Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production

Shingo Noguchi, Sastia P. Putri, Ethan I. Lan, Walter A. Laviña, Yudai Dempo, Takeshi Bamba, James C. Liao, Eiichiro Fukusaki

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO2 into 1-butanol. Efforts to optimize the 1-butanol pathway in Synechococcus elongatus PCC 7942 have focused on the improvement of the CoA-dependent pathway thus, probing the in vivo metabolic state of the CoA-dependent pathway is essential for identifying its limiting steps. In this study, we performed quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway by reversed phase ion-pair liquid chromatography-triple quadrupole mass spectrometry. Using 13C-labelled cyanobacterial cell extract as internal standard, measurement of the intracellular concentration of acyl-CoAs revealed that the reductive reaction of butanoyl-CoA to butanal is a possible rate-limiting step. In addition, improvement of the butanoyl-CoA to butanal reaction resulted in an increased rate of acetyl-CoA synthesis by possibly compensating for the limitation of free CoA species. We inferred that the efficient recycling of free CoA played a key role in enhancing the conversion of pyruvate to acetyl-CoA.

Original languageEnglish
Article number26
Pages (from-to)1-10
Number of pages10
JournalMetabolomics
Volume12
Issue number2
DOIs
Publication statusPublished - Feb 1 2016

Fingerprint

1-Butanol
Coenzyme A
Kinetics
Acetyl Coenzyme A
Synechococcus
Ion chromatography
Clostridium
Liquid chromatography
Recycling
Cyanobacteria
Cell Extracts
Pyruvic Acid
Liquid Chromatography
Mass spectrometry
Mass Spectrometry
Ions

All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Clinical Biochemistry

Cite this

Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production. / Noguchi, Shingo; Putri, Sastia P.; Lan, Ethan I.; Laviña, Walter A.; Dempo, Yudai; Bamba, Takeshi; Liao, James C.; Fukusaki, Eiichiro.

In: Metabolomics, Vol. 12, No. 2, 26, 01.02.2016, p. 1-10.

Research output: Contribution to journalArticle

Noguchi, Shingo ; Putri, Sastia P. ; Lan, Ethan I. ; Laviña, Walter A. ; Dempo, Yudai ; Bamba, Takeshi ; Liao, James C. ; Fukusaki, Eiichiro. / Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production. In: Metabolomics. 2016 ; Vol. 12, No. 2. pp. 1-10.
@article{daa86e416b894dc08157681ef8c77931,
title = "Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production",
abstract = "Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO2 into 1-butanol. Efforts to optimize the 1-butanol pathway in Synechococcus elongatus PCC 7942 have focused on the improvement of the CoA-dependent pathway thus, probing the in vivo metabolic state of the CoA-dependent pathway is essential for identifying its limiting steps. In this study, we performed quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway by reversed phase ion-pair liquid chromatography-triple quadrupole mass spectrometry. Using 13C-labelled cyanobacterial cell extract as internal standard, measurement of the intracellular concentration of acyl-CoAs revealed that the reductive reaction of butanoyl-CoA to butanal is a possible rate-limiting step. In addition, improvement of the butanoyl-CoA to butanal reaction resulted in an increased rate of acetyl-CoA synthesis by possibly compensating for the limitation of free CoA species. We inferred that the efficient recycling of free CoA played a key role in enhancing the conversion of pyruvate to acetyl-CoA.",
author = "Shingo Noguchi and Putri, {Sastia P.} and Lan, {Ethan I.} and Lavi{\~n}a, {Walter A.} and Yudai Dempo and Takeshi Bamba and Liao, {James C.} and Eiichiro Fukusaki",
year = "2016",
month = "2",
day = "1",
doi = "10.1007/s11306-015-0940-2",
language = "English",
volume = "12",
pages = "1--10",
journal = "Metabolomics",
issn = "1573-3882",
publisher = "Springer New York",
number = "2",

}

TY - JOUR

T1 - Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production

AU - Noguchi, Shingo

AU - Putri, Sastia P.

AU - Lan, Ethan I.

AU - Laviña, Walter A.

AU - Dempo, Yudai

AU - Bamba, Takeshi

AU - Liao, James C.

AU - Fukusaki, Eiichiro

PY - 2016/2/1

Y1 - 2016/2/1

N2 - Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO2 into 1-butanol. Efforts to optimize the 1-butanol pathway in Synechococcus elongatus PCC 7942 have focused on the improvement of the CoA-dependent pathway thus, probing the in vivo metabolic state of the CoA-dependent pathway is essential for identifying its limiting steps. In this study, we performed quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway by reversed phase ion-pair liquid chromatography-triple quadrupole mass spectrometry. Using 13C-labelled cyanobacterial cell extract as internal standard, measurement of the intracellular concentration of acyl-CoAs revealed that the reductive reaction of butanoyl-CoA to butanal is a possible rate-limiting step. In addition, improvement of the butanoyl-CoA to butanal reaction resulted in an increased rate of acetyl-CoA synthesis by possibly compensating for the limitation of free CoA species. We inferred that the efficient recycling of free CoA played a key role in enhancing the conversion of pyruvate to acetyl-CoA.

AB - Cyanobacterial 1-butanol production is an important model system for direct conversion of CO2 to fuels and chemicals. Metabolically-engineered cyanobacteria introduced with a heterologous Coenzyme A (CoA)-dependent pathway modified from Clostridium species can convert atmospheric CO2 into 1-butanol. Efforts to optimize the 1-butanol pathway in Synechococcus elongatus PCC 7942 have focused on the improvement of the CoA-dependent pathway thus, probing the in vivo metabolic state of the CoA-dependent pathway is essential for identifying its limiting steps. In this study, we performed quantitative target analysis and kinetic profiling of acyl-CoAs in the CoA-dependent pathway by reversed phase ion-pair liquid chromatography-triple quadrupole mass spectrometry. Using 13C-labelled cyanobacterial cell extract as internal standard, measurement of the intracellular concentration of acyl-CoAs revealed that the reductive reaction of butanoyl-CoA to butanal is a possible rate-limiting step. In addition, improvement of the butanoyl-CoA to butanal reaction resulted in an increased rate of acetyl-CoA synthesis by possibly compensating for the limitation of free CoA species. We inferred that the efficient recycling of free CoA played a key role in enhancing the conversion of pyruvate to acetyl-CoA.

UR - http://www.scopus.com/inward/record.url?scp=84953288420&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84953288420&partnerID=8YFLogxK

U2 - 10.1007/s11306-015-0940-2

DO - 10.1007/s11306-015-0940-2

M3 - Article

VL - 12

SP - 1

EP - 10

JO - Metabolomics

JF - Metabolomics

SN - 1573-3882

IS - 2

M1 - 26

ER -