Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons.

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

Rapid Evaluation of Staple Placement in Stabilized α Helices Using Bacterial Surface Display.

There are a wealth of proteins involved in disease that cannot be targeted by current therapeutics because they are inside cells, inaccessible to most macromolecules, and lack small-molecule binding pockets. Stapled peptides, where two amino acids are covalently linked, form a class of macrocycles that have the potential to penetrate cell membranes and disrupt intracellular protein-protein interactions. However, their discovery relies on solid-phase synthesis, greatly limiting queries into their complex design space involving amino acid sequence, staple location, and staple chemistry. Here, we use stabilized peptide engineering by Escherichia coli display (SPEED), which utilizes noncanonical amino acids and click chemistry for stabilization, to rapidly screen staple location and linker structure to accelerate peptide design. After using SPEED to confirm hotspots in the mdm2-p53 interaction, we evaluated different staple locations and staple chemistry to identify several novel nanomolar and sub-nanomolar antagonists. Next, we evaluated SPEED in the B cell lymphoma 2 (Bcl-2) protein family, which is responsible for regulating apoptosis. We report that novel staple locations modified in the context of BIM, a high affinity but nonspecific naturally occurring peptide, improve its specificity against the highly homologous proteins in the Bcl-2 family. These compounds demonstrate the importance of screening linker location and chemistry in identifying high affinity and specific peptide antagonists. Therefore, SPEED can be used as a versatile platform to evaluate multiple design criteria for stabilized peptide engineering.

GPCR-FEX: A Fluoride-Based Selection System for Rapid GPCR Screening and Engineering.

The directed evolution of proteins comprises a search of sequence space for variants that improve a target phenotype, yet identification of desirable variants is inherently limited by library size and screening ability. Selections that couple protein phenotype to cell viability accelerate identification of promising variants by depleting libraries of undesirable variants en masse. Here, we introduce GPCR-FEX, a stringent selection platform that couples G-protein coupled receptor (GPCR) signaling to expression of a fluoride ion exporter (FEX)-GFP fusion gene and concomitant cellular fluoride tolerance in yeast. The GPCR-FEX platform works to deplete inactive GPCR variants from the library prior to high-throughput fluorescence-based cell sorting for rapid, inexpensive screening of receptor libraries that sample an expanded sequence space. Using this system, FEX1 was placed under the control of either PFUS1 or PFIG1, promoters activated upon agonist binding by the native yeast GPCRs, Ste2p or Ste3p. Addition of a C-terminal degron to FEX1p enhanced the dynamic range of cell growth between agonist-treated and untreated cells. Using deep sequencing to enumerate population members, we show rapid selection of a previously engineered Ste2p receptor mutant strain over wild-type Ste2p in a model library enrichment experiment. Overall, the GPCR-FEX platform provides a mechanism to rapidly engineer GPCRs, which are important cellular sensors for synthetic biology.

The Anaerobic Fungi: Challenges and Opportunities for Industrial Lignocellulosic Biofuel Production.

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation.

Optoacoustic Imaging of Glucagon-like Peptide-1 Receptor with a Near-Infrared Exendin-4 Analog.

Limitations in current imaging tools have long challenged the imaging of small pancreatic islets in animal models. Here, we report the first development and in vivo validation testing of a broad-spectrum and high-absorbance near-infrared optoacoustic contrast agent, E4x12-Cy7. Our near-infrared tracer is based on the amino acid sequence of exendin-4 and targets the glucagon-like peptide-1 receptor (GLP-1R). Cell assays confirmed that E4x12-Cy7 has a high-binding affinity (dissociation constant, Kd, 4.6 ± 0.8 nM). Using the multispectral optoacoustic tomography, we imaged E4x12-Cy7 and optoacoustically visualized ß-cell insulinoma xenografts in vivo for the first time. In the future, similar optoacoustic tracers that are specific for ß-cells and combines optoacoustic and fluorescence imaging modalities could prove to be important tools for monitoring the pancreas for the progression of diabetes.

Directed Evolution Using Stabilized Bacterial Peptide Display.

Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently "undruggable" targets. However, engineering an optimal sequence, stabilizing linker location, and physicochemical properties is a slow and arduous process. By pairing non-natural amino acid incorporation and cell surface click chemistry in bacteria with high-throughput sorting, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Through in situ stabilization on the bacterial surface, we demonstrate rapid isolation of stabilized peptides with improved affinity and novel structures. Several peptides evolved a second loop including one sequence (Kd = 1.8 nM) containing an i, i+4 disulfide bond. NMR structural determination indicated a bent helix in solution and bound to MDM2. The bicyclic peptide had improved protease stability, and we demonstrated that protease resistance could be measured both on the bacterial surface and in solution, enabling the method to test and/or screen for additional drug-like properties critical for biologically active compounds.

Hitting Undruggable Targets: Viewing Stabilized Peptide Development through the Lens of Quantitative Systems Pharmacology.

Stabilized peptide therapeutics have the potential to hit currently undruggable targets, dramatically expanding the druggable genome. However, major obstacles to their development include poor intracellular delivery, rapid degradation, low target affinity, and membrane toxicity. With the emergence of multiple stabilization techniques and screening technologies, the high efficacy of various bioactive peptides has been demonstrated in vitro, albeit with limited success in vivo. We discuss here the chemical and pharmacokinetic barriers to achieving in vivo efficacy, analyze the characteristics of FDA-approved peptide drugs, and propose a developmental tool that considers the molecular properties of stabilized peptides in a comprehensive and quantitative manner to achieve the necessary rates for in vivo delivery to the target, efficacy, and ultimately clinical translation.

A Helix-Stabilizing Linker Improves Subcutaneous Bioavailability of a Helical Peptide Independent of Linker Lipophilicity.

Stabilized peptides address several limitations to peptide-based imaging agents and therapeutics such as poor stability and low affinity due to conformational flexibility. There is also active research in developing these compounds for intracellular drug targeting, and significant efforts have been invested to determine the effects of helix stabilization on intracellular delivery. However, much less is known about the impact on other pharmacokinetic parameters such as plasma clearance and bioavailability. We investigated the effect of different fluorescent helix-stabilizing linkers with varying lipophilicity on subcutaneous (sc) bioavailability using the glucagon-like peptide-1 (GLP-1) receptor ligand exendin as a model system. The stabilized peptides showed significantly higher protease resistance and increased bioavailability independent of linker hydrophilicity, and all subcutaneously delivered conjugates were able to successfully target the islets of Langerhans with high specificity. The lipophilic peptide variants had slower absorption and plasma clearance than their respective hydrophilic conjugates, and the absolute bioavailability was also lower likely due to the longer residence times in the skin. Their ease and efficiency make double-click helix stabilization chemistries a useful tool for increasing the bioavailability of peptide therapeutics, many of which suffer from rapid in vivo protease degradation. Helix stabilization using linkers of varying lipophilicity can further control sc absorption and clearance rates to customize plasma pharmacokinetics.

Antigen specificity can be irrelevant to immunocytokine efficacy and biodistribution.

Cytokine therapy can activate potent, sustained antitumor responses, but collateral toxicity often limits dosages. Although antibody-cytokine fusions (immunocytokines) have been designed with the intent to localize cytokine activity, systemic dose-limiting side effects are not fully ameliorated by attempted tumor targeting. Using the s.c. B16F10 melanoma model, we found that a nontoxic dose of IL-2 immunocytokine synergized with tumor-specific antibody to significantly enhance therapeutic outcomes compared with immunocytokine monotherapy, concomitant with increased tumor saturation and intratumoral cytokine responses. Examination of cell subset biodistribution showed that the immunocytokine associated mainly with IL-2R-expressing innate immune cells, with more bound immunocytokine present in systemic organs than the tumor microenvironment. More surprisingly, immunocytokine antigen specificity and Fcγ receptor interactions did not seem necessary for therapeutic efficacy or biodistribution patterns because immunocytokines with irrelevant specificity and/or inactive mutant Fc domains behaved similarly to tumor-specific immunocytokine. IL-2-IL-2R interactions, rather than antibody-antigen targeting, dictated immunocytokine localization; however, the lack of tumor targeting did not preclude successful antibody combination therapy. Mathematical modeling revealed immunocytokine size as another driver of antigen targeting efficiency. This work presents a safe, straightforward strategy for augmenting immunocytokine efficacy by supplementary antibody dosing and explores underappreciated factors that can subvert efforts to purposefully alter cytokine biodistribution.

Dual-purpose linker for alpha helix stabilization and imaging agent conjugation to glucagon-like peptide-1 receptor ligands.

Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel α-helix-stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enable this technique to potentially be used as a general method for labeling α helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents.