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Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose.
Biotechnol Biofuels. 2019; 12:186.BB

Abstract

Background

Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose.

Results

We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase-hydroxybutyryl-CoA dehydrogenase-crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold.

Conclusions

Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h.

Authors+Show Affiliations

1Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA. 2Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.3Dartmouth College, Hanover, NH 03755 USA.3Dartmouth College, Hanover, NH 03755 USA.2Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA. 4Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 USA.1Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA. 2Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.1Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA. 2Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.1Thayer School of Engineering, Dartmouth College, Hanover, NH 03755 USA. 2Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA. 4Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31367231

Citation

Tian, Liang, et al. "Metabolic Engineering of Clostridium Thermocellum for N-butanol Production From Cellulose." Biotechnology for Biofuels, vol. 12, 2019, p. 186.
Tian L, Conway PM, Cervenka ND, et al. Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose. Biotechnol Biofuels. 2019;12:186.
Tian, L., Conway, P. M., Cervenka, N. D., Cui, J., Maloney, M., Olson, D. G., & Lynd, L. R. (2019). Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose. Biotechnology for Biofuels, 12, 186. https://doi.org/10.1186/s13068-019-1524-6
Tian L, et al. Metabolic Engineering of Clostridium Thermocellum for N-butanol Production From Cellulose. Biotechnol Biofuels. 2019;12:186. PubMed PMID: 31367231.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose. AU - Tian,Liang, AU - Conway,Peter M, AU - Cervenka,Nicholas D, AU - Cui,Jingxuan, AU - Maloney,Marybeth, AU - Olson,Daniel G, AU - Lynd,Lee R, Y1 - 2019/07/23/ PY - 2019/04/30/received PY - 2019/07/05/accepted PY - 2019/8/2/entrez PY - 2019/8/2/pubmed PY - 2019/8/2/medline KW - Cellulosic biofuel KW - Clostridium thermocellum KW - Consolidated bioprocessing KW - Protein engineering KW - n-Butanol SP - 186 EP - 186 JF - Biotechnology for biofuels JO - Biotechnol Biofuels VL - 12 N2 - Background: Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. Results: We tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase-hydroxybutyryl-CoA dehydrogenase-crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Conclusions: Here we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h. SN - 1754-6834 UR - https://www.unboundmedicine.com/medline/citation/31367231/Metabolic_engineering_of_Clostridium_thermocellum_for_n-butanol_production_from_cellulose L2 - https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-019-1524-6 DB - PRIME DP - Unbound Medicine ER -
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