Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E.

2-Stage microfermentations.

Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation.

Phase separation methods for protein purification: A meta-analysis of purification performance and cost-effectiveness.

Protein purifications based on phase separations (e.g., precipitation and liquid-liquid extraction) have seen little adoption in commercial protein drug production.

Precipitation and Extraction Methods for Protein Purification: A Meta-Analysis of Purification Performance and Cost-Effectiveness.

Unlabelled: For protein drug purification, packed-bed chromatography often remains both the predominant method and a bottleneck for cost and scalability. Accordingly, extensive efforts have been made to develop alternatives, such as precipitation and liquid-liquid extraction. Despite decades of development, such methods have been slow to see adoption in commercial processes.

"Multiagent" Screening Improves Directed Enzyme Evolution by Identifying Epistatic Mutations.

Enzyme evolution has enabled numerous advances in biotechnology and synthetic biology, yet still requires many iterative rounds of screening to identify optimal mutant sequences. This is due to the sparsity of the fitness landscape, which is caused by epistatic mutations that only offer improvements when combined with other mutations. We report an approach that incorporates diverse substrate analogues in the screening process, where multiple substrates act like multiple agents navigating the fitness landscape, identifying epistatic mutant residues without a need for testing the entire combinatorial search space.

Integrated autolysis, DNA hydrolysis and precipitation enables an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral and clinical-stage anti-COVID-19 candidate.

Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral currently in clinical trials for COVID-19. Q-Griffithsin is produced at high titer in and purified to anticipated clinical grade without conventional chromatography or the need for any fixed downstream equipment.

A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity.

CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized.

Measuring Oligonucleotide Hydrolysis in Cellular Lysates via Viscosity Measurements.

Cell lysis, a process that releases host oligonucleotides, is required in many biotechnological applications. However, intact oligonucleotides in crude cellular lysates increase the viscosity of lysates, which complicates downstream processes and routine laboratory workflows. To address this, nucleases that hydrolyze the intact oligonucleotides are commonly added, either as purified enzymes or co-expressed in genetically engineered bacterial strains.

Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in .

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial.

Integrated Autolysis, DNA Hydrolysis and Precipitation Enables an Improved Bioprocess for Q-Griffithsin, a Broad-Spectrum Antiviral and Clinical-Stage anti-COVID-19 Candidate.

Unlabelled: Across the biomanufacturing industry, innovations are needed to improve efficiency and flexibility, especially in the face of challenges such as the COVID-19 pandemic. Here we report an improved bioprocess for Q-Griffithsin, a broad-spectrum antiviral currently in clinical trials for COVID-19. Q-Griffithsin is produced at high titer in and purified to anticipated clinical grade without conventional chromatography or the need for any fixed downstream equipment.

Two-stage dynamic deregulation of metabolism improves process robustness & scalability in engineered E. coli.

We report that two-stage dynamic control improves bioprocess robustness as a result of the dynamic deregulation of central metabolism. Dynamic control is implemented during stationary phase using combinations of CRISPR interference and controlled proteolysis to reduce levels of central metabolic enzymes. Reducing the levels of key enzymes alters metabolite pools resulting in deregulation of the metabolic network.

The past, present, and future of enzyme-based therapies.

Enzyme-based therapeutics (EBTs) have the potential to tap into an almost unmeasurable amount of enzyme biodiversity and treat myriad conditions. Although EBTs were some of the first biologics used clinically, the rate of development of newer EBTs has lagged behind that of other biologics. Here, we review the history of EBTs, and discuss the state of each class of EBT, their potential clinical advantages, and the unique challenges to their development.

Optimization of phosphate-limited autoinduction broth for two-stage heterologous protein expression in .

Autoinducible, two-stage protein expression leveraging phosphate-inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date, small-scale production using microtiter plates and shake flasks relies on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, the authors develop a simpler media formulation (AB-2) with just a few autoclavable components.

The bioprocess TEA calculator: An online technoeconomic analysis tool to evaluate the commercial competitiveness of potential bioprocesses.

Techno-economic analysis connects R&D, engineering, and business. By linking process parameters to financial metrics, it allows researchers to understand the factors controlling the potential success of their technologies. In particular, metabolic and bioprocess engineering, as disciplines, are aimed at engineering cells to synthesize products with an ultimate goal of commercial deployment.

BioSamplr: An open source, low cost automated sampling system for bioreactors.

Autosampling from bioreactors reduces error, increases reproducibility and offers improved aseptic handling when compared to manual sampling. Additionally, autosampling greatly decreases the hands-on time required for a bioreactor experiment and enables sampling 24 h a day. We have designed, built and tested a low cost, open source, automated bioreactor sampling system, the BioSamplr.

Dynamic control over feedback regulatory mechanisms improves NADPH flux and xylitol biosynthesis in engineered E. coli.

We report improved NADPH flux and xylitol biosynthesis in engineered E. coli. Xylitol is produced from xylose via an NADPH dependent reductase.

CRISPR-Cas "Non-Target" Sites Inhibit On-Target Cutting Rates.

CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA.

Cas1/2 Endonuclease Complex Modifies Self-Targeting CRISPR/Cascade Spacers Reducing Silencing Guide Stability.

CRISPR-based interference has become common in various applications from genetic circuits to dynamic metabolic control. In , the native CRISPR Cascade system can be utilized for silencing by deletion of the nuclease along with expression of guide RNA arrays, where multiple genes can be silenced from a single transcript. We notice the loss of spacer sequences from guide arrays utilized for dynamic silencing.

Low-Cost, Large-Scale Production of the Anti-viral Lectin Griffithsin.

Griffithsin, a broad-spectrum antiviral lectin, has potential to prevent and treat numerous viruses including HIV, HCV, HSV, SARS-CoV, and SARS-CoV-2. For these indications, the annual demand for Griffithsin could reach billions of doses and affordability is paramount. We report the lab-scale validation of a bioprocess that supports production volumes of >20 tons per year at a cost of goods sold below $3,500/kg.

Improved two-stage protein expression and purification via autoinduction of both autolysis and auto DNA/RNA hydrolysis conferred by phage lysozyme and DNA/RNA endonuclease.

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two-stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted.

Scalable, two-stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion.

We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein.

Media Robustness and Scalability of Phosphate Regulated Promoters Useful for Two-Stage Autoinduction in .

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the evaluation of the media robustness of a small library of PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media.

A Review of the Biotechnological Production of Methacrylic Acid.

Industrial biotechnology can lead to new routes and potentially to more sustainable production of numerous chemicals. We review the potential of biobased routes from sugars to the large volume commodity, methacrylic acid, involving fermentation based bioprocesses. We cover the key progress over the past decade on direct and indirect fermentation based routes to methacrylic acid including both academic as well as patent literature.

A review of lipidation in the development of advanced protein and peptide therapeutics.

The use of biologics (peptide and protein based drugs) has increased significantly over the past few decades. However, their development has been limited by their short half-life, immunogenicity and low membrane permeability, restricting most therapies to extracellular targets and administration by injection. Lipidation is a clinically-proven post-translational modification that has shown great promise to address these issues: improving half-life, reducing immunogenicity and enabling intracellular uptake and delivery across epithelia.

Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity.

Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response.