Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single round of mutagenesis of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis,cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion-of-function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a prokaryotic chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

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The study describes the first glycoside hydrolase that exhibits comparable levels of activity on α- and β-linked saccharide substrates. This enzyme, assigned into GH109, is encoded by the genome of the human gut symbiont Akkermansia muciniphila that is a model primary degrader of the heavily O-glycosylated mucin glycoprotein that coats the epithelial enterocytes.The elusive catalytic acid/base catalyst in GH109 enzymes is identified as a histidine that is presented by a flexible loop that positions it for catalysis on both α- and β-substrates. This dual activity may be an evolutionary adaptation to extend the range of substrates targeted by a single non-canonical NAD+-dependant GH. <br>

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19Allosteric transcription factors (aTFs) have proven widely applicable for 20 biotechnology and synthetic biology as ligand-specific biosensors enabling real-time 21 monitoring, selection and regulation of cellular metabolism. However, both the 22 biosensor specificity and the correlation between ligand concentration and biosensor 23 output signal, also known as the transfer function, often needs to be optimized before 24 meeting application needs. Here, we present a versatile and high-throughput method 25 to evolve and functionalize prokaryotic aTF specificity and transfer functions in a 26 eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single 27 round of directed evolution of the effector-binding domain (EBD) coupled with various 28 2 toggled selection regimes, we robustly select aTF variants of the cis, cis-muconic 29 acid-inducible transcription factor BenM evolved for change in ligand specificity, 30 increased dynamic output range, shifts in operational range, and a complete 31 inversion of function from activation to repression. Importantly, by targeting only the 32 EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are 33 functional when ported back into a non-native prokaryote chassis. The developed 34 platform technology thus leverages aTF evolvability for the development of new host-35 agnostic biosensors with user-defined small-molecule specificities and transfer 36 functions. 37 38 39 40 48 mammalian cell differentiation, and synthetic cell-cell communication devices 2-5 . 49 Given the large number of allosteric transcription factors (aTFs) present in the 50 prokaryotic kingdom 6 , the diversity of chemical structures recognized 7 , and their 51 modular domain structure encoded by a conserved DNA-binding domain (DBD) 52 linked to a diversified effector-binding domain (EBD) 8 , small-molecule biosensors 53 based on aTFs are a particularly valuable class of genetic switches. Ongoing 54biosensor research therefore seeks to prospect new biosensors from genomic 55 resources 9,10 , while also developing general design rules and engineering strategies 56 3 from existing aTFs. Indeed, due to the modular structure of aTFs, several studies 57 have successfully adopted EBD-swapping strategies into platform DBDs to rationally 58 engineer new aTF logic 11-13 , while engineering EBD destabilization for ligand-59 controlled biosensor stability also have proven successful [14][15][16] . However, these 60 rational design strategies for engineering new biosensors suffer from the introduction 61 of cross-talk between ligand specificities, difficulty in creating chimeras from different 62 aTF superfamilies, and the risk of losing allostery 2,11,17 . Ultimately, this may impact 63 several aspects of biosensor performance, such as the operational and dynamic 64 output ranges, the specificity, and mode-of-action -collectively referred to as the 65 biosensor transfer function or logic. 66 Acknowledging that allosteric regulation relies on complex interdom...

The study describes the first glycoside hydrolase that exhibits comparable levels of activity on α- and β-linked saccharide substrates. This enzyme, assigned into GH109, is encoded by the genome of the human gut symbiont Akkermansia muciniphila that is a model primary degrader of the heavily O-glycosylated mucin glycoprotein that coats the epithelial enterocytes.The elusive catalytic acid/base catalyst in GH109 enzymes is identified as a histidine that is presented by a flexible loop that positions it for catalysis on both α- and β-substrates. This dual activity may be an evolutionary adaptation to extend the range of substrates targeted by a single non-canonical NAD+-dependant GH. <br>