Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum

Tian, Liang; Papanek, Beth; Olson, Daniel G.; Rydzak, Thomas; Holwerda, Evert K.; Zheng, Tianyong; Zhou, Jilai; Maloney, Marybeth; Jiang, Nannan; Giannone, Richard J.; Hettich, Robert L.; Guss, Adam M.; Lynd, Lee R.

doi:10.1186/s13068-016-0528-8

Title: Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum

Abstract

Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed. Results We started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yield and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 ± 1.4 g/L ethanol from 60 g/L cellulose. Moreover, the resulting yield was about 0.39 gETOH/gGluc eq, which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains. Conclusions A two step selection method successfully improved the ethanol yield and the titer. Finaly, this evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of thismore » microbe for industrial applications.« less

Authors:: Tian, Liang; Papanek, Beth; Olson, Daniel G.; Rydzak, Thomas; Holwerda, Evert K.; Zheng, Tianyong; Zhou, Jilai; Maloney, Marybeth; Jiang, Nannan; Giannone, Richard J.; Hettich, Robert L.; Guss, Adam M.; Lynd, Lee R.

Publication Date:: Thu Jun 02 00:00:00 EDT 2016

Research Org.:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)

Sponsoring Org.:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES)

OSTI Identifier:: 1618651

Alternate Identifier(s):: OSTI ID: 1286977

Grant/Contract Number:: AC05-00OR22725

Resource Type:: Published Article

Journal Name:: Biotechnology for Biofuels

Additional Journal Information:: Journal Name: Biotechnology for Biofuels Journal Volume: 9 Journal Issue: 1; Journal ID: ISSN 1754-6834

Publisher:: Springer Science + Business Media

Country of Publication:: Netherlands

Language:: English

Subject:: 09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; consolidated bioprocessing; clostridium thermocellum; cellulosic ethanol; adapative evolution

Citation Formats


                    Tian, Liang, Papanek, Beth, Olson, Daniel G., Rydzak, Thomas, Holwerda, Evert K., Zheng, Tianyong, Zhou, Jilai, Maloney, Marybeth, Jiang, Nannan, Giannone, Richard J., Hettich, Robert L., Guss, Adam M., and Lynd, Lee R. Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum.  Netherlands: N. p., 2016. 
Web.  doi:10.1186/s13068-016-0528-8.

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                    Tian, Liang, Papanek, Beth, Olson, Daniel G., Rydzak, Thomas, Holwerda, Evert K., Zheng, Tianyong, Zhou, Jilai, Maloney, Marybeth, Jiang, Nannan, Giannone, Richard J., Hettich, Robert L., Guss, Adam M., & Lynd, Lee R. Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum.  Netherlands.  https://doi.org/10.1186/s13068-016-0528-8

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                    Tian, Liang, Papanek, Beth, Olson, Daniel G., Rydzak, Thomas, Holwerda, Evert K., Zheng, Tianyong, Zhou, Jilai, Maloney, Marybeth, Jiang, Nannan, Giannone, Richard J., Hettich, Robert L., Guss, Adam M., and Lynd, Lee R. Thu .  
"Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum".  Netherlands.  https://doi.org/10.1186/s13068-016-0528-8.

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@article{osti_1618651,

  title        = {Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum},

  author       = {Tian, Liang and Papanek, Beth and Olson, Daniel G. and Rydzak, Thomas and Holwerda, Evert K. and Zheng, Tianyong and Zhou, Jilai and Maloney, Marybeth and Jiang, Nannan and Giannone, Richard J. and Hettich, Robert L. and Guss, Adam M. and Lynd, Lee R.},

  abstractNote = {Background Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed. Results We started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yield and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 ± 1.4 g/L ethanol from 60 g/L cellulose. Moreover, the resulting yield was about 0.39 gETOH/gGluc eq, which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains. Conclusions A two step selection method successfully improved the ethanol yield and the titer. Finaly, this evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications.},

  doi          = {10.1186/s13068-016-0528-8},

  journal      = {Biotechnology for Biofuels},

  number       = 1,

  volume       = 9,

  place        = {Netherlands},

  year         = {Thu Jun 02 00:00:00 EDT 2016},

  month        = {Thu Jun 02 00:00:00 EDT 2016}

}

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Fig. 1 : The ethanol pathway in C. thermocellum strain AG553. Gray crosses represent gene deletions. NAD(P)H producing pathways are in blue, and NAD(P)H consuming steps are in red

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Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, we study a mutant strain of C. thermocellum that was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulosemore »« less
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