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Biomechanical cues are instrumental in guiding embryonic development and cell differentiation. Understanding how these physical stimuli translate into transcriptional programs could provide insight into mechanisms underlying mammalian pre-implantation development. Here, we explore this by exerting microenvironmental control over mouse embryonic stem cells (ESCs). Microfluidic encapsulation of ESCs in agarose microgels stabilized the naïve pluripotency network and specifically induced expression of Plakoglobin (Jup), a vertebrate homologue of β-catenin. Indeed, overexpression of Plakoglobin was sufficient to fully re-establish the naïve pluripotency gene regulatory network under metastable pluripotency conditions, as confirmed by single-cell transcriptome profiling. Finally, we found that in the epiblast, Plakoglobin was exclusively expressed at the blastocyst stage in human and mouse embryos – further strengthening the link between Plakoglobin and naïve pluripotency in vivo. Our work reveals Plakoglobin as a mechanosensitive regulator of naïve pluripotency and provides a paradigm to interrogate the effects of volumetric confinement on cell-fate transitions.Highlights3D agarose spheres stabilize the naïve pluripotency network in mouse ESCs.Volumetric confinement induces expression of Plakoglobin, a vertebrate homologue of β-catenin.Plakoglobin expression in the epiblast is specific to pre-implantation human and mouse embryos.Plakoglobin overexpression maintains naïve pluripotency independently of β-catenin.

Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method—termed SpliMLiB for Split-and-Mix Library on Beads—was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.

Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of non-canonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of intermediates shifts to favor covalently bound Trp adducts, which is associated with increased thermodynamic stability of these species. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a sub-domain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.

Self-assembling protein cages are useful as nanoscale molecular containers for diverse applications in biotechnology and medicine. To expand the utility of such systems, there is considerable interest in customizing the structures of natural cage-forming proteins and designing new ones. Here we report that a circularly permuted variant of lumazine synthase, a cage-forming enzyme from Aquifex aeolicus (AaLS) affords versatile building blocks for the construction of nanocompartments that can be easily produced, tailored, and diversified. The topologically altered protein, cpAaLS, self-assembles into spherical and tubular cage structures with morphologies that can be controlled by the length of the linker connecting the native termini. Moreover, cpAaLS proteins integrate into wild-type and other engineered AaLS assemblies by coproduction in Escherichia coli to form patchwork cages. This coassembly strategy enables encapsulation of guest proteins in the lumen, modification of the exterior through genetic fusion, and tuning of the size and electrostatics of the compartments. This addition to the family of AaLS cages broadens the scope of this system for further applications and highlights the utility of circular permutation as a potentially general strategy for tailoring the properties of cage-forming proteins.

The membrane protein P-glycoprotein (P-gp) plays key roles in the oral bioavailability of drugs, their blood brain barrier passage as well as in multidrug resistance. For new drug candidates it is mandatory to study their interaction with P-gp, according to FDA and EMA regulations. The vast majority of these tests are performed using confluent cell layers of P-gp overexpressing cell lines that render these tests laborious. In this study, we introduce a cell-free microfluidic assay for the rapid testing of drug- P-gp interactions. Cell-derived vesicles are prepared from MDCKII-MDR1 overexpressing cells and immobilized on the surface of a planar microfluidic device. The drug is delivered continuously to the vesicles and calcein accumulation is monitored by means of a fluorescence assay and total internal reflection fluorescence (TIRF) microscopy. Only small amounts of compounds (~10 μl) are required in concentrations of 5, 25 and 50 μM for a test that provides within 5 min information on the apparent dissociation constant of the drug and P-gp. We tested 10 drugs on-chip, 9 of which are inhibitors or substrates of P-glycoprotein and one negative control. We benchmarked the measured apparent dissociation constants against an alternative assay on a plate reader and reference data from FDA. These comparisons revealed good correlations between the logarithmic apparent dissociation constants (R2 = 0.95 with ATPase assay, R2 = 0.93 with FDA data) and show the reliability of the rapid on-chip test. The herein presented assay has an excellent screening window factor (Z’-factor) of 0.8, and is suitable for high-throughput testing. ISSN:0724-8741 ISSN:1573-904X

We report that l-threonine may substitute for l-serine in the β-substitution reaction of an engineered subunit of tryptophan synthase from Pyrococcus furiosus, yielding (2S,3S)-β-methyltryptophan (β-MeTrp) in a single step. The trace activity of the wild-type β-subunit on this substrate was enhanced more than 1000-fold by directed evolution. Structural and spectroscopic data indicate that this increase is correlated with stabilization of the electrophilic aminoacrylate intermediate. The engineered biocatalyst also reacts with a variety of indole analogues and thiophenol for diastereoselective C–C, C–N, and C–S bond-forming reactions. This new activity circumvents the 3-enzyme pathway that produces β-MeTrp in nature and offers a simple and expandable route to preparing derivatives of this valuable building block.

Many enzymes perform desirable biochemical transformations, but are not suitable to use as biocatalysts outside of the cell. In particular, enzymes from heteromeric complexes typically have decreased activity when removed from their protein partners. We used directed evolution to restore the catalytic efficiency of the tryptophan synthase β-subunit (TrpB), which synthesizes l-tryptophan from l-serine and indole, surpassing the activity of the native complex. Experiments show that activating mutations promote catalysis through the same mechanism as partner protein binding, establishing that isolated subunits may be readily reactivated through directed evolution. Engineering TrpB for stand-alone function restored high activity with indole analogs, providing a simplified enzyme platform for the biocatalytic production of noncanonical amino acids.