Design and Characterization of a Generalist Biosensor for Indole Derivatives.

Transcription factor (TF)-based biosensors are useful synthetic biology tools for applications in a variety of areas of biotechnology. A major challenge of biosensor circuits is the limited repertoire of identified and well-characterized TFs for applications of interest, in addition to the challenge of optimizing selected biosensors. In this work, we implement the IclR family repressor TF TtgV from Pseudomonas putida DOT-T1E as an indole-derivative biosensor in Escherichia coli. We optimize the genetic circuit utilizing different components, providing insights into biosensor design and expanding on previous studies investigating this TF. We discover novel physiologically relevant ligands of TtgV, such as skatole. The broad specificity of TtgV makes it a useful target for directed evolution and protein engineering toward desired specificity. TtgV, as an indole-derivative biosensor, is a promising genetic component for the detection of compounds with biological activities relevant to health and the gut microbiome.

Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.

Computation-guided transcription factor biosensor specificity engineering for adipic acid detection.

Transcription factor (TF)-based biosensors that connect small-molecule sensing with readouts such as fluorescence have proven to be useful synthetic biology tools for applications in biotechnology. However, the development of specific TF-based biosensors is hindered by the limited repertoire of TFs specific for molecules of interest since current construction methods rely on a limited set of characterized TFs. In this study, we present an approach for engineering the specificity of TFs through a computation-based workflow using molecular docking that enables targeted alteration of TF ligand specificity. Using this method, we engineer the LysR family BenM TF to alter its specificity from its cognate ligand cis,cis-muconic acid to adipic acid through a single amino acid substitution identified by our computational workflow. When implemented in a cell-free system, the engineered biosensor shows higher ligand sensitivity, expanding the potential applications of this circuit. We further investigate ligand binding through molecular dynamics to analyze the substitution, elucidating the impact of modulating a single amino acid position on the mechanism of BenM ligand binding. This study represents the first application of biomolecular modeling methods for altering BenM specificity and for gaining insights into how mutations influence the structural dynamics of BenM. Such methods can potentially be applied to other TFs to alter specificity and analyze the dynamics responsible for these changes, highlighting the applicability of computational tools for informing experiments. In addition, our developed adipic acid biosensor can be applied for the identification and engineering of enzymes to produce adipic acid.

Ni(II)-binding affinity of CcNikZ-II and its homologs: the role of the HH-prong and variable loop revealed by structural and mutational studies.

Extracytoplasmic Ni(II)-binding proteins (NiBPs) are molecular shuttles involved in cellular nickel uptake. Here, we determined the crystal structure of apo CcNikZ-II at 2.38 Å, which revealed a Ni(II)-binding site comprised of the double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Mutagenesis of the site identified Glu60 and His511 as critical for high affinity Ni(II)-binding. Phylogenetic analysis showed 15 protein clusters with two groups containing the HH-prong. Metal-binding assays with 11 purified NiBPs containing this feature yielded higher Ni(II)-binding affinities. Replacement of the wild type v-loop with those from other NiBPs improved the affinity by up to an order of magnitude. This work provides molecular insights into the determinants for Ni(II) affinity and paves way for NiBP engineering.

An acetyltransferase effector conserved across Legionella species targets the eukaryotic eIF3 complex to modulate protein translation.

The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF "core" effectors in Legionella's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.

Mechanistic plasticity in ApmA enables aminoglycoside promiscuity for resistance.

The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed ß-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed ß-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification.

Functional diversification despite structural congruence in the HipBST toxin-antitoxin system of Legionella pneumophila.

IMPORTANCE: Toxin-antitoxin (TA) systems are parasitic genetic elements found in almost all bacterial genomes. They are exchanged horizontally between cells and are typically poorly conserved across closely related strains and species. Here, we report the characterization of a tripartite TA system in the bacterial pathogen Legionella pneumophila that is highly conserved across Legionella species genomes. This system (denoted HipBSTLp) is a distant homolog of the recently discovered split-HipA system in Escherichia coli (HipBSTEc). We present bioinformatic, molecular, and structural analyses of the divergence between these two systems and the functionality of this newly described TA system family. Furthermore, we provide evidence to refute previous claims that the toxin in this system (HipTLp) possesses bifunctionality as an L. pneumophila virulence protein. Overall, this work expands our understanding of the split-HipA system architecture and illustrates the potential for undiscovered biology in these abundant genetic elements.

A simple and robust serum-free media for the proliferation of muscle cells.

Cultivated meat production requires an efficient, robust and highly optimized serum-free cell culture media for the needed upscaling of muscle cell expansion. Existing formulations of serum-free media are complex, expensive and have not been optimized for muscle cells. Thus, we undertook this work to develop a simple and robust serum-free media for the proliferation of bovine satellite cells (SCs) through Design of Experiment (DOE) and Response Surface Methodology (RSM) using precise and high-throughput image-based cytometry. Proliferative attributes were investigated with transcriptomics and long-term performance was validated using multiple live assays. Here we formulated a media based on three highly optimized components; FGF2 (2 ng/mL), fetuin (600 µg/mL) and BSA (75 µg/mL) which together with an insulin-transferrin-selenium (1x) supplement, sustained the proliferation of bovine SCs, porcine SCs and murine C2C12 muscle cells. Remarkably, cells cultured in our media named Tri-basal 2.0+ performed better than cell cultured in 10% FBS, with respect to proliferation. Hence, the optimized Tri-basal 2.0+ enhanced serum-free cell attachment and long-term proliferation, providing an alternative solution to the use of FBS in the production of cultivated meat.

Molecular mechanism of plasmid-borne resistance to sulfonamide antibiotics.

The sulfonamides (sulfas) are the oldest class of antibacterial drugs and inhibit the bacterial dihydropteroate synthase (DHPS, encoded by folP), through chemical mimicry of its co-substrate p-aminobenzoic acid (pABA). Resistance to sulfa drugs is mediated either by mutations in folP or acquisition of sul genes, which code for sulfa-insensitive, divergent DHPS enzymes. While the molecular basis of resistance through folP mutations is well understood, the mechanisms mediating sul-based resistance have not been investigated in detail. Here, we determine crystal structures of the most common Sul enzyme types (Sul1, Sul2 and Sul3) in multiple ligand-bound states, revealing a substantial reorganization of their pABA-interaction region relative to the corresponding region of DHPS. We use biochemical and biophysical assays, mutational analysis, and in trans complementation of E. coli ΔfolP to show that a Phe-Gly sequence enables the Sul enzymes to discriminate against sulfas while retaining pABA binding and is necessary for broad resistance to sulfonamides. Experimental evolution of E. coli results in a strain harboring a sulfa-resistant DHPS variant that carries a Phe-Gly insertion in its active site, recapitulating this molecular mechanism. We also show that Sul enzymes possess increased active site conformational dynamics relative to DHPS, which could contribute to substrate discrimination. Our results reveal the molecular foundation for Sul-mediated drug resistance and facilitate the potential development of new sulfas less prone to resistance.

Structural analysis of a hormone-bound Striga strigolactone receptor.

Strigolactones (SLs) regulate many aspects of plant development, but ambiguities remain about how this hormone is perceived because SL-complexed receptor structures do not exist. We find that when SL binds the Striga receptor, ShHTL5, a series of conformational changes relative to the unbound state occur, but these events are not sufficient for signalling. Ligand-complexed receptors, however, form internal tunnels that posit an explanation for how SL exits its receptor after hydrolysis.

Ent2 Governs Morphogenesis and Virulence in Part through Regulation of the Cdc42 Signaling Cascade in the Fungal Pathogen Candida albicans.

The ability to transition between yeast and filamentous growth states is critical for virulence of the leading human fungal pathogen Candida albicans. Large-scale genetic screens have identified hundreds of genes required for this morphological switch, but the mechanisms by which many of these genes orchestrate this developmental transition remain largely elusive. In this study, we characterized the role of Ent2 in governing morphogenesis in C. albicans. We showed that Ent2 is required for filamentous growth under a wide range of inducing conditions and is also required for virulence in a mouse model of systemic candidiasis. We found that the epsin N-terminal homology (ENTH) domain of Ent2 enables morphogenesis and virulence and does so via a physical interaction with the Cdc42 GTPase-activating protein (GAP) Rga2 and regulation of its localization. Further analyses revealed that overexpression of the Cdc42 effector protein Cla4 can overcome the requirement for the ENTH-Rga2 physical interaction, indicating that Ent2 functions, at least in part, to enable proper activation of the Cdc42-Cla4 signaling pathway in the presence of a filament-inducing cue. Overall, this work characterizes the mechanism by which Ent2 regulates hyphal morphogenesis in C. albicans, unveils the importance of this factor in enabling virulence in an in vivo model of systemic candidiasis and adds to the growing understanding of the genetic control of a key virulence trait. IMPORTANCE Candida albicans is a leading human fungal pathogen that can cause life-threatening infections in immunocompromised individuals, with mortality rates of ~40%. The ability of this organism to grow in both yeast and filamentous forms is critical for the establishment of systemic infection. Genomic screens have identified many genes required for this morphological transition, yet our understanding of the mechanisms that regulate this key virulence trait remains incomplete. In this study, we characterized Ent2 as a core regulator of C. albicans morphogenesis. We show that Ent2 regulates hyphal morphogenesis through an interaction between its ENTH domain and the Cdc42 GAP, Rga2, which signals through the Cdc42-Cla4 signaling pathway. Finally, we show that the Ent2 protein, and specifically its ENTH domain, is required for virulence in a mouse model of systemic candidiasis. Overall, this work identifies Ent2 as a key regulator of filamentation and virulence in C. albicans.

Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

Systems engineering of Escherichia coli for n-butane production.

Rising concerns about climate change and sustainable energy have attracted efforts towards developing environmentally friendly alternatives to fossil fuels. Biosynthesis of n-butane, a highly desirable petro-chemical, fuel additive and diluent in the oil industry, remains a challenge. In this work, we first engineered enzymes Tes, Car and AD in the termination module to improve the selectivity of n-butane biosynthesis, and ancestral reconstruction and a synthetic RBS significantly improved the AD abundance. Next, we did ribosome binding site (RBS) calculation to identify potential metabolic bottlenecks, and then mitigated the bottleneck with RBS engineering and precursor propionyl-CoA addition. Furthermore, we employed a model-assisted strain design and a nonrepetitive extra-long sgRNA arrays (ELSAs) and quorum sensing assisted CRISPRi to facilitate a dynamic two-stage fermentation. Through systems engineering, n-butane production was increased by 168-fold from 0.04 to 6.74 mg/L. Finally, the maximum n-butane production from acetate was predicted using parsimonious flux balance analysis (pFBA), and we achieved n-butane production from acetate produced by electrocatalytic CO reduction. Our findings pave the way for selectively producing n-butane from renewable carbon source.

Structural and functional analysis of EntV reveals a 12 amino acid fragment protective against fungal infections.

Fungal pathogens are a continuing challenge due to few effective antifungals and a rise in resistance. In previous work, we described the inhibition of Candida albicans virulence following exposure to the 68 amino acid bacteriocin, EntV, secreted by Enterococcus faecalis. Here, to optimize EntV as a potential therapeutic and better understand its antifungal features, an X-ray structure is obtained. The structure consists of six alpha helices enclosing a seventh 16 amino acid helix (α7). The individual helices are tested for antifungal activity using in vitro and nematode infection assays. Interestingly, α7 retains antifungal, but not antibacterial activity and is also effective against Candida auris and Cryptococcus neoformans. Further reduction of α7 to 12 amino acids retains full antifungal activity, and excellent efficacy is observed in rodent models of C. albicans oropharyngeal, systemic, and venous catheter infections. Together, these results showcase EntV-derived peptides as promising candidates for antifungal therapeutic development.

Recombinant production of growth factors for application in cell culture.

Culturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as "cellular agriculture". These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF2, IGF1, PDGF-BB, and TGF-ß1 in Escherichia coli. Using this E. coli expression system, we produced soluble GF orthologs from species including bovine, chicken, and salmon. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering.

Advances in engineering and optimization of transcription factor-based biosensors for plug-and-play small molecule detection.

Transcription factor (TF)-based biosensors have been applied in biotechnology for a variety of functions, including protein engineering, dynamic control, environmental detection, and point-of-care diagnostics. Such biosensors are promising analytical tools due to their wide range of detectable ligands and modular nature. However, designing biosensors tailored for applications of interest with the desired performance parameters, including ligand specificity, remains challenging. Biosensors often require significant engineering and tuning to meet desired specificity, sensitivity, dynamic range, and operating range parameters. Another limitation is the orthogonality of biosensors across hosts, given the role of the cellular context. Here, we describe recent advances and examples in the engineering and optimization of TF-based biosensors for plug-and-play small molecule detection. We highlight novel developments in TF discovery and biosensor design, TF specificity engineering, and biosensor tuning, with emphasis on emerging computational methods.

Molecular analysis and essentiality of Aro1 shikimate biosynthesis multi-enzyme in Candida albicans.

In the human fungal pathogen Candida albicans, ARO1 encodes an essential multi-enzyme that catalyses consecutive steps in the shikimate pathway for biosynthesis of chorismate, a precursor to folate and the aromatic amino acids. We obtained the first molecular image of C. albicans Aro1 that reveals the architecture of all five enzymatic domains and their arrangement in the context of the full-length protein. Aro1 forms a flexible dimer allowing relative autonomy of enzymatic function of the individual domains. Our activity and in cellulo data suggest that only four of Aro1's enzymatic domains are functional and essential for viability of C. albicans, whereas the 3-dehydroquinate dehydratase (DHQase) domain is inactive because of active site substitutions. We further demonstrate that in C. albicans, the type II DHQase Dqd1 can compensate for the inactive DHQase domain of Aro1, suggesting an unrecognized essential role for this enzyme in shikimate biosynthesis. In contrast, in Candida glabrata and Candida parapsilosis, which do not encode a Dqd1 homolog, Aro1 DHQase domains are enzymatically active, highlighting diversity across Candida species.

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family.

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.

A novel strigolactone receptor antagonist provides insights into the structural inhibition, conditioning, and germination of the crop parasite Striga.

Crop parasites of the Striga genera are a major biological deterrent to food security in Africa and are one of the largest obstacles to poverty alleviation on the continent. Striga seeds germinate by sensing small-molecule hormones, strigolactones (SLs), that emanate from host roots. Although SL receptors (Striga hermonthica HYPOSENSITIVE TO LIGHT [ShHTL]) have been identified, discerning their function has been difficult because these parasites cannot be easily grown under laboratory conditions. Moreover, many Striga species are obligate outcrossers that are not transformable, hence not amenable to genetic analysis. By combining phenotypic screening with ShHTL structural information and hybrid drug discovery methods, we discovered a potent SL perception inhibitor for Striga, dormirazine (DOZ). Structural analysis of this piperazine-based antagonist reveals a novel binding mechanism, distinct from that of known SLs, blocking access of the hormone to its receptor. Furthermore, DOZ reduces the flexibility of protein-protein interaction domains important for receptor signaling to downstream partners. In planta, we show, via temporal additions of DOZ, that SL receptors are required at a specific time during seed conditioning. This conditioning is essential to prime seed germination at the right time; thus, this SL-sensitive stage appears to be critical for adequate receptor signaling. Aside from uncovering a function for ShHTL during seed conditioning, these results suggest that future Ag-Biotech Solutions to Striga infestations will need to carefully time the application of antagonists to exploit receptor availability and outcompete natural SLs, critical elements for successful parasitic plant invasions.