Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed “essential raft”, where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.

Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host–microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labeling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

Ligand-directed chemistry (LDchem) sialyltransferases (ST) probes with an O-nitrobenzoxadiazole warhead enable selective fluorescent labelling of a wide range of recombinant STs and native lipooligosaccharide ST (Lst) in Neisseria gonorrhoeae.

Sialic acids (Sia) on glycoproteins and glycosphingolipids act as receptors for many respiratory viruses, including human influenza A viruses and respiroviruses, which typically bind to α2-6- and α2-3-linked Sia (2-6Sia and 2-3Sia), respectively. In view of this binding discrepancy and as the exact sialoglycoconjugates supporting respirovirus infection remain unclear, we assessed the receptor requirements for infection and binding of several respiroviruses and avian Newcastle disease virus (NDV). To this end, we employed a library of isogenic HEK293 cells displaying defined sialoglycoconjugates. Respirovirus infection was shown to strictly depend on 2-3Sia, with a preference for the terminal Siaα2-3Galβ1-4GlcNAc sialoglycotope regardless of the underlying glycoconjugate structure, whereas NDV showed a broader sialoglycotope usage. Notably, 2-6Sia enhanced respirovirus infection in low 2-3Sia contexts via heteromultivalent surface binding, which resulted in prolonged virus–receptor interactions. This heteromultivalent binding is expected to aid respirovirus infection of the 2-6Sia-rich human upper respiratory tract.

Sialyltransferases (ST) are enzymes found in among others mammals and bacteria that are responsible for producing sialylated glycans, which are known to play important roles in human health and disease. An example of the latter being the opportunistic molecular mimicry by pathogenic bacteria. However, chemical tools to study sialyltransferases have been limited to non-covalent inhibitors and probes that do not allow isolation and profiling of these important enzymes. Here we report the first ever covalent probes for ST by utilising ligand-directed chemistry. These affinity-based probes are armed with a simple to synthesise but robust O-nitrobenzoxadiazole (O-NBD) warhead, which is a lysine specific SNAr electrophilic warhead with a desirable turn-on fluorescence property. We demonstrate their high specificity when applied to label both recombinant sialyltransferases as well as native lipooligosaccharide sialyltransferase (Lst) in Neisseria gonorrhoeae, a relevant human pathogen. This new class of modular covalent ST probes, and their future iterations, could pave the way for new detailed studies of sialyltransferases in their native environment.

Covalent chemical probes are powerful tools for protein profiling and inhibitor development, but their synthesis often poses significant challenges. Here, we introduce chlorotriazine (ClTriZ) as a novel covalent warhead that enables efficient and modular synthesis of covalent probes via a fully water compatible, “double-click” direct-to-biology (D2B) platform. The ClTriZ warhead displays a labelling rate of kL = (9.0 ± 0.3) x 10-5 s−1, comparable to the established ligand-directed chemistry warheads, and when integrated into the platform supports rapid generation and screening of covalent probes and inhibitors directly with recombinant proteins or in cell lysates without purification. Applying this platform, we identified proximity-induced, context-dependent probes that selectively label multiple targets, including avidin, cereblon (CRBN) and sialyltransferases. We focused further on sialyltransferases, using the platform to rapidly iterate and identify highly ligand-specific probes that selectively label the endogenous sialyltransferase Lst in live Neisseria gonorrhoeae. Notably, these ClTriZ probes also enabled the first reported irreversible inhibition of a sialyltransferase, PmST1, with kinact / KI = 56.2 ± 6.1 M-1 s-1, overcoming the lack of covalent intermediates in SN2/SNi catalytic mechanisms, characteristic of this enzyme class. Overall, our platform provides a versatile and high-throughput route to develop covalent probes and inhibitors, with broad potential across diverse glycosyltransferases and beyond to other protein families.