Controlling metabolic flux by toehold-mediated strand displacement.

To maximize desired products in engineered cellular factories it is often necessary to optimize metabolic flux. While a number of works have focused on metabolic pathway enhancement through genetic regulators and synthetic scaffolds, these approaches require time-intensive design and optimization with limited versatility and capacity for scale-up. Recently, nucleic-acid nanotechnology has emerged as an encouraging approach to overcome these limitations and create systems for modular programmable control of metabolic flux. Using toehold-mediated strand displacement (TMSD), nucleic acid constructs can be made into dynamic devices that recognize specific biomolecular triggers for conditional control of gene regulation as well as design of dynamic synthetic scaffolds. This review will consider the various approaches that have been used thus far to control metabolic flux using toehold-gated devices.

Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.

Riboregulated toehold-gated gRNA for programmable CRISPR-Cas9 function.

Predictable control over gene expression is essential to elicit desired synthetic cellular phenotypes. Although CRISPR-Cas9 offers a simple RNA-guided method for targeted transcriptional control, it lacks the ability to integrate endogenous cellular information for efficient signal processing. Here, we present a new class of riboregulators termed toehold-gated gRNA (thgRNA) by integrating toehold riboswitches into sgRNA scaffolds, and demonstrate their programmability for multiplexed regulation in Escherichia coli with minimal cross-talks.

Control of the Yeast Mating Pathway by Reconstitution of Functional α-Factor Using Split Intein-Catalyzed Reactions.

Synthetic control strategies using signaling peptides to regulate and coordinate cellular behaviors in multicellular organisms and synthetic consortia remain largely underdeveloped because of the complexities necessitated by heterologous peptide expression. Using recombinant proteins that exploit split intein-mediated reactions, we presented here a new strategy for reconstituting functional signaling peptides capable of eliciting desired cellular responses in S. cerevisiae. These designs can potentially be tailored to any signaling peptides to be reconstituted, as the split inteins are promiscuous and both the peptides and the reactions are amenable to changes by directed evolution and other protein engineering tools, thereby offering a general strategy to implement synthetic control strategies in a large variety of applications.

Synthetic scaffolds for pathway enhancement.

Controlling local concentrations of reactants, intermediates, and enzymes in synthetic pathways is critical for achieving satisfactory productivity of any desired products. An emerging approach to exert control over local concentrations is the use of synthetic biomolecular scaffolds to co-localize key molecules of synthetic pathways. These scaffolds bring the key molecules into close proximity by recruiting pathway enzymes via ligand binding and/or physically sequestrating enzymes and metabolites into isolated compartments. Novel scaffolds made of proteins, nucleic acids, and micro-compartments with increasingly complex architecture have recently been explored and applied to a variety of pathways, with varying degrees of success. Despite these strides, precise assembly of synthetic scaffolds remains a difficult task, particularly in vivo, where interactions both intended and unexpected can lead to unpredictable results. Additionally, because heterologous enzymes often have lowered activities in their new hosts, an ideal scaffold should provide a flexible platform that can adapt to kinetic imbalances in different contexts. In this review, we discuss some of the notable advances in the creation of these synthetic scaffolds and highlight the current challenges in their application.

Bioengineering strategies to generate artificial protein complexes.

For many applications, increasing synergy between distinct proteins through organization is important for the specificity, regulation, and overall reaction efficiency. Although there are many examples of protein complexes in nature, a generalized method to create these complexes remains elusive. Many conventional techniques such as random chemical conjugation, physical adsorption onto surfaces, and encapsulation within matrices are imprecise approaches and can lead to deactivation of protein native functionalities. More "bio-friendly" approaches such as genetically fused proteins and biological scaffolds often can result in low yields and low complex stability. Alternatively, site-specific protein conjugation or ligation can generate artificial protein complexes that preserve the native functionalities of protein domains and maintain stability through covalent bonds. In this review, we describe three distinct methods to synthesize artificial protein complexes (genetic incorPoration of unnatural amino acids to introduce bio-orthogonal azide and alkyne groups to proteins, split-intein based expressed protein ligation, and sortase mediated ligation) and highlight interesting applications for each technique.

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.

Biomolecular scaffolds for enhanced signaling and catalytic efficiency.

Proteins inherently are not designed to be standalone entities. Whether it is a multi-step biochemical reaction or a signaling event that triggers several other cascading events, proteins are naturally designed to function cohesively. Several natural systems have been developed through evolution to co-localize the functional proteins of the same pathway in order to ensure efficient communication of signals or intermediates. This review focuses on some selected examples of where synthetic scaffolds inspired by nature have been used to enhance the overall biological pathway performance. Applications encompass both in vivo and in vitro systems that address two key biological events in cell signaling and biosynthesis will be discussed.