JBEI - Thomas Eng et al

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Thomas C.glu isopentenol production targeted proteins_2019-01-07_16-39-19.sky.zip (6 MB)2019-01-089414115648
Engineering Corynebacterium glutamicum to produce the biogasoline isopentenol from plant biomass hydrolysates

  • Organism: Corynebacterium glutamicum ATCC 13032
  • Instrument: 6460 Triple Quadrupole LC/MS
  • SpikeIn: No
  • Keywords: C. glutamicum, isoprenoid pathway, targeted proteomics
  • Lab head: Chris Petzold
Abstract
Background. Many microbial hosts used for the rapid discovery and development of metabolic pathways can have sensitivities to final products and process reagents. Isopentenol, a biogasoline candidate, has an established heterologous gene pathway but is toxic to many microbial hosts. Reagents used in the pretreatment of plant biomass, such as ionic liquids, also inhibit growth of many host strains. We explored the use of Corynebacterium glutamicum as an alternative host to address these constraints. Results. We found C. glutamicum ATCC 13032 to be tolerant to both the final product, isopentenol, as well to three classes ionic liquids. A heterologous mevalonate-based isopentenol pathway was engineered in C. glutamicum. Isopentenol titers were improved via three routes: media optimization; substitution of an NADH-dependent HmgR homolog from Silicibacter pomeroyi; and development of a C. glutamicum ∆poxB ∆ldhA host chassis.
Experiment Description
We describe the successful expression of a heterologous pathway in the gram-positive industrial microorganism, C. glutamicum, for the production of the biogasoline candidate, isopentenol. Targeted proteomics for the heterologous pathway proteins indicated that the 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein, HmgR, as a potential rate-limiting enzyme in this synthetic pathway. We identified and optimized the critical genetic and media parameters required to produce 1.25 g/L isopentenol in defined minimal media with D-glucose as the carbon source and similar titers (1g/ L) using sorghum biomass hydrolysates as carbon source.
Sample Description
Whole cell protein extraction and targeted proteomic analysis of isoprenoid pathway proteins.
Created on 1/8/19, 3:55 PM