Publications by authors named "Luke N Latimer"

6 Publications

  • Page 1 of 1

Exploration of Acetylation as a Base-Labile Protecting Group in for an Indigo Precursor.

ACS Synth Biol 2020 10 18;9(10):2775-2783. Epub 2020 Sep 18.

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

Biochemical protecting groups are observed in natural metabolic pathways to control reactivity and properties of chemical intermediates; similarly, they hold promise as a tool for metabolic engineers to achieve the same goals. Protecting groups come with costs: lower yields from carbon, metabolic load to the production host, deprotection catalyst costs and kinetics limitations, and wastewater treatment of the group. Compared to glycosyl biochemical protection, such as glucosyl groups, acetylation can mitigate each of these costs. As an example application where these benefits could be valuable, we explored acetylation protection of indoxyl, the reactive precursor to the clothing dye, indigo. First, we demonstrated denim dyeing with chemically sourced indoxyl acetate by deprotection with base, showing results comparable to industry-standard denim dyeing. Second, we modified an production host for improved indoxyl acetate stability by the knockout of 14 endogenous hydrolases. Cumulatively, these knockouts yielded a 67% reduction in the indoxyl acetate hydrolysis rate from 0.22 mmol/g DCW/h to 0.07 mmol/g DCW/h. To biosynthesize indoxyl acetate, we identified three promiscuous acetyltransferases which acetylate indoxyl . Indoxyl acetate titer, while low, was improved 50%, from 43 μM to 67 μM, in the hydrolase knockout strain compared to wild-type . Unfortunately, low millimolar concentrations of indoxyl acetate proved to be toxic to the production host; however, the principle of acetylation as a readily cleavable and low impact biochemical protecting group and the engineered hydrolase knockout production host should prove useful for other metabolic products.
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http://dx.doi.org/10.1021/acssynbio.0c00297DOI Listing
October 2020

DANPY (dimethylaminonaphthylpyridinium): an economical and biocompatible fluorophore.

Org Biomol Chem 2019 04;17(15):3765-3780

Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195, USA.

Dyes with nonlinear optical (NLO) properties enable new imaging techniques and photonic systems. We have developed a dye (DANPY-1) for photonics applications in biological substrates such as nucleic acids; however, the design specification also enables it to be used for visualizing biomolecules. It is a prototype dye demonstrating a water-soluble, NLO-active fluorophore with high photostability, a large Stokes shift, and a favorable toxicity profile. A practical and scalable synthetic route to DANPY salts has been optimized featuring: (1) convergent Pd-catalyzed Suzuki coupling with pyridine 4-boronic acid, (2) site-selective pyridyl N-methylation, and (3) direct recovery of crystalline intermediates without chromatography. We characterize the optical properties, biocompatibility, and biological staining behavior of DANPY-1. In addition to stability and solubility across a range of polar media, the DANPY-1 chromophore shows a first hyperpolarizability similar to common NLO dyes such as Disperse Red 1 and DAST, a large two-photon absorption cross section for its size, substantial affinity to nucleic acids in vitro, an ability to stain a variety of cellular components, and strong sensitivity of its fluorescence properties to its dielectric environment.
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http://dx.doi.org/10.1039/c8ob02536cDOI Listing
April 2019

Engineering Saccharomyces cerevisiae for co-utilization of D-galacturonic acid and D-glucose from citrus peel waste.

Nat Commun 2018 11 29;9(1):5059. Epub 2018 Nov 29.

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

Pectin-rich biomasses, such as citrus peel and sugar beet pulp, hold promise as inexpensive feedstocks for microbial fermentations as enzymatic hydrolysis of their component polysaccharides can be accomplished inexpensively to yield high concentrations of fermentable sugars and D-galacturonic acid (D-galUA). In this study, we tackle a number of challenges associated with engineering a microbial strain to convert pectin-rich hydrolysates into commodity and specialty chemicals. First, we engineer D-galUA utilization into yeast, Saccharomyces cerevisiae. Second, we identify that the mechanism of D-galUA uptake into yeast is mediated by hexose transporters and that consumption of D-galUA is inhibited by D-glucose. Third, we enable co-utilization of D-galUA and D-glucose by identifying and expressing a heterologous transporter, GatA, from Aspergillus niger. Last, we demonstrate the use of this transporter for production of the platform chemical, meso-galactaric acid, directly from industrial Navel orange peel waste.
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http://dx.doi.org/10.1038/s41467-018-07589-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265301PMC
November 2018

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

Biotechnol Bioeng 2017 06 20;114(6):1301-1309. Epub 2017 Feb 20.

Department of Bioengineering, University of California, 2151 Berkeley Way, Berkeley, California 94720.

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ∼70% increase in biomass yield and ∼240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26262DOI Listing
June 2017

Application of a Palladium-Catalyzed C-H Functionalization/Indolization Method to Syntheses of cis-Trikentrin A and Herbindole B.

Angew Chem Int Ed Engl 2016 09 29;55(39):11824-8. Epub 2016 Aug 29.

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

We describe herein formal syntheses of the indole alkaloids cis-trikentrin A and herbindole B from a common meso-hydroquinone intermediate prepared by a ruthenium-catalyzed [2+2+1+1] cycloaddition that has not been used previously in natural product synthesis. Key steps include a sterically demanding Buchwald-Hartwig amination as well as a unique C(sp(3) )-H amination/indole formation. Studies toward a selective desymmetrization of the meso-hydroquinone are also reported.
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http://dx.doi.org/10.1002/anie.201605475DOI Listing
September 2016

Employing a combinatorial expression approach to characterize xylose utilization in Saccharomyces cerevisiae.

Metab Eng 2014 Sep 13;25:20-9. Epub 2014 Jun 13.

Energy Biosciences Institute, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Fermentation of xylose, a major constituent of lignocellulose, will be important for expanding sustainable biofuel production. We sought to better understand the effects of intrinsic (genotypic) and extrinsic (growth conditions) variables on optimal gene expression of the Scheffersomyces stipitis xylose utilization pathway in Saccharomyces cerevisiae by using a set of five promoters to simultaneously regulate each gene. Three-gene (xylose reductase, xylitol dehydrogenase (XDH), and xylulokinase) and eight-gene (expanded with non-oxidative pentose phosphate pathway enzymes and pyruvate kinase) promoter libraries were enriched under aerobic and anaerobic conditions or with a mutant XDH with altered cofactor usage. Through characterization of enriched strains, we observed (1) differences in promoter enrichment for the three-gene library depending on whether the pentose phosphate pathway genes were included during the aerobic enrichment; (2) the importance of selection conditions, where some aerobically-enriched strains underperform in anaerobic conditions compared to anaerobically-enriched strains; (3) improved growth rather than improved fermentation product yields for optimized strains carrying the mutant XDH compared to the wild-type XDH.
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http://dx.doi.org/10.1016/j.ymben.2014.06.002DOI Listing
September 2014