Uncoupling fermentative synthesis of molecular hydrogen from biomass formation in Thermotoga maritima

Raghuveer Singh, Derrick White, Yaşar Demirel, Robert Kelly, Kenneth Noll, Paul H Blum

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2 through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ldh) with a truncated ldh fused to a kanamycin resistance cassette expressed from a native PgroESL promoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2 at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded by malK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the malK3 mutation into a clean genetic background also conferred increased H2 production, confirming that the mutant allele was sufficient for increased H2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2 production, changing fermentation efficiency and shifting energy management.

Original languageEnglish (US)
Article numbere00998-18
JournalApplied and environmental microbiology
Volume84
Issue number17
DOIs
StatePublished - Sep 1 2018

Fingerprint

Thermotoga maritima
Maltose
maltose
Biomass
hydrogen
Hydrogen
mutation
fermentation
synthesis
biomass
hydrogen production
substrate
Mutation
Fermentation
genetic marker
lactates
Lactic Acid
organic acid
excretion
recombination

Keywords

  • Adaptive laboratory evolution
  • Bacterial genetics
  • Fermentation
  • Hydrogen
  • Hydrogen
  • Hyperthermophile
  • MalK
  • Thermotoga maritima
  • Transient gene inactivation

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Cite this

Uncoupling fermentative synthesis of molecular hydrogen from biomass formation in Thermotoga maritima. / Singh, Raghuveer; White, Derrick; Demirel, Yaşar; Kelly, Robert; Noll, Kenneth; Blum, Paul H.

In: Applied and environmental microbiology, Vol. 84, No. 17, e00998-18, 01.09.2018.

Research output: Contribution to journalArticle

Singh, Raghuveer ; White, Derrick ; Demirel, Yaşar ; Kelly, Robert ; Noll, Kenneth ; Blum, Paul H. / Uncoupling fermentative synthesis of molecular hydrogen from biomass formation in Thermotoga maritima. In: Applied and environmental microbiology. 2018 ; Vol. 84, No. 17.
@article{2aa2f00b7fa34bcaad6e047f6427a238,
title = "Uncoupling fermentative synthesis of molecular hydrogen from biomass formation in Thermotoga maritima",
abstract = "When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2 through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ldh) with a truncated ldh fused to a kanamycin resistance cassette expressed from a native PgroESL promoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2 at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded by malK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the malK3 mutation into a clean genetic background also conferred increased H2 production, confirming that the mutant allele was sufficient for increased H2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2 production, changing fermentation efficiency and shifting energy management.",
keywords = "Adaptive laboratory evolution, Bacterial genetics, Fermentation, Hydrogen, Hydrogen, Hyperthermophile, MalK, Thermotoga maritima, Transient gene inactivation",
author = "Raghuveer Singh and Derrick White and Yaşar Demirel and Robert Kelly and Kenneth Noll and Blum, {Paul H}",
year = "2018",
month = "9",
day = "1",
doi = "10.1128/AEM.00998-18",
language = "English (US)",
volume = "84",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "17",

}

TY - JOUR

T1 - Uncoupling fermentative synthesis of molecular hydrogen from biomass formation in Thermotoga maritima

AU - Singh, Raghuveer

AU - White, Derrick

AU - Demirel, Yaşar

AU - Kelly, Robert

AU - Noll, Kenneth

AU - Blum, Paul H

PY - 2018/9/1

Y1 - 2018/9/1

N2 - When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2 through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ldh) with a truncated ldh fused to a kanamycin resistance cassette expressed from a native PgroESL promoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2 at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded by malK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the malK3 mutation into a clean genetic background also conferred increased H2 production, confirming that the mutant allele was sufficient for increased H2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2 production, changing fermentation efficiency and shifting energy management.

AB - When carbohydrates are fermented by the hyperthermophilic anaerobe Thermotoga maritima, molecular hydrogen (H2) is formed in strict proportion to substrate availability. Excretion of the organic acids acetate and lactate provide an additional sink for removal of excess reductant. However, mechanisms controlling energy management of these metabolic pathways are largely unexplored. To investigate this topic, transient gene inactivation was used to block lactate production as a strategy to produce spontaneous mutant cell lines that overproduced H2 through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded by ldh) with a truncated ldh fused to a kanamycin resistance cassette expressed from a native PgroESL promoter. Passage of the unstable recombinant resulted in loss of the genetic marker and recovery of evolved cell lines, including strain Tma200. Relative to the wild type, and considering the mass balance of fermentation substrate and products, Tma200 grew more slowly, produced H2 at levels above the physiologic limit, and simultaneously consumed less maltose while oxidizing it more efficiently. Whole-genome resequencing indicated that the ABC maltose transporter subunit, encoded by malK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of the malK3 mutation into a clean genetic background also conferred increased H2 production, confirming that the mutant allele was sufficient for increased H2 synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2 production, changing fermentation efficiency and shifting energy management.

KW - Adaptive laboratory evolution

KW - Bacterial genetics

KW - Fermentation

KW - Hydrogen

KW - Hydrogen

KW - Hyperthermophile

KW - MalK

KW - Thermotoga maritima

KW - Transient gene inactivation

UR - http://www.scopus.com/inward/record.url?scp=85052563902&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85052563902&partnerID=8YFLogxK

U2 - 10.1128/AEM.00998-18

DO - 10.1128/AEM.00998-18

M3 - Article

VL - 84

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 17

M1 - e00998-18

ER -