Publications by authors named "William C DeLoache"

7 Publications

  • Page 1 of 1

An exclusive metabolic niche enables strain engraftment in the gut microbiota.

Nature 2018 05 9;557(7705):434-438. Epub 2018 May 9.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.

The dense microbial ecosystem in the gut is intimately connected to numerous facets of human biology, and manipulation of the gut microbiota has broad implications for human health. In the absence of profound perturbation, the bacterial strains that reside within an individual are mostly stable over time . By contrast, the fate of exogenous commensal and probiotic strains applied to an established microbiota is variable, generally unpredictable and greatly influenced by the background microbiota. Therefore, analysis of the factors that govern strain engraftment and abundance is of critical importance to the emerging field of microbiome reprogramming. Here we generate an exclusive metabolic niche in mice via administration of a marine polysaccharide, porphyran, and an exogenous Bacteroides strain harbouring a rare gene cluster for porphyran utilization. Privileged nutrient access enables reliable engraftment of the exogenous strain at predictable abundances in mice harbouring diverse communities of gut microbes. This targeted dietary support is sufficient to overcome priority exclusion by an isogenic strain , and enables strain replacement. We demonstrate transfer of the 60-kb porphyran utilization locus into a naive strain of Bacteroides, and show finely tuned control of strain abundance in the mouse gut across multiple orders of magnitude by varying porphyran dosage. Finally, we show that this system enables the introduction of a new strain into the colonic crypt ecosystem. These data highlight the influence of nutrient availability in shaping microbiota membership, expand the ability to perform a broad spectrum of investigations in the context of a complex microbiota, and have implications for cell-based therapeutic strategies in the gut.
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http://dx.doi.org/10.1038/s41586-018-0092-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126907PMC
May 2018

Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.

Nat Commun 2016 Mar 30;7:11152. Epub 2016 Mar 30.

Department of Bioengineering, UC Berkeley, Berkeley, California 94720, USA.

Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways. Here we focus on the Saccharomyces cerevisiae peroxisome and develop a sensitive high-throughput assay for peroxisomal cargo import. We identify an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly sequestering non-native cargo proteins. Additionally, we perform the first systematic in vivo measurements of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay. Finally, we apply these new insights to compartmentalize a two-enzyme pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titre. This work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.
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http://dx.doi.org/10.1038/ncomms11152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476825PMC
March 2016

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

ACS Synth Biol 2015 Nov 15;4(11):1205-16. Epub 2015 Jul 15.

Department of Bioengineering, §Department of Chemical and Biomolecular Engineering, and ⊥Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.

Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.
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http://dx.doi.org/10.1021/acssynbio.5b00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654675PMC
November 2015

An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose.

Nat Chem Biol 2015 Jul 18;11(7):465-71. Epub 2015 May 18.

Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA.

Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.
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http://dx.doi.org/10.1038/nchembio.1816DOI Listing
July 2015

A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly.

ACS Synth Biol 2015 Sep 1;4(9):975-86. Epub 2015 May 1.

Department of Bioengineering, University of California , Berkeley, California 94720, United States.

Saccharomyces cerevisiae is an increasingly attractive host for synthetic biology because of its long history in industrial fermentations. However, until recently, most synthetic biology systems have focused on bacteria. While there is a wealth of resources and literature about the biology of yeast, it can be daunting to navigate and extract the tools needed for engineering applications. Here we present a versatile engineering platform for yeast, which contains both a rapid, modular assembly method and a basic set of characterized parts. This platform provides a framework in which to create new designs, as well as data on promoters, terminators, degradation tags, and copy number to inform those designs. Additionally, we describe genome-editing tools for making modifications directly to the yeast chromosomes, which we find preferable to plasmids due to reduced variability in expression. With this toolkit, we strive to simplify the process of engineering yeast by standardizing the physical manipulations and suggesting best practices that together will enable more straightforward translation of materials and data from one group to another. Additionally, by relieving researchers of the burden of technical details, they can focus on higher-level aspects of experimental design.
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http://dx.doi.org/10.1021/sb500366vDOI Listing
September 2015

Compartmentalizing metabolic pathways in organelles.

Nat Biotechnol 2013 Apr;31(4):320-1

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http://dx.doi.org/10.1038/nbt.2549DOI Listing
April 2013

Spatial organization of enzymes for metabolic engineering.

Metab Eng 2012 May 18;14(3):242-51. Epub 2011 Sep 18.

Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.

As synthetic pathways built from exogenous enzymes become more complicated, the probability of encountering undesired interactions with host organisms increases, thereby lowering product titer. An emerging strategy to combat this problem is to spatially organize pathway enzymes into multi-protein complexes, where high local concentrations of enzymes and metabolites may enhance flux and limit problematic interactions with the cellular milieu. Co-localizing enzymes using synthetic scaffolds has improved titers for multiple pathways. While lacking physical diffusion barriers, scaffolded systems could concentrate intermediates locally through a mechanism analogous to naturally occurring microdomains. A more direct strategy for compartmentalizing pathway components would be to encapsulate them within protein shells. Several classes of shells have been loaded with exogenous proteins and expressed successfully in industrial hosts. A critical challenge for achieving ideal pathway compartmentalization with protein shells will likely be evolving pores to selectively limit intermediate diffusion. Eventually, these tools should enhance our ability to rationally design metabolic pathways.
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http://dx.doi.org/10.1016/j.ymben.2011.09.003DOI Listing
May 2012