Positive Selection Screens for Programmable Endonuclease Activity Using I-SceI.

Positive selection screens are high-throughput assays to characterize novel enzymes from environmental samples and enrich for more powerful variants from libraries in applications such as biodiversity mining and directed evolution. However, overly stringent selection can limit the power of these screens due to a high false-negative rate. To create a more flexible and less restrictive screen for novel programmable DNA endonucleases, we developed a novel I-SceI-based platform. In this system, mutant E. coli genomes are cleaved upon induction of I-SceI to inhibit cell growth. Growth is rescued in an activity-dependent manner by plasmid curing or cleavage of the I-SceI expression plasmid via endonuclease candidates. More active candidates more readily proliferate and overtake growth of less active variants leading to enrichment. While demonstrated here with Cas9, this protocol can be readily adapted to any programmable DNA endonuclease and used to characterize single candidates or to enrich more powerful variants from pooled candidates or libraries.

Genetically Engineered Protein-Based Bioadhesives with Programmable Material Properties.

Silk-amyloid-mussel foot protein (SAM) hydrogels made from recombinant fusion proteins containing ß-amyloid peptide, spider silk domain, and mussel foot protein (Mfp) are attractive bioadhesives as they display a unique combination of tunability, biocompatibility, bioabsorbability, strong cohesion, and underwater adhesion to a wide range of biological surfaces. To design tunable SAM hydrogels for tailored surgical repair applications, an understanding of the relationships between protein sequence and hydrogel properties is imperative. Here, we fabricated SAM hydrogels using fusion proteins of varying lengths of silk-amyloid repeats and Mfps to characterize their structure and properties. We found that increasing silk-amyloid repeats enhanced the hydrogel's ß-sheet content (r = 0.74), leading to higher cohesive strength and toughness. Additionally, increasing the Mfp length beyond the half-length of the full Mfp sequence (1/2 Mfp) decreased the ß-sheet content (r = -0.47), but increased hydrogel surface adhesion. Among different variants, the hydrogel made of 16xKLV-2Mfp displayed a high ultimate strength of 3.0 ± 0.3 MPa, an ultimate strain of 664 ± 119%, and an attractive underwater adhesivity of 416 ± 20 kPa to porcine skin. Collectively, the sequence-structure-property relationships learned from this study will be useful to guide the design of future protein adhesives with tunable characteristics for tailored surgical applications.

Engineering Alkaline-Stable Barley Stripe Mosaic Virus-Like Particles for Efficient Surface Modification.

Viruses and virus-like particles are powerful templates for materials synthesis because of their capacity for precise protein engineering and diverse surface functionalization. We recently developed a recombinant bacterial expression system for the production of barley stripe mosaic virus-like particles (BSMV VLPs). However, the applicability of this biotemplate was limited by low stability in alkaline conditions and a lack of chemical handles for ligand attachment. Here, we identify and validate novel residues in the BSMV Caspar carboxylate clusters that mediate virion disassembly through repulsive interactions at high pH. Point mutations of these residues to create attractive interactions that increase rod length ~2 fold, with an average rod length of 91 nm under alkaline conditions. To enable diverse chemical surface functionalization, we also introduce reactive lysine residues at the C-terminus of BSMV coat protein, which is presented on the VLP surface. Chemical conjugation reactions with this lysine proceed more quickly under alkaline conditions. Thus, our alkaline-stable VLP mutants are more suitable for rapid surface functionalization of long nanorods. This work validates novel residues involved in BSMV VLP assembly and demonstrates the feasibility of chemical functionalization of BSMV VLPs for the first time, enabling novel biomedical and chemical applications.

Protein-Based Hydrogels and Their Biomedical Applications.

Hydrogels made from proteins are attractive materials for diverse medical applications, as they are biocompatible, biodegradable, and amenable to chemical and biological modifications. Recent advances in protein engineering, synthetic biology, and material science have enabled the fine-tuning of protein sequences, hydrogel structures, and hydrogel mechanical properties, allowing for a broad range of biomedical applications using protein hydrogels. This article reviews recent progresses on protein hydrogels with special focus on those made of microbially produced proteins. We discuss different hydrogel formation strategies and their associated hydrogel properties. We also review various biomedical applications, categorized by the origin of protein sequences. Lastly, current challenges and future opportunities in engineering protein-based hydrogels are discussed. We hope this review will inspire new ideas in material innovation, leading to advanced protein hydrogels with desirable properties for a wide range of biomedical applications.

Microbial Synthesis of High-Molecular-Weight, Highly Repetitive Protein Polymers.

High molecular weight (MW), highly repetitive protein polymers are attractive candidates to replace petroleum-derived materials as these protein-based materials (PBMs) are renewable, biodegradable, and have outstanding mechanical properties. However, their high MW and highly repetitive sequence features make them difficult to synthesize in fast-growing microbial cells in sufficient amounts for real applications. To overcome this challenge, various methods were developed to synthesize repetitive PBMs. Here, we review recent strategies in the construction of repetitive genes, expression of repetitive proteins from circular mRNAs, and synthesis of repetitive proteins by ligation and protein polymerization. We discuss the advantages and limitations of each method and highlight future directions that will lead to scalable production of highly repetitive PBMs for a wide range of applications.

Repurposing the Homing Endonuclease I-SceI for Positive Selection and Development of Gene-Editing Technologies.

Prokaryote genomes encode diverse programmable DNA endonucleases with significant potential for biotechnology and gene editing. However, these endonucleases differ significantly in their properties, which must be screened and measured. While positive selection screens based on ccdB and barnase have been developed to evaluate such proteins, their high levels of toxicity make them challenging to use. Here, we develop and validate a more robust positive selection screen based on the homing endonuclease I-SceI. Candidate endonucleases target and cure the I-SceI expression plasmid preventing induction of I-SceI-mediated double strand DNA breaks that lead to cell death in E. coli. We validated this screen to measure the relative activity of SpCas9, xCas9, and eSpCas9 and demonstrated an ability to enrich for more active endonuclease variants from a mixed population. This system may be applied in high throughput to rapidly characterize novel programmable endonucleases and be adapted for directed evolution of endonuclease function.

NgAgo possesses guided DNA nicking activity.

Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.

Engineering Tobacco Mosaic Virus and Its Virus-Like-Particles for Synthesis of Biotemplated Nanomaterials.

Biomolecules are increasingly attractive templates for the synthesis of functional nanomaterials. Chief among them is the plant tobacco mosaic virus (TMV) due to its high aspect ratio, narrow size distribution, diverse biochemical functionalities presented on the surface, and compatibility with a number of chemical conjugations. These properties are also easily manipulated by genetic modification to enable the synthesis of a range of metallic and non-metallic nanomaterials for diverse applications. This article reviews the characteristics of TMV and related viruses, and their virus-like particle (VLP) derivatives, and how these may be manipulated to extend their use and function. A focus of recent efforts has been on greater understanding and control of the self-assembly processes that drive biotemplate formation. How these features have been exploited in engineering applications such as, sensing, catalysis, and energy storage are briefly outlined. While control of VLP surface features is well-established, fewer tools exist to control VLP self-assembly, which limits efforts to control template uniformity and synthesis of certain templated nanomaterials. However, emerging advances in synthetic biology, machine learning, and other fields promise to accelerate efforts to control template uniformity and nanomaterial synthesis enabling more widescale industrial use of VLP-based biotemplates.

Extracellular Pgk1 enhances neurite outgrowth of motoneurons through Nogo66/NgR-independent targeting of NogoA.

NogoA inhibits neurite outgrowth of motoneurons (NOM) through interaction with its receptors, Nogo66/NgR. Inhibition of Nogo receptors rescues NOM, but not to the extent exhibited by NogoA-knockout mice, suggesting the presence of other pathways. We found that NogoA-overexpressing muscle cells reduced phosphoglycerate kinase 1 (Pgk1) secretion, resulting in inhibiting NOM. Apart from its glycolytic role and independent of the Nogo66 pathway, extracellular Pgk1 stimulated NOM by triggering a reduction of p-Cofilin-S3, a growth cone collapse marker, through decreasing a novel Rac1-GTP/p-Pak1-T423/p-P38-T180/p-MK2-T334/p-Limk1-S323/p-Cofilin-S3 molecular pathway. Not only did supplementary Pgk1 enhance NOM in defective cells, but injection of Pgk1 rescued denervation in muscle-specific NogoA-overexpression of zebrafish and an Amyotrophic Lateral Sclerosis mouse model, SOD1 G93A. Thus, Pgk1 secreted from muscle is detrimental to motoneuron neurite outgrowth and maintenance.

Leveraging anaerobic fungi for biotechnology.

Early-branching anaerobic fungi are critical for hydrolyzing untreated lignocellulose in the digestive tracts of large herbivorous animals. While these fungi were discovered more than 40 years ago, they remain understudied and underexploited. Recent advances in -omics technologies, however, have enabled studies that reveal significant biosynthetic potential within anaerobic fungal genomes for diverse biotechnological applications. Applications range from enhanced second-generation bioenergy platforms to improved animal health. However, developing gut fungi for these applications will require significant advances in genome engineering technologies for these organisms. Here, we review the biotechnological abilities of anaerobic fungi and highlight challenges that must be addressed to develop them for a range of biotechnological applications.