Dataset Information

Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum.

ABSTRACT: Thermoanaerobacterium saccharolyticum is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer. T. saccharolyticum uses the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis. However, the genes and enzymes used in each step of the EMP pathway in T. saccharolyticum are not completely known. In T. saccharolyticum, both pyruvate kinase (PYK) and pyruvate phosphate dikinase (PPDK) are highly expressed based on transcriptomic and proteomic data. Both enzymes catalyze the formation of pyruvate from phosphoenolpyruvate (PEP). PYK is typically the last step of EMP glycolysis pathway while PPDK is reversible and is found mostly in C4 plants and some microorganisms. It is not clear what role PYK and PPDK play in T. saccharolyticum metabolism and fermentation pathways and whether both are necessary. In this study we deleted the ppdk gene in wild type and homoethanologen strains of T. saccharolyticum and showed that it is not essential for growth or ethanol production.

SUBMITTER: Cui J

PROVIDER: S-EPMC6997586 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum.

Cui Jingxuan J Maloney Marybeth I MI Olson Daniel G DG Lynd Lee R LR

Metabolic engineering communications 20200123

Thermoanaerobacterium saccharolyticum is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer. T. saccharolyticum uses the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis. However, the genes and enzymes used in each step of the EMP pathway in T. saccharolyticum are not completely known. In T. saccharolyticum, both pyruvate kinase (PYK) and pyruvate phosphate ...[more]

PMID: 32025490

Similar Datasets

Project description:BackgroundThermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation.ResultsHere we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results.ConclusionThese data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

Project description:UnlabelledNfnAB catalyzes the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+). The NfnAB complex has been hypothesized to be the main enzyme for ferredoxin oxidization in strains of Thermoanaerobacterium saccharolyticum engineered for increased ethanol production. NfnAB complex activity was detectable in crude cell extracts of T. saccharolyticum. Activity was also detected using activity staining of native PAGE gels. The nfnAB gene was deleted in different strains of T. saccharolyticum to determine its effect on end product formation. In wild-type T. saccharolyticum, deletion of nfnAB resulted in a 46% increase in H2 formation but otherwise little change in other fermentation products. In two engineered strains with 80% theoretical ethanol yield, loss of nfnAB caused two different responses: in one strain, ethanol yield decreased to about 30% of the theoretical value, while another strain had no change in ethanol yield. Biochemical analysis of cell extracts showed that the ΔnfnAB strain with decreased ethanol yield had NADPH-linked alcohol dehydrogenase (ADH) activity, while the ΔnfnAB strain with unchanged ethanol yield had NADH-linked ADH activity. Deletion of nfnAB caused loss of NADPH-linked ferredoxin oxidoreductase activity in all cell extracts. Significant NADH-linked ferredoxin oxidoreductase activity was seen in all cell extracts, including those that had lost nfnAB. This suggests that there is an unidentified NADH:ferredoxin oxidoreductase (distinct from nfnAB) playing a role in ethanol formation. The NfnAB complex plays a key role in generating NADPH in a strain that had become reliant on NADPH-ADH activity.ImportanceThermophilic anaerobes that can convert biomass-derived sugars into ethanol have been investigated as candidates for biofuel formation. Many anaerobes have been genetically engineered to increase biofuel formation; however, key aspects of metabolism remain unknown and poorly understood. One example is the mechanism for ferredoxin oxidation and transfer of electrons to NAD(P)(+). The electron-bifurcating enzyme complex NfnAB is known to catalyze the reversible transfer of electrons from reduced ferredoxin and NADH to 2 NADP(+) and is thought to play key roles linking NAD(P)(H) metabolism with ferredoxin metabolism. We report the first deletion of nfnAB and demonstrate a role for NfnAB in metabolism and ethanol formation in Thermoanaerobacterium saccharolyticum and show that this may be an important feature among other thermophilic ethanologenic anaerobes.

Dataset Information

Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum.

Publications

Conversion of phosphoenolpyruvate to pyruvate in <i>Thermoanaerobacterium saccharolyticum</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets