Advancing protein display on bacterial spores through an extensive survey of coat components.

The profound stability of bacterial spores makes them a promising platform for biotechnological applications like biocatalysis, bioremediation, drug delivery, etc. However, though the spore is composed of >40 proteins, only ∼12 have been explored as fusion carriers for protein display. Here, we assessed the suitability of 33 spore proteins (SPs) as enzyme display carriers by direct allele tagging at native genomic loci.

Rapid spectrophotometric detection for optimized production of landomycins and characterization of their therapeutic potential.

Microbial-derived natural products remain a major source of structurally diverse bioactive compounds and chemical scaffolds that have the potential as new therapeutics to target drug-resistant pathogens and cancers. In particular, genome mining has revealed the vast number of cryptic or low-yield biosynthetic gene clusters in the genus Streptomyces. However, low natural product yields-improvements to which have been hindered by the lack of high throughput methods-have slowed the discovery and development of many potential therapeutics.

Specific codons control cellular resources and fitness.

As cellular engineering progresses from simply overexpressing proteins to imparting complex phenotypes through multigene expression, judicious appropriation of cellular resources is essential. Since codon use is degenerate and biased, codons may control cellular resources at a translational level. We investigate how partitioning transfer RNA (tRNA) resources by incorporating dissimilar codon usage can drastically alter interdependence of expression level and burden on the host.

Rapid spectrophotometric detection for optimized production of landomycins and characterization of their therapeutic potential.

Microbial derived natural products remain a major source of structurally diverse bioactive compounds and chemical scaffolds that have potential as new therapeutics to target drug resistant pathogens and cancers. In particular, genome mining has revealed the vast number of cryptic or low yield biosynthetic gene clusters in the genus . Here, we describe our efforts to improve yields of landomycins - angucycline family polyketides under investigation as cancer therapeutics - by a genetically modified 136.

Integration of metabolism and regulation reveals rapid adaptability to growth on non-native substrates.

Engineering synthetic heterotrophy is a key to the efficient bio-based valorization of renewable and waste substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades-yet the answer to what makes their utilization inherently recalcitrant remains elusive. Through implementation of a semi-synthetic regulon, we find that harmonizing cellular and engineering objectives are a key to obtaining highest growth rates and yields with minimal metabolic engineering effort.

Towards universal synthetic heterotrophy using a metabolic coordinator.

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3p), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates.

Flow cytometric evaluation of yeast-bacterial cell-cell interactions.

Synthetic cell-cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody-antigen pairs to a yeast-bacteria coincubation format and use flow cytometry to evaluate cell-cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting to enrich a specific yeast-displayed nanobody within a mixed yeast-display population.

A Silk-Based Platform to Stabilize Phenylalanine Ammonia-lyase for Orally Administered Enzyme Replacement Therapy.

Phenylalanine ammonia-lyase (PAL) has gained attention in recent years for the treatment of phenylketonuria (PKU), a genetic disorder that affects ∼1 in 15 000 individuals globally. However, the enzyme is easily degraded by proteases, unstable at room temperature, and currently administered in PKU patients as daily subcutaneous injections. We report here the stabilization of the PAL from , which is currently used to formulate pegvaliase, through incorporation in a silk fibroin matrix.

In-depth Sequence-Function Characterization Reveals Multiple Pathways to Enhance Enzymatic Activity.

Deep mutational scanning (DMS) has recently emerged as a powerful method to study protein sequence-function relationships but it has not been well-explored as a guide to enzyme engineering and identifying pathways by which their catalytic cycle may be improved. We report such a demonstration in this work using a Phenylalanine ammonia-lyase (PAL), which deaminates L-phenylalanine to -cinnamic acid and has widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from (AvPAL*) has garnered significant attention as the active ingredient in Pegvaliase, the only FDA-approved drug treating classical Phenylketonuria (PKU).

Cheating the Cheater: Suppressing False-Positive Enrichment during Biosensor-Guided Biocatalyst Engineering.

Transcription factor (TF)-based biosensors are very desirable reagents for high-throughput enzyme and strain engineering campaigns. Despite their potential, they are often difficult to deploy effectively as the small molecules being detected can leak out of high-producer cells, into low-producer cells, and activate the biosensor therein. This crosstalk leads to the overrepresentation of false-positive/cheater cells in the enriched population.

Analysis of metabolic network disruption in engineered microbial hosts due to enzyme promiscuity.

Increasing understanding of metabolic and regulatory networks underlying microbial physiology has enabled creation of progressively more complex synthetic biological systems for biochemical, biomedical, agricultural, and environmental applications. However, despite best efforts, confounding phenotypes still emerge from unforeseen interplay between biological parts, and the design of robust and modular biological systems remains elusive. Such interactions are difficult to predict when designing synthetic systems and may manifest during experimental testing as inefficiencies that need to be overcome.

A quantitative model for metabolic intervention using gut microbes.

As medicine shifts toward precision-based and personalized therapeutics, utilizing more complex biomolecules to treat increasingly difficult and rare conditions, microorganisms provide an avenue for realizing the production and processing necessary for novel drug pipelines. More so, probiotic microbes can be co-opted to deliver therapeutics by oral administration as living drugs, able to survive and safely transit the digestive tract. As living therapeutics are in their nascency, traditional pharmacokinetic-pharmacodynamic (PK-PD) models for evaluating drug candidates are not appropriate for this novel platform.

Engineered lactobacilli display anti-biofilm and growth suppressing activities against Pseudomonas aeruginosa.

Biofilms are an emerging target for new therapeutics in the effort to address the continued increase in resistance and tolerance to traditional antimicrobials. In particular, the distinct nature of the biofilm growth state often means that traditional antimcirobials, developed to combat planktonic cells, are ineffective. Biofilm treatments are designed to both reduce pathogen load at an infection site and decrease the development of resistance by rendering the embedded organisms more susceptible to treatment at lower antimicrobial concentrations.

Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in .

Poly-γ-glutamic acid (PGA) produced by many species is a polymer with many distinct and desirable characteristics. However, the multi-subunit enzymatic complex responsible for its synthesis, PGA Synthetase (PGS), has not been well characterized yet, in native nor in recombinant contexts. Elucidating structural and functional properties are crucial for future engineering efforts aimed at altering the catalytic properties of this enzyme.

Directed evolution of Anabaena variabilis phenylalanine ammonia-lyase (PAL) identifies mutants with enhanced activities.

There is broad interest in engineering phenylalanine ammonia-lyase (PAL) for its biocatalytic applications in industry and medicine. While site-specific mutagenesis has been employed to improve PAL stability or substrate specificity, combinatorial techniques are poorly explored. Here, we report development of a directed evolution technique to engineer PAL enzymes.

Quantifying and Engineering Mucus Adhesion of Probiotics.

Mucus in the gastrointestinal (GI) tract is the primary point-of-interaction between humans and their gut microbiota. This intimates that mucus not only ensures protection against endogenous and exogenous opportunists but also provisions for the human microbiota to reside and flourish. With the emergence of living therapeutics, engineered microbes can deliver and produce increasingly complex medicine, and controlling the mucoadhesive properties of different microbial chassis can dictate dose-response in a patient.

Galactose to tagatose isomerization at moderate temperatures with high conversion and productivity.

There are many industrially-relevant enzymes that while active, are severely limited by thermodynamic, kinetic, or stability issues (isomerases, lyases, transglycosidases). In this work, we study Lactobacillus sakei L-arabinose isomerase (LsLAI) for D-galactose to D-tagatose isomerization-that is limited by all three reaction parameters. The enzyme demonstrates low catalytic efficiency, low thermostability at temperatures > 40 °C, and equilibrium conversion < 50%.

Towards creating an extended metabolic model (EMM) for E. coli using enzyme promiscuity prediction and metabolomics data.

Background: Metabolic models are indispensable in guiding cellular engineering and in advancing our understanding of systems biology. As not all enzymatic activities are fully known and/or annotated, metabolic models remain incomplete, resulting in suboptimal computational analysis and leading to unexpected experimental results. We posit that one major source of unaccounted metabolism is promiscuous enzymatic activity.

Establishing synthesis pathway-host compatibility via enzyme solubility.

Current pathway synthesis tools identify possible pathways that can be added to a host to produce the desired target molecule through the exploration of abstract metabolic and reaction network space. However, not many of these tools explore gene-level information required to physically realize the identified synthesis pathways, and none explore enzyme-host compatibility. Developing tools that address this disconnect between abstract reactions/metabolic design space and physical genetic sequence design space will enable expedited experimental efforts that avoid exploring unprofitable synthesis pathways.

Pentose Metabolism in Saccharomyces cerevisiae: The Need to Engineer Global Regulatory Systems.

Extending the host substrate range of industrially relevant microbes, such as Saccharomyces cerevisiae, has been a highly-active area of research since the conception of metabolic engineering. Yet, rational strategies that enable non-native substrate utilization in this yeast without the need for combinatorial and/or evolutionary techniques are underdeveloped. Herein, this review focuses on pentose metabolism in S.

A semi-synthetic regulon enables rapid growth of yeast on xylose.

Nutrient assimilation is the first step that allows biological systems to proliferate and produce value-added products. Yet, implementation of heterologous catabolic pathways has so far relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. In contrast, natural systems prefer nutrient-responsive gene regulation (called regulons) that control multiple cellular functions necessary for cell survival and growth.

Synthetic biology in probiotic lactic acid bacteria: At the frontier of living therapeutics.

The trillions of microbes hosted by humans can dictate health or illness depending on a multitude of genetic, environmental, and lifestyle factors that help define the human ecosystem. As the human microbiota is characterized, so can the interconnectivity of microbe-host-disease be realized and manipulated. Designing microbes as therapeutic agents can not only enable targeted drug delivery but also restore homeostasis within a perturbed microbial community.

Synthetic Biology Approaches to Engineer Probiotics and Members of the Human Microbiota for Biomedical Applications.

An increasing number of studies have strongly correlated the composition of the human microbiota with many human health conditions and, in several cases, have shown that manipulating the microbiota directly affects health. These insights have generated significant interest in engineering indigenous microbiota community members and nonresident probiotic bacteria as biotic diagnostics and therapeutics that can probe and improve human health. In this review, we discuss recent advances in synthetic biology to engineer commensal and probiotic lactic acid bacteria, bifidobacteria, and Bacteroides for these purposes, and we provide our perspective on the future potential of these technologies.

Selection Finder (SelFi): A computational metabolic engineering tool to enable directed evolution of enzymes.

Directed evolution of enzymes consists of an iterative process of creating mutant libraries and choosing desired phenotypes through screening or selection until the enzymatic activity reaches a desired goal. The biggest challenge in directed enzyme evolution is identifying high-throughput screens or selections to isolate the variant(s) with the desired property. We present in this paper a computational metabolic engineering framework, Selection Finder (SelFi), to construct a selection pathway from a desired enzymatic product to a cellular host and to couple the pathway with cell survival.

Toward a genetic tool development pipeline for host-associated bacteria.

Bacteria reside in externally accessible niches on and in multicellular organisms, often forming mutualistic relationships with their host. Recent studies have linked the composition of these microbial communities with alterations in the host's health, behavior, and development, yet the causative mediators of host-microbiota interactions remain poorly understood. Advances in understanding and engineering these interactions require the development of genetic tools to probe the molecular interactions driving the structure and function of microbial communities as well as their interactions with their host.