Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (
            <i>araA</i>
            ) in the Hyperthermophilic Bacterium Thermotoga maritima

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is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In, three putative genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from inactivation, the mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile The occurrence of three ABC transporter ATPase subunits all annotated as was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Identification of the ATPase Subunit of the Primary Maltose Transporter in the Hyperthermophilic Anaerobe Thermotoga maritima

Singh

White

Blum

2017

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“…maritima is transformable using replicating plasmids (38). Therefore, chromosomal recombination was pursued as a means to generate useful mutations as demonstrated previously (39,40). As efforts to disrupt ldh by double crossover were not successful, indicating that the gene was essential and therefore unlike its homologs in other H 2 -producing microbes (41), transient gene disruption was used instead.…”

Section: Isolation Of H 2 -Resistant and H 2 -Overproducing Cell Linementioning

confidence: 99%

Uncoupling Fermentative Synthesis of Molecular Hydrogen from Biomass Formation in Thermotoga maritima

Singh

White

Demi̇rel

et al. 2018

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When carbohydrates are fermented by the hyperthermophilic anaerobeThermotoga 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 H2through mutation of unpredicted genetic targets. Single-crossover homologous chromosomal recombination was used to disrupt lactate dehydrogenase (encoded byldh) with a truncatedldhfused to a kanamycin resistance cassette expressed from a native PgroESLpromoter. 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 H2at 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 bymalK3, had undergone repeated mutation, and high-temperature anaerobic [14C]maltose transport assays demonstrated that the rate of maltose transport was reduced. Transfer of themalK3mutation into a clean genetic background also conferred increased H2production, confirming that the mutant allele was sufficient for increased H2synthesis. These data indicate that a reduced rate of maltose uptake was accompanied by an increase in H2production, changing fermentation efficiency and shifting energy management.IMPORTANCEBiorenewable energy sources are of growing interest to mitigate climate change, but like other commodities with nominal value, require innovation to maximize yields. Energetic considerations constrain production of many biofuels, such as molecular hydrogen (H2) because of the competing needs for cell mass synthesis and metabolite formation. Here we describe cell lines of the extremophileThermotoga maritimathat exceed the physiologic limits for H2formation arising from genetic changes in fermentative metabolism. These cell lines were produced using a novel method called transient gene inactivation combined with adaptive laboratory evolution. Genome resequencing revealed unexpected changes in a maltose transport protein. Reduced rates of sugar uptake were accompanied by lower rates of growth and enhanced productivity of H2.

“…T. kodakarensis and A. fulgidus are archaea, whereas T. maritima is a bacterium. The complete genome sequences of these organisms have been determined (26)(27)(28), and gene manipulation systems have been established for T. kodakarensis (29)(30)(31)(32)(33) and T. maritima (34). We have been examining the metabolism of T. kodakarensis in detail and have identified a number of enzymes and pathways that differ from the conventional pathways identified in bacteria and eukaryotes (35)(36)(37).…”

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confidence: 99%

An In Vitro Enzyme System for the Production of myo -Inositol from Starch

Fujisawa

Fujinaga

Atomi

2017

We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus, and IMPase from the hyperthermophilic bacterium Thermotoga maritima. The enzymes were individually produced in Escherichia coli and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD ϩ , resulting in the production of myo-inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD ϩ was added every 2 h, we observed the production of 2.9 g myo-inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from T. maritima into the system and observed myo-inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively. IMPORTANCE myo-Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce myo-inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase, and myo-inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of myo-inositol. myo-Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo-inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo-inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of myo-inositol.