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The rapid growth of sequence databases over the past two decades means that protein engineers faced with optimizing a protein for any given task will often have immediate access to a vast number of related protein sequences. These sequences encode information about the evolutionary history of the protein and the underlying sequence requirements to produce folded, stable, and functional protein variants. Methods that can take advantage of this information are an increasingly important part of the protein engineering tool kit. In this Perspective, we discuss the utility of sequence data in protein engineering and design, focusing on recent advances in three main areas: the use of ancestral sequence reconstruction as an engineering tool to generate thermostable and multifunctional proteins, the use of sequence data to guide engineering of multipoint mutants by structure-based computational protein design, and the use of unlabeled sequence data for unsupervised and semisupervised machine learning, allowing the generation of diverse and functional protein sequences in unexplored regions of sequence space. Altogether, these methods enable the rapid exploration of sequence space within regions enriched with functional proteins and therefore have great potential for accelerating the engineering of stable, functional, and diverse proteins for industrial and biomedical applications.

Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, our understanding of loop functions is mainly limited to the catalytic loops. To understand the function of remote loops in enzymes, we studied Glycoside hydrolase family 19 (GH19) chitinase - an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discovered that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrated that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that Nature applies this new strategy at the molecular level to achieve a complex biological function while maintaining the original activity one in the catalytic pocket, suggesting an alternative way to design new enzyme function.

SAR11 bacteria are the most abundant members of the global ocean microbiome and have a broad impact on ocean ecosystems. To thrive in their competitive oligotrophic environments, these bacteria rely on solute-binding proteins (SBPs) that facilitate nutrient uptake through ABC transporters. Nonetheless, previous studies have been unable to access the molecular mechanisms and functions of these transporters because they rely heavily on homology-based predictions. These mechanisms and functions are essential to understand biogeochemical cycling in the ocean, including assimilation of dissolved organic matter (DOM). Here, by doing a biochemical study of the collective behavior of all SBPs in a SAR11 bacterium, we discover that these transporters have unprecedented binding affinity (Kd≥30 pM) and unexpectedly high binding specificity, revealing molecular mechanisms for oligotrophic adaptation. Our study uncovers new carbon sources for the SAR11 bacteria and provides an accurate biogeographical map of nutrient uptake in the ocean. Our results show how functional adaptation at the molecular level in ubiquitous marine bacteria impacts global patterns of DOM assimilation and provides insight into the contribution of different compounds to oceanic nutrient cycles.

The tRNA modification m1G37, introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria because it suppresses translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question through experimental evolution of trmD mutant strains of Escherichia coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via duplication or mutation of the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that proS may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.

Nucleobase-containing coenzymes are considered the relics of an early RNA-based world that preceded the emergence of protein domains. Despite the importance of coenzyme-protein synergisms, their emergence and evolution remain poorly understood. An excellent target to address this issue is the Rossman fold, the most catalytically diverse and abundant protein architecture in Nature. Here, we investigatedted the two largest Rossman lineages, namely the nicotinamide adenine dinucleotide phosphate (NAD(P))-binding and the S-adenosyl methionine (SAM)-dependent superfamilies. With the aim to identify the evolutionary changes that lead to a switch in coenzyme specificity on these superfamilies, we performed structural and sequence-based Hidden Markov Models to systematically search for key motifs in their coenzyme-binding pockets. Our analyses revealed how insertions and deletions (InDels) reshaped the ancient β1−loop−α1 coenzyme-binding structure of NAD(P) into the well-defined SAM-binding β1−loop−α1 structure. To prove this observation experimentally, we removed an InDel of three amino acids from the NAD(P) coenzyme pocket and solved the structure of the resulting mutant, revealing the characteristic features of the SAM-binding pocket. To confirm the binding to SAM, we performed isothermal titration calorimetry measurements, validating the successful coenzyme switch. Molecular dynamics simulations also corroborated the role of InDels in abolishing NAD-binding and acquiring SAM binding. Our results uncovered how Nature utilized insertions and deletions to switch coenzyme specificity, and in turn, functionalities between these superfamilies. This work also establishes how protein structures could have been recycled through the course of evolution to adopt different coenzymes and confer different chemistries.Significance StatementCofactors are ubiquitous molecules necessary to drive about half of the enzymatic reactions in Nature. Among them, organic cofactors (coenzymes) that contain nucleotide moieties are believed to be relics of a hypothetical RNA world. Understanding coenzyme-binding transitions sheds light onto the emergence of the first enzymes and their chemical diversity. Rossmann enzymes bind to 7 out of 10 nucleotide coenzymes, representing an ideal target to study how different coenzyme specificities emerged and evolved. Here we demonstrated how insertions and deletions reshape coenzyme-specificity in Rossmann enzymes by retracing the emergence of the SAM-binding function from an NAD-binding ancestor. This work constitutes the first example of an evolutionary bridge between redox and methylation reactions, providing a new strategy to engineer coenzyme specificity.

We have studied adenosine binding specificities of two bacterial DNA methyltransferase Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While these DNA methyltransferases have similar cofactor binding pocket interactions, experimental data showed different specificity for novel cofactors ((SNM) (S-guanosyl-L-methionine (SGM), S-cytidyl-L-methionine (SCM), S-uridyl-L-methionine (SUM)). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-L-methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data proves a catalytically productive methylation by M.HhaI binding pocket for all the SNM. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.Table of ContentTOC: Methylation by DNA methyltransferase is nucleobase dependent. While M.Taq1 is specific for SAM, M.HhaI is promiscuous for other SAM analogs

Connecting protein sequence to function is becoming increasingly relevant since high-throughput sequencing studies accumulate large amounts of genomic data. In order to go beyond the existing database annotation, it is fundamental to understand the mechanisms underlying functional inheritance and divergence. If the homology relationship between proteins is known, can we determine whether the function diverged?In this work, we analyze different possibilities of protein sequence evolution after gene duplication and identify “residue inversions”, i.e., sites where the relationship between the ancestry and the functional signal is decoupled. Residues in these sites are masked from being recognized by other prediction tools. Still, they play a role in functional divergence and could indicate a shift in protein function. We develop a method to specifically recognize residue inversions in a phylogeny and test it on real and simulated datasets. In a dataset built from the Epidermal Growth Factor Receptor (EGFR) sequences found in 88 fish species, we identify 19 positions that went through inversion after gene duplication, mostly located at the ligand-binding extracellular domain.Our work uncovers a rare event of protein divergence that has direct implications in protein functional annotation and sequence evolution as a whole. The developed method is optimized to work with large protein datasets and can be readily included in a targeted protein analysis pipeline.

The tRNA modification m1G37, which is introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria due to its role in suppressing translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question by performing experimental evolution of trmD mutant strains of E. coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that the ProRS enzyme may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in protein translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.

Genetic variations expand the conformational landscape of proteins and may underlie cryptic functions able to influence protein adaptability under unfavorable conditions. Cryptic functions usually associate with regions of increased frustration or even intrinsically disordered, whose role as drivers of innovation is increasingly being recognized. Hence, the balance between protein stability and controlled disorder translates in the dichotomy between conservation and innovability, and should be regarded as the more comprehensive measure of protein fitness. In this context, understanding how genetic variations affect protein fitness is not trivial, since cryptic functions behind frustrated regions are not easily detectable. Herein, we used as model the pyridoxal 5-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population and considered a lower limit of AGT fitness. By solving the structure of AGT-Mi, we could show that three distinct regions, that are structured in AGT-Ma, are disordered in AGT-Mi. Molecular dynamics shows that AGT-Mi samples more flexible conformations than AGT-Ma, supporting a plasticity effect propagated to all the structure. In-depth biochemical characterisation of mutants from a small library of variants encompassing the three regions reproduces the fitness window between AGT-Ma and AGT-Mi. Cellular studies highlight the consequences of this plasticity at functional level and suggest the existence of cryptic functions likely related to protein-protein interactions. These results establish that naturally-occurring genetic variations tip the balance between protein stability and frustration to encode cryptic functions that expand the potential innovability of the protein.

Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium., Nature Communications, 12 (1), ISSN:2041-1723

How do proteins evolve? How do changes in sequence mediate changes in protein structure, and in turn in function? This question has multiple angles, ranging from biochemistry and biophysics to evolutionary biology. This review provides a brief integrated view of some key mechanistic aspects of protein evolution. First, we explain how protein evolution is primarily driven by randomly acquired genetic mutations and selection for function, and how these mutations can even give rise to completely new folds. Then, we also comment on how phenotypic protein variability, including promiscuity, transcriptional and translational errors, may also accelerate this process, possibly via “plasticity‐first” mechanisms. Finally, we highlight open questions in the field of protein evolution, with respect to the emergence of more sophisticated protein systems such as protein complexes, pathways, and the emergence of pre‐LUCA enzymes.

Methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine from L-methionine and ATP, is an ancient, highly conserved enzyme present in all three domains of life. Although the MAT enzymes of each domain are believed to share a common ancestor, the sequences of archaeal MATs show a high degree of divergence from the sequences of bacterial and eukaryotic MATs. However, the structural and functional consequences of this sequence divergence are not well understood. Here, we use structural bioinformatics analysis and ancestral sequence reconstruction to highlight the consequences of archaeal MAT divergence. We show that the dimer interface containing the active site, which would be expected to be well conserved across all three domains, diverged considerably between the bacterial/eukaryotic MATs and archaeal MATs. Furthermore, the characterization of reconstructed ancestral archaeal MATs showed that they probably had low substrate specificity which expanded during their evolutionary trajectory hinting towards the observation that all the modern day MAT enzymes from the three-kingdom probably originated from a common specific ancestor and then archaea MATs diverged in sequence, structure and substrate specificity. Altogether, our results show that the archaea MAT is an ideal system for studying an enzyme family which evolved to display high degrees of divergence at the sequence/structural levels and yet are capable of performing the same catalytic reactions as their orthologous counterparts.

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT-Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT-Mi. An in-depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT-Ma and AGT-Mi. Finally, co-immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein-protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well-defined fitness window, thus expanding the adaptability potential of the protein.

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5′-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.

The Epidermal Growth Factor Receptor (EGFR) is a membrane-anchored tyrosine kinase that is able to selectively respond to multiple extra-cellular stimuli. Previous studies have indicated that the modularity of this system is affected by ligand-induced differences in the stability of the dimerized receptor in a process known as “Biased signaling”. However, this hypothesis has not been explored using single-mutant ligands thus far. Herein, we developed a new approach to identify residues responsible for functional divergence combining the conservation and co-evolution information of ortholog and paralog genes encoding the epidermal growth factor (EGF) ligand. Then, we mutated these residues and assessed the mutants’ effects on the receptor by employing a combination of molecular dynamics (MD) and biochemical techniques. Although the EGF mutants had comparable binding affinities to the wild type ligand for EGFR, the EGF mutants induced a different phosphorylation and cell growth pattern in multiple cell lines. The MD simulations of the EGF mutants show a long-range effect on the receptor dimer interface. For the first time in this study, a single mutation in EGF is shown to be enough to alter the activation of the pathway at the cellular level. These results also support the theory of biased signaling in the tyrosine kinase receptor system and demonstrate a promising new way to study ligand-receptor interactions.

Dan Tawfik suddenly left us on 4 May, 2021. His scientific intuition led him to articulate, and solve, many key questions related to protein chemistry and molecular evolution. Although science, particularly for his students, postdoctoral fellows and colleagues, is dimmer after his loss, his legacy will persist.

The epidermal growth factor receptor (EGFR) is a membrane-anchored tyrosine kinase that is able to selectively respond to multiple extracellular stimuli. Previous studies have indicated that the modularity of this system may be caused by ligand-induced differences in the stability of the receptor dimer. However, this hypothesis has not been explored using single-mutant ligands thus far. Herein, we developed a new approach to identify residues responsible for functional divergence by selecting residues in the epidermal growth factor (EGF) ligand that are conserved among orthologs yet divergent between paralogs. Then, we mutated these residues and assessed the mutants' effects on the receptor using a combination of molecular dynamics (MD) and biochemical techniques. Although the EGF mutants had binding affinities for the EGFR comparable with the WT ligand, the EGF mutants showed differential patterns of receptor phosphorylation and cell growth in multiple cell lines. The MD simulations of the EGF mutants indicated that mutations had long-range effects on the receptor dimer interface. This study shows for the first time that a single mutation in the EGF is sufficient to alter the activation of the EGFR signaling pathway at the cellular level. These results also support that biased ligand–receptor signaling in the tyrosine kinase receptor system can lead to differential downstream outcomes and demonstrate a promising new method to study ligand–receptor interactions.

Field-effect transistor (FET) biosensors based on low-dimensional materials are capable of highly sensitive and specific label-free detection of various analytes. In this work, a FET biosensor based on graphene decorated with gold nanoparticles (Au NPs) was fabricated for lactose detection in a liquid-gate measurement configuration. This graphene device is functionalized with a carbohydrate recognition domain (CRD) of the human galectin-3 (hGal-3) protein to detect the presence of lactose from the donor effect of lectin – glycan affinity binding on the graphene. Although the detection of lactose is important because of its ubiquitous presence in food and for disease related applications (lactose intolerance condition), in this work we exploit the lectin/carbohydrate interaction to develop a device that in principle could specifically detect very low concentrations of any carbohydrate. The biosensor achieved an effective response to lactose concentrations over a dynamic range from 1 fM to 1 pM (10−15 to 10−12 mol L−1) with a detection limit of 200 aM, a significant enhancement over previous electrochemical graphene devices. The FET sensor response is also specific to lactose at aM concentrations, indicating the potential of a combined lectin and graphene FET (G-FET) sensor to detect carbohydrates at high sensitivity and specificity for disease diagnosis.

Methyltransferases (MTases) are superfamilies of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM), a nucleoside-based cofactor, to a wide variety of substrates such as DNA, RNA, proteins, small molecules, and lipids. Depending upon their structural features, the MTases can be further classified into different classes; we consider exclusively the largest class of MTases, the Rossmann-fold MTases. It has been shown that the nucleoside cofactor-binding Rossmann enzymes, particularly the nicotinamide adenine dinucleotide (NAD)-, flavin adenine dinucleotide (FAD)-, and SAM-binding MTases enzymes, share common binding motifs that include a Gly-rich loop region that interacts with the cofactor and a highly conserved acidic residue (Asp/Glu) that interacts with the ribose moiety of the cofactor. Here, we observe that the Gly-rich loop region of the Rossmann MTases adapts a specific type II′ β-turn in the proximity of the cofactor (

S-Adenosylmethionine (SAM) is an essential methylation cofactor. The origins of SAM methylation are complex, seemingly demanding the simultaneous emergence of an enzyme that makes SAM and enzyme(s) that utilize it. We report that both ATP and adenosine spontaneously react with methionine to yield SAM, thus suggesting that SAM could have emerged by chance. SAM methylation thus exemplifies how metabolites and pathways can co-emerge through the gradual recruitment of individual enzymes in reverse order.

DNA methyltransferases (MTases) constitute an attractive target for protein engineering, thus opening the road to new ways of manipulating DNA in a unique and selective manner. Here, we review various aspects of MTase engineering, both methodological and conceptual, and also discuss future directions and challenges. Bacterial MTases that are part of restriction/modification (R/M) systems offer a convenient way for the selection of large gene libraries, both in vivo and in vitro. We review these selection methods, their strengths and weaknesses, and also the prospects for new selection approaches that will enable the directed evolution of mammalian DNA methyltransferases (Dnmts). We explore various properties of MTases that may be subject to engineering. These include engineering for higher stability and soluble expression (MTases, including bacterial ones, are prone to misfolding), engineering of the DNA target specificity, and engineering for the usage of S-adenosyl-L-methionine (AdoMet) analogs. Directed evolution of bacterial MTases also offers insights into how these enzymes readily evolve in nature, thus yielding MTases with a huge spectrum of DNA target specificities. Engineering for alternative cofactors, on the other hand, enables modification of DNA with various groups other than methyl and thus can be employed to map and redirect DNA epigenetic modifications.

Supported transition-metal catalysts offer the promise of catalyst reuse in order to make chemical transformations more environmentally friendly and less expensive; however, catalysts that are supported on insoluble scaffolds often exhibit significantly reduced selectivities and rates. A capture/release strategy that unites the benefits of heterogeneous and homogeneous catalysis would overcome these current shortcomings. Herein, we report on a novel capture-and-release flow system that takes advantage of a non-covalent pyrenesingle wall nanotube (SWNT) interaction. We demonstrate that a Pd complex containing one or two pyrene arms is captured and released from a SWNT column at different rates and can be utilized for the homogeneous catalysis of Suzuki and Heck reactions.

A detailed protocol for the large-scale synthesis of carbohydrate and dihydrolipoic acid (DHLA)-coated CdSe/ZnS and CdTe/ZnS nanoparticles using continuous flow reactors is described here. Three continuous flow microreaction systems, operating at three different temperatures, are used for the synthesis of mannose-, galactose- or DHLA-functionalized quantum dots (QDs). In the first step of synthesis, the CdSe and CdTe nanoparticles are prepared. The size and spectral properties of the CdSe core of the nanoparticles are controlled by adjustment of the residence time and the temperature. As a second step, the zinc sulfide capping under homogenous conditions is carried out at a substantially lower temperature than is required for nanoparticle growth in batch processes. Finally, the trioctylphosphine/oleic acid ligand is effectively replaced with either carbohydrate PEG-thiol moieties or DHLA at 60 °C. This new protocol allows the synthesis of biologically active fluorescent QDs in 4 d.

Dimethyl sulfide (DMS) is produced in oceans in vast amounts (10(7) tons/year) and mediates a wide range of processes from regulating marine life forms to cloud formation. Nonetheless, none of the enzymes that produce DMS from dimethylsulfoniopropionate (DMSP) has been adequately characterized. We describe the expression and purification of DddD from the marine bacterium Marinomonas sp. MWYL1 and its biochemical characterization. We identified DMSP and acetyl-coenzyme A to be DddD's native substrates and Asp602 as the active site residue mediating the CoA-transferase prior to lyase activity. These findings shed light on the biochemical utilization of DMSP in the marine environment.

Homogeneous continuous flow RAFT polymerizations with N-isopropylacrylamide (NIPAM) as monomer and a trithiocarbonate as chain transfer agent are investigated. Polymerization rates are considerably increased compared to reactions in batch due to more uniform heating of the solution. The fast reaction did not influence the well-defined architecture of the polymer products. PNIPAM with molecular weights of 20 kDa was produced in minutes rather than hours. The continuous polymerization is readily scalable at low operating costs.

Biocompatible glyco-dendronized poly-l-lysine (PLL) polymers carry either three or nine mannose- or galactose-bearing dendrons that selectively bind, and thus can be used to detect, bacteria. Central to the synthesis of glyco-dendronized polymers was the development of a continuous flow [2 + 2] photocycloaddition reaction to connect the dendrons and PLL. Glycodendronized polymers cluster bacteria by binding to cell-surface carbohydrate receptors and thereby result in an easy read-out using microscopic analyses.