Biology subjects

Trivedi, V. D.

Publications and source records attributed to Trivedi, V. D..

9 recordsLinked to original sources

bioRxiv2025

Pharmacophore-driven antibody discovery on the yeast surface

Protein-small molecule hybrids are structures capable of combining the inhibitory properties of small molecules and the specificities of binding proteins. However, discovery of such synergistic conjugates is a substantial engineering challenge. Here, we describe pharmacophore-driven antibody discovery as a high throughput approach to hybrid discovery. In this approach, we use a yeast display antibody library containing reactive noncanonical amino acids (ncAAs) and further diversify it by conjugating the library to four sulfonamide pharmacophores. Yeast display binding screens with each of the resulting billion-member hybrid collections against bovine carbonic anhydrase (bCA) yielded diverse collections of hybrids. Individual hybrids exhibited double digit nanomolar binding affinities, and frequently exhibited inhibitory properties in solution, despite the fact that the screens were based solely on binding phenotypes. Deep sequencing of sorted populations revealed that enrichments were strongly pharmacophore-dependent. In particular, screens with a potent pharmacophore led to collections of hybrids varying substantially in pharmacophore attachment point and antibody sequence features, while screens with moderate or weak pharmacophores led to collections with much narrower sets of enriched attachment points and antibody sequence features. Identification of the most frequently isolated CDR-H3 sequences and clustering CDR-H3 sequences by similarity within enriched populations provided further evidence for pharmacophore-dependent sorting outcomes. Experimental binding assays in which the pharmacophore warhead used during screening was replaced by another warhead indicated that isolated clones can tolerate alternative pharmacophores, but tend to prefer the warhead used during screening. Overall, these efforts demonstrate the utility of introducing pharmacophores into antibody libraries along with several lines of evidence that screening outcomes are pharmacophore-driven. These findings advance our understanding of hybrid discovery and highlight opportunities to pursue hybrids as research tools and potential therapeutic leads. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/682880v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@11c2a65org.highwire.dtl.DTLVardef@237055org.highwire.dtl.DTLVardef@a3fb4borg.highwire.dtl.DTLVardef@1c71700_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Engineered antimicrobial-derived peptides to manipulate mixed microbial systems

Due to complexity of microbial communities and coarseness of currently available manipulation techniques (e.g. transplantation, antibiotic-treatment), it is often difficult to fully elucidate the interactions between members that define community structure and function from the top-down. Thus, it is imperative to be able to observe and manipulate subpopulations within microbial communities to enable a fine-detail understanding of the full-spectrum and mechanism of community functions. However, there is a technological gap that prevents targeted manipulation of subpopulations within intact microbial mixtures and communities. In this work, we develop molecular probes to manipulate specific subpopulations within multispecies microbial populations and validate these methods using model synthetic populations in vitro. We leverage the narrow-spectrum of class II peptide bacteriocin (a bacterially-synthesized antimicrobial peptide), pediocin PA-1, as a model to develop molecular probes. We first demonstrate the narrow-spectrum activity of pediocin and quantify its potency against a panel of bacteria. Next, we conjugate chemical handles on the bacteriocin and show that the binding spectrum is largely unchanged. Finally, using truncated variants, also conjugated to chemical handles, we show functional non-bactericidal binders that largely maintain their specificity. Finally, with the unmodified and modified bacteriocins, we show that specific bacteria can be depleted through killing or cell sorting within a mixture of highly similar bacteria. While we developed the system with a single bacteriocin, we expect that the elucidated design rules may be applicable to a variety of natural bacteriocins to develop a generalized approach for manipulating specific bacterial members within a community. Development of such molecular probes would be transformative to advancing mechanistic underpinnings of microbial community and microbiota structure-function relationships.

synthetic biology↗

bioRxiv2022

Towards universal synthetic heterotrophy using a metabolic coordinator

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as potential inputs to biotechnological processes. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pS25144.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies confirm that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

synthetic biology↗

bioRxiv2022

Integration of metabolism and regulation reveals rapid adaptability to growth on non-native substrates.

Engineering synthetic heterotrophy (i.e., growth on non-native substrates) is key to the efficient bio-based valorization of renewable and waste substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades - yet the answer to what makes their utilization inherently recalcitrant remains elusive. Through implementation of a semi-synthetic regulon, we find that harmonizing cellular and engineering objectives are key to obtaining highest growth rates and yields with minimal metabolic engineering effort. Concurrently, results indicate that "extrinsic" factors - specifically, upstream genes that direct flux of pentoses into central carbon metabolism - are rate-limiting. We also reveal that yeast metabolism is innately highly adaptable to rapid growth on non-native substrates and that systems metabolic engineering (i.e., flux balancing, directed evolution, functional genomics, and network modeling) is largely unnecessary. We posit that the need for extensive engineering espoused by prior works is a consequence of unfortunate (albeit avoidable) antagonism between engineering and cellular objectives. We also found that deletion of endogenous genes to promote growth demonstrate inconsistent outcomes that are genetic-context- and condition-dependent. For the most part, these knockouts also lead to deleterious pleiotropic effects that decrease the robustness of strains against inhibitors and stressors associated with bioprocessing. Thus, at best, perturbation of "intrinsic" factors (e.g., native metabolic, regulatory genes) results in incremental and inconsistent benefits. At worst, they are detrimental. Overall, this work provides insight into the limitations and pitfalls to realizing efficient synthetic heterotrophy using traditional/systems metabolic engineering approaches, demonstrates the innate adaptability of yeast for metabolism of non-native substrates, and provides an alternate, novel, holistic (and yet minimalistic) approach based on integrating non-native metabolic genes with a native regulon system.

synthetic biology↗

bioRxiv2022

A silk-based platform to stabilize phenylalanine ammonia-lyase (PAL) for orally administered enzyme replacement therapy

Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects [~]1 in 15,000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and is currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from Anabaena variabilis, which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix. The combination with silk stabilizes PAL at 37 {degrees}C. In addition, in vitro studies showed that inclusion in a silk matrix preserves the biological activity of the enzyme in simulated intestinal fluid, enabling the oral administration of pegvaliase for the treatment of PKU.

bioengineering↗

bioRxiv2021

Cheating the cheater: Suppressing false positive enrichment during biosensor-guided biocatalyst engineering

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false positive/cheater cells in the enriched population. While the host cell can be engineered to minimize crosstalk (e.g., by deleting responsible transporters), this is not easily applicable to all molecules of interest, particularly those that can diffuse passively. One such biosensor recently reported for trans-cinnamic acid (tCA) suffers from crosstalk when used for phenylalanine ammonia-lyase (PAL) enzyme engineering by directed evolution. We report that desensitizing the biosensor (i.e., increasing the limit of detection, LOD) suppresses cheater population enrichment. Further we show that, if we couple the biosensor-based screen with an orthogonal pre-screen that eliminates a large fraction of true negatives, we can successfully reduce the cheater population during the fluorescence-activated cell sorting (FACS). Using the approach developed here, we were successfully able to isolate PAL variants with ~70% high kcat after a single sort. These mutants have tremendous potential in Phenylketonuria (PKU) treatment and flavonoid production.

synthetic biology↗

bioRxiv2021

In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity.

Phenylalanine ammonia-lyases (PALs) deaminate L-phenylalanine to trans-cinnamic acid and ammonium and have idespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis (Trichormus variabilis) has garnered significant attention as the active ingredient in Pegvaliase(R), the only FDA-approved drug treating classical phenylketonuria (PKU). Although an extensive body of literature exists on structure, substrate-specificity, and catalytic mechanism, protein-wide sequence determinants of function remain unknown, which limits the ability to rationally engineer these enzymes. Previously, we developed a high-throughput screen (HTS) for PAL, and here, we leverage it to create a detailed sequence-function landscape of PAL by performing deep mutational scanning (DMS). Our method revealed 79 hotspots that affected a positive change in enzyme fitness, many of which have not been reported previously. Using fitness values and structure-function analysis, we picked a subset of residues for comprehensive single- and multi-site saturation mutagenesis to improve the catalytic activity of PAL and identified combinations of mutations that led to improvement in reaction kinetics in cell-free and cellular contexts. To understand the mechanistic role of the most beneficial mutations, we performed QM/MM and MD and observed that different mutants confer improved catalytic activity via different mechanisms, including stabilizing first transition and intermediate states and improving substrate diffusion into the active site, and decreased product inhibition. Thus, this work provides a comprehensive sequence-function relationship for PAL, identifies positions that improve PAL activity when mutated and assesses their mechanisms of action.

bioengineering↗

bioRxiv2020

Charged Gram-positive species sequester and decrease the potency of pediocin PA-1 in mixed microbial settings

Antimicrobial peptides (AMPs) have gained attention recently due to increasing antibiotic resistance amongst pathogens. Most AMPs are cationic in nature and their preliminary interactions with the negatively charged cell surface is mediated by electrostatic attraction. This is followed by pore formation, which is either receptor-dependent or -independent and leads to cell death. Typically, AMPs are characterized by their killing activity using bioactivity assays to determine host range and degree of killing. However, cell surface binding is independent from killing. Most of the studies performed to-date have attempted to quantify the peptide binding using artificial membranes. Here, we use the narrow-spectrum class IIa bacteriocin AMP pediocin PA-1 conjugated to a fluorescent dye as a probe to monitor cell surface binding. We developed a flow cytometry-based assay to quantify the strength of binding in target and non-target species. Through our binding assays, we found a strong positive correlation between cell surface charge and pediocin PA-1 binding. Interestingly, we also found inverse correlation between zeta potential and pediocin PA-1 binding, the correlation coefficient for which improved when only Gram-positives were considered. We also show the effect of the presence of protein, salt, polycationic species, and other non-target species on the binding of pediocin PA-1 to the target organism. We conclude that the of presence of highly charged non-target species, as well as solutes, can decrease the binding, and the apparent potency, of pediocin PA-1. Thus, these outcomes are highly significant to the use of pediocin PA-1 and related AMPs in mixed microbial settings such as those found in the gut microbiota.

molecular biology↗

bioRxiv2020

Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities

There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry (fine-chemicals and natural product synthesis) and medicine (phenylketoruria/PKU and cancer treatment). While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, a more comprehensive mutational landscape has yet to be explored for this class of enzymes. Here, we report development of a directed evolution technique to engineer PAL enzymes. Central to this approach is a high-throughput enrichment that couples E. coli growth to PAL activity. Using the clinically-relevant PAL from Anabaena variabilis, which is used on the formulation of pegvaliase for PKU therapy, we identified mutations at residues previously unknown as relevant for function that increase turnover frequency almost twofold after only a single round of engineering. This work demonstrates the power our technique for ammonia-lyase enzyme engineering.

bioengineering↗