared their structures when used in photodynamic inactivation (PDI) against Staphylococcus aureus, Escherichia coli, and Candida albicans under different conditions. The PSs containing carboxylic acids and butyl groups were highly effective against S. aureus and C. albicans following our PDI protocol. Overall, our results indicate that these nature-inspired PSs are a promising alternative to selectively inactivate microorganisms using PDI.

Blood-feeding arthropods produce antiinflammatory salivary proteins called evasins that function through inhibition of chemokine-receptor signaling in the host. Herein, we show that the evasin ACA-01 from the Amblyomma cajennense tick can be posttranslationally sulfated at two tyrosine residues, albeit as a mixture of sulfated variants. Homogenously sulfated variants of the proteins were efficiently assembled via a semisynthetic native chemical ligation strategy. Sulfation significantly improved the binding affinity of ACA-01 for a range of proinflammatory chemokines and enhanced the ability of ACA-01 to inhibit chemokine signaling through cognate receptors. Comparisons of evasin sequences and structural data suggest that tyrosine sulfation serves as a receptor mimetic strategy for recognizing and suppressing the proinflammatory activity of a wide variety of mammalian chemokines. As such, the incorporation of this posttranslational modification (PTM) or mimics thereof into evasins may provide a strategy to optimize tick salivary proteins for antiinflammatory applications.