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. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

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. Additionally, we discuss key remaining technical barriers that, if addressed, could increase the diversity and rate of the development of EBTs.

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. Deregulated networks are less sensitive to environmental conditions improving process robustness. Process robustness in turn leads to predictable scalability, minimizing the need for traditional process optimization. We validate process robustness and scalability of strains and bioprocesses synthesizing the important industrial chemicals alanine, citramalate and xylitol. Predictive high throughput approaches that translate to larger scales are critical for metabolic engineering programs to truly take advantage of the rapidly increasing throughput and decreasing costs of synthetic biology.

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

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. AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate the adoption of phosphate-limited two-stage protein expression protocols.

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. We utilize 2-stage dynamic metabolic control to compare two approaches to optimize xylitol biosynthesis, a stoichiometric approach, wherein competitive fluxes are decreased, and a regulatory approach wherein the levels of key regulatory metabolites are reduced. The stoichiometric and regulatory approaches lead to a 20-fold and 90-fold improvement in xylitol production, respectively. Strains with reduced levels of enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, led to altered metabolite pools resulting in the activation of the membrane bound transhydrogenase and an NADPH generation pathway, consisting of pyruvate ferredoxin oxidoreductase coupled with NADPH dependent ferredoxin reductase, leading to increased NADPH fluxes, despite a reduction in NADPH pools. These strains produced titers of 200 g/L of xylitol from xylose at 86% of theoretical yield in instrumented bioreactors. We expect dynamic control over the regulation of the membrane bound transhydrogenase as well as NADPH production through pyruvate ferredoxin oxidoreductase to broadly enable improved NADPH dependent bioconversions or production via NADPH dependent metabolic pathways.

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

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 E. coli 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. Expression was both media and promoter dependent. Of the 16 promoters tested, 4 were identified to have tightly controlled expression, which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlights the impact of the environment on the performance of biological parts and the importance of robustness testing in synthetic biology.

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. After cell harvest, the addition of detergent (0.1% Triton X-100) and a single 30 min freeze-thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high-throughput cultivation in microtiter plates, and larger scale stirred-tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.

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. Despite its great potential, lipidation remains an underutilized strategy in the clinical translation of lead biologics. We review how lipidation can overcome common challenges in biologics development as well as highlight gaps in our understanding of the effect of lipidation on therapeutic efficacy, where increased research and development efforts may lead to next-generation drugs.