Talk Abstracts

Quantum Mechanics Driven Machine Learning for Enzyme Design: Application to Carboligases

Harnessing enzymes as natural biocatalysts is a useful and increasingly sustainable method for synthesizing complex chemicals opposed to traditional petrochemical refinement. To that end, many enzymes are naturally promiscuous, with the tendency to catalyze multiple reactions. Understanding and predicting the scope of new-to-nature reactions an enzyme can catalyze is an important step towards advancing biosynthesis techniques. To fulfill this need, this study will use quantum mechanical calculations to inform a machine learning model to predict enzyme activity on non-native substrates. The current work will focus on carboligases, a class of enzymes capable of catalyzing a carbon-carbon bond condensation reaction.

Excreted Fungal Melanin as a Mediator for Photosynthetic Symbioses

Melanin is a complex biomolecule that endows fungi with broad resistance to abiotic and biotic stress. Polyextremotolerant fungi are the only group of fungi that constitutively produce melanin and incorporate it into their entire cell wall. This unique feature allows these fungi to colonize ecosystems such as the surfaces of rocks and desert soils. The drawback to living in these oligotrophic locations is that there is no organic matter for these fungi to degrade. Instead, it has been hypothesized, that polyextremotolerant fungi engage in lichen-like symbioses with the algae and cyanobacteria located in these ecosystems to obtain the carbon and nitrogen that are essential for growth. We have isolated, whole genome sequenced, annotated, and physiologically characterized a novel polyextremotolerant fungal species named Exophiala viscosa. E. viscosa was isolated from a biological soil crust, where it is associated with many algal and cyanobacterial species. One unique feature of E. viscosa is that it releases melanin into its environment under certain conditions. This feature makes E. viscosa a potential source of extracellular melanin, leading to increased abiotic stress resistance of the microbes in its surrounding community. However, we do not know what metabolic or genetic triggers induce this melanin excretion. We hypothesize that symbiotic algae and cyanobacteria play a role in inducing melanin excretion. We have established two separate genetic systems in E. viscosa, an induced and a CRISPR-mediated system, which has allowed us to create non-melanized forms of E. viscosa. By culturing E. viscosa or its non-melanized mutants with algae and cyanobacteria also isolated from the same ecosystem, we can determine if E. viscosa can survive solely on carbon and nitrogen sources provided by the photosynthetic microbes, and if the melanin provided by E. viscosa is vital to their symbiosis.

Engineering MS2 Bacteriophage Virus-Like Particle for Targeted Drug Delivery into Hepatocellular Carcinoma.

Hepatocellular carcinoma, comprising 90% of liver cancers, exhibits a dismal five-year survival rate of 30%. Current treatment confronts limitations such as drug resistance and severe side effects. Nanotechnology, particularly Virus-Like Particles(VLPs), are a promising avenue for delivering therapeutics exclusively into the liver through targeted drug delivery, reducing side effects and enabling advanced treatments, such as gene therapy. To this end, we identified and engineered targeted drug delivery nanocarriers based on the native bacteriophage MS2 VLP. Initially, we screened sixteen MS2 VLP mutants that are assembly competent and contain an externally facing cysteine that could be conjugated with targeting ligands of interest via maleimide-thiol conjugation. Next, we selected variants that maintain the capsid quaternary structure after undergoing conjugation with maleimide-GalNAc, the ligand that binds to ASGPR receptors predominately encountered in hepatocytes. Finally, we demonstrated that MS2 VLP-GalNAc constructs are selectively internalized by cells that express ASGPR receptors. This research aims to advance the development of MS2 VLPs as a versatile platform for targeted drug delivery and the research outcomes are foundational for advances in therapeutic interventions for hepatocellular carcinoma.

Using cell-free prototyping and plant genome engineering to produce plant-based metabolites

Plants produce an incredible diversity of complex metabolites useful to humans as fragrances, insecticides, bioactives, and medicines. Furthermore, plants can be imagined as a distributed manufacturing system where seeds encode the blueprints for low-capital bioreactors made from light and carbon dioxide. However, additional synthetic biology tools are needed to control and engineer plant metabolism. The Dudley lab at UW-Madison will exploit cell-free protein synthesis as a tool to screen enzyme variants and pathway combinations to accelerate slow design-build-test cycles. Additionally, our lab is using genome engineering to knock out deleterious gene to make improved "chassis" plants for metabolite production and discovery of unknown metabolic pathways. This has proved useful for producing monoterpene indole alkaloids (e.g the cancer drug vinblastine) in the wild tobacco Nicotiana benthamiana.

Spiky gold nanoparticles, a nanoscale approach to enhanced T-cell activation

While existing synthetic technologies for ex vivo T-cell activation face challenges like suboptimal expansion rates and low effectiveness, nanoscale artificial antigen-presenting cells (aAPCs) hold great promise for enhanced T-cell based therapies. In particular, gold nanoparticles (AuNPs), known for their biocompatibility, ease of synthesis and versatile surface chemistry are strong candidates for nanoscale aAPCs. In this study, we developed spiky gold nanoparticles with branched geometries to present activating ligands to primary human T-cells. The special structure of spiky AuNPs enhances biomolecule loading capacity and significantly improves T-cell activation through multivalent binding of co-stimulatory ligands and receptors. Our spiky AuNPs outperform existing benchmarks such as Dynabeads and soluble activators by promoting greater polyclonal expansion of T-cells, boosting sustained cytokine production, and generating highly functional T-cells with reduced exhaustion. In addition, spiky AuNPs effectively activate and expand CD19 CAR-T cells while demonstrating increased in vitro cytotoxicity against target cells using fewer effector cells than Dynabeads. This study underscores the potential of spiky AuNPs as a powerful tool bringing new opportunities to adoptive cell therapy applications.

An Engineered Variant of E. coli Nissle Unveils Enhanced Transformation Efficiency and Versatility in Probiotic Engineering

In recent years, the spotlight has increasingly focused on the gastrointestinal microflora, driving scientific, veterinary, and medical research interest. Consequently, probiotics, as live biotherapeutic agents, have garnered substantial attention. Among these, E. coli Nissle (EcN), a non-pathogenic gut isolate bacterium, has gained popularity. However, a formidable bottleneck faced universally in harnessing EcN’s potential has been its poor transformation efficiency, relative to other bacterial strains. In this study, we present a novel engineered strain of EcN, developed through adaptive laboratory evolution, showcasing a remarkable enhancement in transformation efficiency. This new strain has been comprehensively characterized in comparison to the wildtype EcN, encompassing assessments of growth under gut-mimicking duress conditions, motility, hydrophobicity, and plasmid replication. Since EcN is known to compete with pathogenic strains in the gut for iron, the competition dynamics and iron consumption of the strain were also significant factors to consider. Furthermore, we conducted a genome sequencing and comparative analysis, confirming the engineering of a robust EcN strain. This development heralds a groundbreaking frontier in probiotic engineering, endowing EcN with superlative potential while preserving its biological functionality.

High-throughput evolutionary function discovery of the fluoride riboswitch

Central to cellular processes is RNA. As such, RNA is central to various biotechnology development. For example, small molecules targeting RNA structure have been implemented for antivirals, antibiotics, and cancer therapeutics. Additionally, the field of synthetic biology has adapted a subtype of structured RNAs called riboswitches, which switch their structure depending on ligand binding and regulate gene expression. Riboswitch discovery has been greatly aided in comparative genomics to find evolutionary conserved RNA structures that bind ligand. Here, we expand upon these computational techniques to identify the evolution of the second half of the riboswitch responsible for controlling gene expression. We then bring comparative genomics out of the computer by developing a high-throughput RNA-sequencing assay to test every unique occurrence of the fluoride riboswitch. We compare the accuracy of computational predictions and validate hits of interest. These findings have lead to development of new hypothesizes around the evolution of the RNA sequence-structure-function relationship with implications for biotechnological innovation.

Breaking through evolutionary constraints using synthetic biology to engineer phage therapeutics

Bacteriophages are a powerful platform to target and destroy bacteria, but evolutionary constraints have prevented unlocking their full potential as therapeutic agents. Here, we demonstrate how systematic, large-scale investigation of phages reveals patterns and insights that enable engineering of bacteriophages to overcome these evolutionary constraints to optimize phage activity and eliminate bacterial pathogens they cannot naturally infect. Large scale investigations in novel environments, such as in microgravity onboard the International Space Station, reveal important trends that enable this engineering. We demonstrate the effectiveness of this approach in vivo in a murine model to resolve an extensively drug resistant E. cloacae infection.

A modular toolkit for environmental Rhodococcus, Gordonia, and Nocardia enables complex metabolic manipulation

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome, but largely lack genetic tools comparable to those available in model species such as E. coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208 and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux towards the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.

Purified, modified tRNAs for enhanced cell-free translation

Transfer RNAs (tRNAs) act as the mediators of translation by delivering amino acids to the elongating ribosome. In vivo, tRNAs are extensively adorned with post-transcriptional modifications (PTMs) which can serve as identity elements for charging, enable faithful decoding, or enhance interactions with the translational machinery. For convenience, individual tRNAs are usually generated using in vitro transcription, rendering them devoid of these important modifications. To enable the use of modified tRNAs in cell-free translation systems, we have developed a method which couples the overexpression of tRNAs to a hybridizing-probe purification process. Using this method, we purified native E. coli tRNAs bearing PTMs in good yield, high purity, and which are almost completely modified. Further, we were able to describe the modification profile of tRNAOpt from Methanocaldococcus jannaschii which is important for genetic code expansion efforts. We were also able to show modified tRNAOpt outperforms its unmodified counterpart in cell-free translation reactions requiring amber codon suppression. Our results highlight the importance of PTMs while offering a convenient way to generate these critical reagents.

Dissecting Lignin Degradation Mechanisms of Rhodopseudomonas Palustris

Lignin is a complex and abundant biopolymer that constitutes a significant proportion of the structural material of many plants. Roughly 30% of global plant biomass is composed of lignin, however, the phenolic and crosslinked structure of lignin biopolymer and lignin breakdown products prevent the efficient and cost-effective conversion to more valuable products. As a result, most lignin valorization reaction schemes are economically infeasible, and most produced lignin is instead burned as fuel. To effectively utilize lignin as a carbon substrate for bioreactors, lignin catabolism requires complex biochemistry for its degradation, which is typically supplied from a community of microorganisms and fungi. Rhodopsuedomonas palustris (hereafter, R. palustris), a non-model, gram-negative soil bacterium with a wide array of unique metabolic features, has demonstrated its ability to catabolize many lignin (Alsiyabi et al., 2021; Brown et al., 2020, 2022; Immethun et al., 2022). With its unique biochemistry that allows it to produce many sought-after chemicals such as bioplastic and biofuels, it has the potential to become a metabolic engineering chassis. From analysis of growth data collected in this study, R. palustris can catabolize multiple lignin breakdown products (LBPs), including p-coumarate, sodium ferulate, p-coumaryl, coniferyl, and sinapyl alcohols aerobically and anaerobically. Following from the study reported in literature on the catabolism of p-coumarate, this work elucidates the pathways in which other major breakdown products of lignin are catabolized by R. palustris. In this study, we investigate these pathways through an analogous “multi-omics” perspective by combining both proteomic and transcriptomic profiles of R. palustris cultured with several LBPs. A supplemental metabolomics and CRISPRi study are then used to confirm the metabolites and reactions present in these pathways. Overall, this study will advance the understanding of the complex carbon metabolism of R. palustris and assist in enabling sustainable biochemical production through lignin valorization.

TBD

Engineering cytokines to target lipid-laden macrophages

Cytokine therapies have the potential to revolutionize treatment for immunologic diseases but are limited by their poor pharmacokinetic profiles, off-target effects, and pleiotropic nature. To overcome these challenges, engineered cytokine platforms have the potential to target specific tissue environments and immune cell types to provide local immunomodulation with minimal side effects. Here, we developed a cytokine platform that targets metabolically dysfunctional macrophages in the context of atherosclerosis, a paradigm chronic inflammatory disease with high prevalence. Since macrophages that comprise atherosclerotic plaques engulf large amounts of low-density lipoprotein (LDL) and become pro-inflammatory lipid-laden “foam cells,” we engineered a protein fusion in which one side is an antibody fragment (Fab) that binds to LDL and the other side is the anti-inflammatory cytokine IL-10. Fab-IL-10 constructs attach to LDL in the bloodstream of hypercholesterolemic mice, hitchhike a ride to inflamed regions, preferentially target macrophages, and successfully reduce inflammation. Since foam cells are prevalent in many immune-mediated diseases, this platform technology could be widely applicable to multiple cytokine therapies and diverse disease settings.

Developing Light-Controlled Synthetic Zinc Finger Transcription Factors for Budding Yeast

Synthetic zinc finger transcription factors (TFs) are powerful tools that have been used to create multigene control, logic gates, and cooperativity. However, previous studies of these TFs have not fully explored optogenetics which enables rapid and precise control of nuclear localization dynamics. Based on computational modeling results of said dynamics, we constructed optogenetically controlled synthetic zinc finger TFs with differing nuclear import/export rates and nuclear TF saturation levels by balancing nuclear localization signals, nuclear export signals, and anchor proteins that sequester the TFs to the plasma membrane. In accordance with our models, a) TFs with faster import and slower export resulted in pulsatile light input yielding higher reporter expression than continuous light, and b) TFs with slower import and faster export resulted in the opposite expression pattern, showing the power of our model guided design strategy. Furthermore, we altered the number of transcription factor binding sites (TFBSs) in the cognate synthetic promoter and observed a non-linear relationship between basal level, induced expression level, and the number of TFBSs. This study lays the groundwork for designing more advanced gene expression systems with broad implications for synthetic biology and bioengineering by incorporating synthetic zinc finger TFs and cognate promoters into optogenetics.

Utilization of olefin synthase to identify the substrate and chain length selection of fatty acyl-AMP ligases (FAALs) in cyanobacteria Synechococcus sp. strain PCC 7002

Cyanobacteria are widespread Gram-negative bacteria that generate numerous bioactive secondary metabolites via complex biosynthetic enzymatic machinery. Many cyanobacterial natural products contain unique chemical structures that may provide novel mechanisms of action for clinical applications, but not many of the biosynthetic transformations from cyanobacteria are well characterized. Fatty acyl-AMP ligases (FAALs) are pivotal biosynthetic domains that divert fatty acyl chains from primary metabolism for incorporation into natural product scaffolds. FAAL domains are often linked with multidomain polyketide synthases (PKS) and incorporate free fatty acids directly. However, cyanobacteria typically do not maintain a pool of free fatty acids to act as FAAL substrates, and it is unclear what the alternative substrate for FAALs is in cyanobacteria or how they select specific acyl chain lengths for incorporation. The model cyanobacteria Synechococcus sp. strain PCC 7002 contains a unique type I PKS olefin synthase (OlsWT) that produces 1-nonadecene, an α-olefin hydrocarbon that links primary fatty acid metabolism to secondary metabolism. We developed PCC 7002 as a heterologous host to facilitate the expression and study of FAAL domains in cyanobacteria to uncover their substrates and chain length selection. We hypothesized that the linker regions between the FAAL and the PKS domains are crucial for the transfer of specific fatty acid chain lengths and that chain length modulation can be achieved by maintaining appropriate linker elements. We successfully expressed two Ols homologs, Ols04 and Ols08, in PCC 7002 for the generation of 1-pentadecene and 1-heptadecene α-olefins. Through additional genetic manipulations, we demonstrated that free fatty acids liberated from the lipid and thylakoid membranes during lipid remodeling are the likely substrates for Ols enzymes. We also evaluated the OlsWT substrate via in vitro chrome azurol S (CAS) assay to demonstrate a chain length specific activation of octadecanoic acid. Future work will focus on using the model OlsWT in PCC 7002 to identify and modulate the gate-keeping linker regions that govern fatty acid integration into secondary metabolites. This work helps to uncover the key structural elements in the model OlsWT for future bioengineering of more complex natural product enzymes that utilize identical biosynthetic logic.