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Title: Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol

Authors:
ORCiD logo; ORCiD logo;
Publication Date:
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1355946
Grant/Contract Number:
AR0000432
Resource Type:
Journal Article: Published Article
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Related Information: CHORUS Timestamp: 2018-05-02 04:30:08; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English

Citation Formats

Rohlhill, Julia, Sandoval, Nicholas R., and Papoutsakis, Eleftherios T. Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol. United States: N. p., 2017. Web. doi:10.1021/acssynbio.7b00114.
Rohlhill, Julia, Sandoval, Nicholas R., & Papoutsakis, Eleftherios T. Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol. United States. doi:10.1021/acssynbio.7b00114.
Rohlhill, Julia, Sandoval, Nicholas R., and Papoutsakis, Eleftherios T. Tue . "Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol". United States. doi:10.1021/acssynbio.7b00114.
@article{osti_1355946,
title = {Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol},
author = {Rohlhill, Julia and Sandoval, Nicholas R. and Papoutsakis, Eleftherios T.},
abstractNote = {},
doi = {10.1021/acssynbio.7b00114},
journal = {ACS Synthetic Biology},
number = ,
volume = ,
place = {United States},
year = {Tue May 09 00:00:00 EDT 2017},
month = {Tue May 09 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1021/acssynbio.7b00114

Citation Metrics:
Cited by: 3works
Citation information provided by
Web of Science

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  • Escherichia coli cells grown to logarithmic phase in, and plated on, rich medium (yeast extract-nutrient broth) were more resistant to X rays, ultraviolet (uv) radiation, and methyl methanesulfonate (MMS) than cells grown in, and plated on, minimal medium. We have called this enhanced survival capability medium-dependent resistance (MDR). The magnitude of MDR observed after oxic X irradiation was greater than that observed after anoxic X irradiation, uv irradiation, or MMS treatment. MDR was not observed in stationary-phase cells with X or uv radiation. MDR was associated with an increased ability to repair X-ray-induced DNA single-strand breaks, and with reduced X-ray-inducedmore » DNA degradation and protein synthesis retardation. Postirradiation protein synthesis was concluded to be critical in allowing the high X-ray survival associated with MDR, because of the large radiosensitization caused by a postirradiation growth medium shift down or treatment with rifampicin (RIF). recA protein must be at least one of the proteins whose synthesis is critical to MDR, as judged by the absence of MDR or a RIF effect in X-irradiated recA and lexA mutants. The results with X-irradiated temperature-conditional recA cells suggest that it is only after cells have been damaged that the recA gene plays a role in MDR.« less
  • Escherichia coli cells grown to logarithmic phase in, and plated on, rich medium (yeast extract-nutrient broth) were more resistant to X rays, ultraviolet (uv) radiation, and methyl methanesulfonate (MMS) than cells grown in, and plated on, minimal medium. We have called this enhanced survival capability medium-dependent resistance (MDR). The magnitude of MDR observed after oxic X irradiation was greater than that observed after anoxic X irradiation, uv irradiation, or MMS treatment. MDR was not observed in stationary-phase cells with X or uv radiation. MDR was associated with an increased ability to repair X-ray-induced DNA single-strand breaks, and with reduced X-ray-inducedmore » DNA degradation and protein synthesis retardation. Postirradiation protein synthesis was concluded to be critical in allowing the high X-ray survival associated with MDR, because of the large radiosensitization caused by a postirradiation growth medium shift down or treatment with rifampicin (RIF), recA protein must be at least one of the proteins whose synthesis is critical to MDR, as judged by the absence of MDR or a RIF effect in X-irradiated recA and lexA mutants. The results with X-irradiated temperature-conditional recA cells suggest that it is only after cells have been damaged that the recA gene plays a role in MDR.« less
  • Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene ( pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determinedmore » by 13C-labeling in intracellular metabolites. As a result, introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.« less
  • Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using 13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolyticmore » intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. In conclusion, by incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.« less