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Title: Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus

Abstract

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO3 and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO2. Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO2-sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M.more » sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. Here, while further efforts to improve 3HP production by regulating gene dosage,« less

Authors:
 [1];  [1];  [2];  [1];  [1];  [1];  [1];  [2];  [1]
  1. North Carolina State Univ., Raleigh, NC (United States)
  2. Univ. of Georgia, Athens, GA (United States)
Publication Date:
Research Org.:
North Carolina State Univ., Raleigh, NC (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1422399
Grant/Contract Number:  
AR0000081
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 113; Journal Issue: 12; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; biotin protein ligase; carboxylase; carbon dioxide; Pyrococcus furiosus; 3-hydroxypropionate; Metallosphaera sedula

Citation Formats

Lian, Hong, Zeldes, Benjamin M., Lipscomb, Gina L., Hawkins, Aaron B., Han, Yejun, Loder, Andrew J., Nishiyama, Declan, Adams, Michael W. W., and Kelly, Robert M. Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus. United States: N. p., 2016. Web. doi:10.1002/bit.26033.
Lian, Hong, Zeldes, Benjamin M., Lipscomb, Gina L., Hawkins, Aaron B., Han, Yejun, Loder, Andrew J., Nishiyama, Declan, Adams, Michael W. W., & Kelly, Robert M. Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus. United States. doi:10.1002/bit.26033.
Lian, Hong, Zeldes, Benjamin M., Lipscomb, Gina L., Hawkins, Aaron B., Han, Yejun, Loder, Andrew J., Nishiyama, Declan, Adams, Michael W. W., and Kelly, Robert M. Sat . "Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus". United States. doi:10.1002/bit.26033. https://www.osti.gov/servlets/purl/1422399.
@article{osti_1422399,
title = {Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus},
author = {Lian, Hong and Zeldes, Benjamin M. and Lipscomb, Gina L. and Hawkins, Aaron B. and Han, Yejun and Loder, Andrew J. and Nishiyama, Declan and Adams, Michael W. W. and Kelly, Robert M.},
abstractNote = {Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO3– and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO2. Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO2-sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. Here, while further efforts to improve 3HP production by regulating gene dosage,},
doi = {10.1002/bit.26033},
journal = {Biotechnology and Bioengineering},
number = 12,
volume = 113,
place = {United States},
year = {2016},
month = {6}
}

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Works referenced in this record:

Malonyl-Coenzyme A Reductase in the Modified 3-Hydroxypropionate Cycle for Autotrophic Carbon Fixation in Archaeal Metallosphaera and Sulfolobus spp.
journal, October 2006

  • Alber, B.; Olinger, M.; Rieder, A.
  • Journal of Bacteriology, Vol. 188, Issue 24, p. 8551-8559
  • DOI: 10.1128/JB.00987-06

A unique biotin carboxyl carrier protein in archaeon Sulfolobus tokodaii
journal, February 2006


Cloning, expression and characterization of the δ-carbonic anhydrase of Thalassiosira weissflogii (Bacillariophyceae)
journal, September 2012


Carbonic anhydrase is an ancient enzyme widespread in prokaryotes
journal, December 1999

  • Smith, K. S.; Jakubzick, C.; Whittam, T. S.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 26
  • DOI: 10.1073/pnas.96.26.15184

Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production
journal, December 2002


Parallel evolution of transcriptome architecture during genome reorganization
journal, July 2011

  • Yoon, S. H.; Reiss, D. J.; Bare, J. C.
  • Genome Research, Vol. 21, Issue 11, p. 1892-1904
  • DOI: 10.1101/gr.122218.111

Substrate specificity of archaeon Sulfolobus tokodaii biotin protein ligase
journal, May 2006

  • Sueda, Shinji; Li, Yan-Qiu; Kondo, Hiroki
  • Biochemical and Biophysical Research Communications, Vol. 344, Issue 1
  • DOI: 10.1016/j.bbrc.2006.03.118

Engineering a Hyperthermophilic Archaeon for Temperature-Dependent Product Formation
journal, April 2012

  • Basen, M.; Sun, J.; Adams, M. W. W.
  • mBio, Vol. 3, Issue 2, Article No. e00053-12
  • DOI: 10.1128/mBio.00053-12

Chemical synthesis using synthetic biology
journal, August 2009

  • Carothers, James M.; Goler, Jonathan A.; Keasling, Jay D.
  • Current Opinion in Biotechnology, Vol. 20, Issue 4
  • DOI: 10.1016/j.copbio.2009.08.001

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO 2 -based 3-hydroxypropionate production : 3-Hydroxypropionate Production by
journal, June 2015

  • Hawkins, Aaron B.; Lian, Hong; Zeldes, Benjamin M.
  • Biotechnology and Bioengineering, Vol. 112, Issue 8
  • DOI: 10.1002/bit.25584

Carboxylases in Natural and Synthetic Microbial Pathways
journal, October 2011

  • Erb, Tobias J.
  • Applied and Environmental Microbiology, Vol. 77, Issue 24
  • DOI: 10.1128/AEM.05702-11

Metabolic Engineering of Escherichia coli: Increase of NADH Availability by Overexpressing an NAD+-Dependent Formate Dehydrogenase
journal, July 2002

  • Berríos-Rivera, Susana; Bennett, George N.; San, Ka-Yiu
  • Metabolic Engineering, Vol. 4, Issue 3, p. 217-229
  • DOI: 10.1006/mben.2002.0227

Aqueous Carbonic Acid (H2CO3)
journal, June 2010


Prokaryotic carbonic anhydrases
journal, October 2000


Polyhedral organelles compartmenting bacterial metabolic processes
journal, March 2006


Kinetic and Spectroscopic Characterization of the Gamma-Carbonic Anhydrase from the Methanoarchaeon Methanosarcina thermophila
journal, October 1999

  • Alber, Birgit E.; Colangelo, Christopher M.; Dong, Jun
  • Biochemistry, Vol. 38, Issue 40
  • DOI: 10.1021/bi9828876

Structure and catalytic mechanism of the β-carbonic anhydrases
journal, February 2010


Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases
journal, February 2011

  • Lipscomb, Gina L.; Stirrett, Karen; Schut, Gerrit J.
  • Applied and Environmental Microbiology, Vol. 77, Issue 7, p. 2232-2238
  • DOI: 10.1128/AEM.02624-10

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide
journal, March 2013

  • Keller, M. W.; Schut, G. J.; Lipscomb, G. L.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 15, p. 5840-5845
  • DOI: 10.1073/pnas.1222607110

Autotrophic carbon fixation in archaea
journal, May 2010

  • Berg, Ivan A.; Kockelkorn, Daniel; Ramos-Vera, W. Hugo
  • Nature Reviews Microbiology, Vol. 8, Issue 6, p. 447-460
  • DOI: 10.1038/nrmicro2365

Biological conversion of carbon dioxide and hydrogen into liquid fuels and industrial chemicals
journal, June 2013

  • Hawkins, Aaron S.; McTernan, Patrick M.; Lian, Hong
  • Current Opinion in Biotechnology, Vol. 24, Issue 3, p. 376-384
  • DOI: 10.1016/j.copbio.2013.02.017

A 3-Hydroxypropionate/4-Hydroxybutyrate Autotrophic Carbon Dioxide Assimilation Pathway in Archaea
journal, December 2007

  • Berg, Ivan A.; Kockelkorn, Daniel; Buckel, Wolfgang
  • Science, Vol. 318, Issue 5857, p. 1782-1786
  • DOI: 10.1126/science.1149976

“Short-chain” α-1,4-glucan phosphorylase having a truncated N-terminal domain: Functional expression and characterization of the enzyme from Sulfolobus solfataricus
journal, November 2009

  • Mueller, Mario; Takemasa, Ryo; Schwarz, Alexandra
  • Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1794, Issue 11
  • DOI: 10.1016/j.bbapap.2009.08.006

Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon Pyrococcus furiosus
journal, January 2001


Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula : Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation
journal, January 2003


Observation of a calcium-binding site in the γ-class carbonic anhydrase from Pyrococcus horikoshii
journal, September 2008

  • Jeyakanthan, Jeyaraman; Rangarajan, Sarani; Mridula, P.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 64, Issue 10
  • DOI: 10.1107/S0907444908024323

Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli
journal, March 2011

  • Shen, Claire R.; Lan, Ethan I.; Dekishima, Yasumasa
  • Applied and Environmental Microbiology, Vol. 77, Issue 9, p. 2905-2915
  • DOI: 10.1128/AEM.03034-10

Biotinylation in the hyperthermophile Aquifex aeolicus. Isolation of a cross-linked BPL:BCCP complex
journal, March 2003


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

  • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
  • Nature Methods, Vol. 6, Issue 5, p. 343-345
  • DOI: 10.1038/nmeth.1318

Multiple Roles of Carbonic Anhydrase in Cellular Transport and Metabolism
journal, October 1996


Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals
journal, November 2015

  • Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J.
  • Frontiers in Microbiology, Vol. 6
  • DOI: 10.3389/fmicb.2015.01209

Alcohol Selectivity in a Synthetic Thermophilic n -Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes
journal, August 2015

  • Loder, Andrew J.; Zeldes, Benjamin M.; Garrison, G. Dale
  • Applied and Environmental Microbiology, Vol. 81, Issue 20
  • DOI: 10.1128/AEM.02028-15