Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox-and ATP-neutral, CO 2 -fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose) ؊1 . A previously engineered glucose-tolerant, C 2 -independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter ؊1 at a malate yield of 0.42 mol (mol glucose)؊1 . Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on 13 C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Oxidation of succinate to fumarate is an energetically difficult step in the biochemical pathway of propionate oxidation by syntrophic methanogenic cultures. Therefore, the effect of fumarate on propionate oxidation by two different propionate-oxidizing cultures was investigated. When the methanogens in a newly enriched propionate-oxidizing methanogenic culture were inhibited by bromoethanesulfonate, fumarate could act as an apparent terminal electron acceptor in propionate oxidation. "3C-nuclear magnetic resonance experiments showed that propionate was carboxylated to succinate while fumarate was partly oxidized to acetate and partly reduced to succinate. Fumarate alone was fermented to succinate and CO2. Bacteria growing on fumarate were enriched and obtained free of methanogens. Propionate was metabolized by these bacteria when either fumarate or Methanospirillum hungatii was added. In cocultures with Syntrophobacter wolinii, such effects were not observed upon addition of fumarate. Possible slow growth of S. wolinii on fumarate could not be demonstrated because of the presence of a Desulfovibrio strain which grew rapidly on fumarate in both the absence and presence of sulfate.

~~ ~~ ~-~ ~~ ~~ ~A mutant of Aspergillus niger unable to grow on D-XylOSe and L-arabinose has been isolated. Genetic analysis revealed that the mutation is located on linkage group IV. Enzymic analysis revealed a deficiency in D-XylUlOSe kinase activity. After transfer of growing mycelium to a medium containing either D-xylose or L-arabinose, the mutant accumulates large amounts of arabitol and xylitol, as shown by I3C NMR spectroscopy. These data and an analysis of enzyme activities induced by D-XylOSe and L-arabinose in the wild-type strain led to the following catabolic pathway for D-xylose : D-xylosexylitol -D-xylulose -D-xylulose 5-phosphate; and for L-arabinose :The reduction steps of the sugars to the corresponding polyols are all NADPH dependent. The oxidation steps of the polyols to the sugars are all NAD+ dependent. Fractionation of cell-free extracts gave information about the specificity of the enzymes and showed that all the reactions are catalysed by different enzymes.

NMR analysis of 13 C-labelling patterns showed that the Embden-Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H 2 per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10(7) cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10(7) cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10(4) cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. "In vivo" 13C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification.

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by 13 C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-13 C]propionate was converted to [2-13 C]acetate, with no [1-13 C]acetate formed. Butyrate from [3-13 C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-13 C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-13 C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-13 C-labeled propionate yielded both [1-13 C]acetate and [2-13 C]acetate. When 13 C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, 13 C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.In methanogenic environments propionate is oxidized by acetogenic bacteria to acetate and carbon dioxide (16,18). Methanogenic archaea make this reaction energetically favorable by removing reducing equivalents either as hydrogen or as formate (1,3,19). Syntrophic propionate oxidation mainly occurs via the randomizing methyl-malonyl-coenzyme A (CoA) pathway, as was demonstrated for several Syntrophobacter species (6, 7, 11), as well as for mixed methanogenic cultures (2,5,8,13,14,15,22). However, other pathways of propionate degradation are possible as well, such as a nonrandomizing pathway via butyrate (9,22,23). In these studies, evidence was provided that part of the propionate is carboxylated to butyrate which is then degraded to acetate. Alternative possible pathways of propionate conversion were recently documented by Textor et al. (21).Recently, a novel syntrophic propionate-oxidizing bacterium was isolated which may possess a propionate-degradation pathway via butyrate (10). Cocultures of Smithella propionica and a hydrogen-and formate-utilizing methanogen produce less methane and more acetate than cocultures with Syntrophobacter strains. In addition, the cocultures with S. propionica produce small amounts of butyrate. It was suggested that this organism dismutates propionate to acetate and butyrate followed by syntrophic ␤-oxidation of butyrate to acetate. We report here the results of 13 C-nuclear magnetic resonance (NMR) studies to elucidate the pathway of propionate oxidation in S. propionica. MATERIALS AND METHODSOrganisms and cultivation. Methanospirillum hungatei JF1 T was obtained from the Deutsche Sammlung von Mikroorganism...

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-(13)C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct (13)C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the (13)C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine.

The genome sequence of Archaeoglobus fulgidus VC16 encodes three CO dehydrogenase genes. Here we explore the capacity of A. fulgidus to use CO as growth substrate. Archaeoglobus fulgidus VC16 was successfully adapted to growth medium that contained sulfate and CO. In the presence of CO and sulfate the culture OD(660) increased to 0.41 and sulfide, carbon dioxide, acetate and formate were formed. Accumulation of formate was transient. Similar results, except that no sulfide was formed, were obtained when sulfate was omitted. Hydrogen was never detected. Under the conditions tested, the observed concentrations of acetate (18 mM) and formate (8.2 mM) were highest in cultures without sulfate. Proton NMR spectroscopy indicated that CO2, and not CO, is the precursor of formate and the methyl group of acetate. Methylviologen-dependent formate dehydrogenase activity (1.4 micromol formate oxidized min(-1) mg(-1)) was detected in cell-free extracts and expected to have a role in formate reuptake. It is speculated that formate formation proceeds through hydrolysis of formyl-methanofuran or formyl-tetrahydromethanopterin. This study demonstrates that A. fulgidus can grow chemolithoautotrophically with CO as acetogen, and is not strictly dependent on the presence of sulfate, thiosulfate or other sulfur compounds as electron acceptor.