Quantifying complexity in metabolic engineering using the LASER database

We previously introduced the LASER database (Learning Assisted Strain EngineeRing, https://bitbucket.org/jdwinkler/laser_release) (Winkler et al. 2015) to serve as a platform for understanding past and present metabolic engineering practices. Over the past year, LASER has been expanded by 50% to include over 600 engineered strains from 450 papers, including their growth conditions, genetic modifications, and other information in an easily searchable format. Here, we present the results of our efforts to use LASER as a means for defining the complexity of a metabolic engineering “design”. We evaluate two complexity metrics based on the concepts of construction difficulty and novelty. No correlation is observed between expected product yield and complexity, allowing minimization of complexity without a performance trade-off. We envision the use of such complexity metrics to filter and prioritize designs prior to implementation of metabolic engineering efforts, thereby potentially reducing the time, labor, and expenses of large-scale projects. Possible future developments based on an expanding LASER database are then discussed. Keywords: Metabolic engineering, Synthetic biology, Standardization, Design tool

The LASER database: Formalizing design rules for metabolic engineering

The ability of metabolic engineers to conceptualize, implement, and evaluate strain designs has dramatically increased in the last decade. Unlike other engineering fields, no centralized, open-access, and easily searched repository exists for cataloging these designs and the lessons learned from their construction and evaluation. To address this issue, we have developed a repository for metabolic engineering strain designs, known as LASER (Learning Assisted Strain EngineeRing, laser.colorado.edu) and a formal standard for disseminating designs to metabolic engineers. Curation of every available genetically-defined E. coli and S. cerevisiae strain from 310 metabolic engineering papers published over the last 21 years yields a total of 417 designs containing a total of 2661 genetic modifications. This collection has been deposited in LASER and represents the known bibliome of genetically defined and tested metabolic engineering designs in the academic literature. Properties of LASER designs and the analysis pipeline are examined to provide insight into LASER capabilities. Several future research directions utilizing LASER capabilities are discussed to highlight the potential of the LASER database for metabolic engineering. Keywords: Metabolic engineering, Synthetic biology, Standardization, Design tool

The Resistome: A Comprehensive Database of <i>Escherichia coli</i> Resistance Phenotypes

The microbial ability to resist stressful environmental conditions and chemical inhibitors is of great industrial and medical interest. Much of the data related to mutation-based stress resistance, however, is scattered through the academic literature, making it difficult to apply systematic analyses to this wealth of information. To address this issue, we introduce the Resistome database: a literature-curated collection of <i>Escherichia coli</i> genotypes–phenotypes containing over 5,000 mutants that resist hundreds of compounds and environmental conditions. We use the Resistome to understand our current state of knowledge regarding resistance and to detect potential synergy or antagonism between resistance phenotypes. Our data set represents one of the most comprehensive collections of genomic data related to resistance currently available. Future development will focus on the construction of a combined genomic–transcriptomic–proteomic framework for understanding <i>E. coli</i>’s resistance biology. The Resistome can be downloaded at https://bitbucket.org/jdwinkler/resistome_release/overview

Rapid and Efficient One-Step Metabolic Pathway Integration in <i>E. coli</i>

Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in <i>Escherichia coli</i>. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the <i>E. coli</i> genome as a stable testing platform

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1-2wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1–2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies