Phosphoketolase flux in Clostridium acetobutylicum during growth on L-arabinose.

Clostridium acetobutylicum's metabolic pathways have been studied for decades due to its metabolic diversity and industrial value, yet many details of its metabolism continue to emerge. The flux through the recently discovered pentose phosphoketolase pathway (PKP) in C. acetobutylicum has been determined for growth on xylose but transcriptional analysis indicated the pathway may have a greater contribution to arabinose metabolism. To elucidate the role of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (XFP), and the PKP in C. acetobutylicum, experimental and computational metabolic isotope analyses were performed under growth conditions of glucose or varying concentrations of xylose and arabinose. A positional bias in labelling between carbons 2 and 4 of butyrate was found and posited to be due to an enzyme isotope effect of the thiolase enzyme. A correction for the positional bias was applied, which resulted in reduction of residual error. Comparisons between model solutions with low residual error indicated flux through each of the two XFP reactions was variable, while the combined flux of the reactions remained relatively constant. PKP utilization increased with increasing xylose concentration and this trend was further pronounced during growth on arabinose. Mutation of the gene encoding XFP almost completely abolished flux through the PKP during growth on arabinose and resulted in decreased acetate/butyrate ratios. Greater flux through the PKP during growth on arabinose when compared with xylose indicated the pathway's primary role in C. acetobutylicum is arabinose metabolism.

Fermentation of oxidized hexose derivatives by Clostridium acetobutylicum.

BACKGROUND: Clostridium acetobutylicum fermentations are promising for production of commodity chemicals from heterogeneous biomass due to the wide range of substrates the organism can metabolize. Much work has been done to elucidate the pathways for utilization of aldoses, but little is known about metabolism of more oxidized substrates. Two oxidized hexose derivatives, gluconate and galacturonate, are present in low cost feedstocks, and their metabolism will contribute to overall metabolic output of these substrates. RESULTS: A complete metabolic network for glucose, gluconate, and galacturonate utilization was generated using online databases, previous studies, genomic context, and experimental data. Gluconate appears to be metabolized via the Entner-Doudoroff pathway, and is likely dehydrated to 2-keto-3-deoxy-gluconate before phosphorylation to 2-keto-3-deoxy-6-P-gluconate. Galacturonate appears to be processed via the Ashwell pathway, converging on a common metabolite for gluconate and galacturonate metabolism, 2-keto-3-deoxygluconate. As expected, increasingly oxidized substrates resulted in increasingly oxidized products with galacturonate fermentations being nearly homoacetic. Calculations of expected ATP and reducing equivalent yields and experimental data suggested galacturonate fermentations were reductant limited. Galacturonate fermentation was incomplete, which was not due solely to product inhibition or the inability to utilize low concentrations of galacturonate. Removal of H2 and CO2 by agitation resulted in faster growth, higher cell densities, formation of relatively more oxidized products, and higher product yields for cultures grown on glucose or gluconate. In contrast, cells grown on galacturonate showed reduced growth rates upon agitation, which was likely due to loss in reductant in the form of H2. The growth advantage seen on agitated glucose or gluconate cultures could not be solely attributed to improved ATP economics, thereby indicating other factors are also important. CONCLUSIONS: The metabolic network presented in this work should facilitate similar reconstructions in other organisms, and provides a further understanding of the pathways involved in metabolism of oxidized feedstocks and carbohydrate mixtures. The nearly homoacetic fermentation during growth on galacturonate indicates further optimization of this and related organisms could provide a route to an effective biologically derived acetic acid production platform. Furthermore, the pathways could be targeted to decrease production of undesirable products during fermentations of heterogeneous biomass.

5-Flucytosine Longitudinal Antifungal Susceptibility Testing of Cryptococcus neoformans: A Substudy of the EnACT Trial Testing Oral Amphotericin.

Background: The EnACT trial was a phase 2 randomized clinical trial conducted in Uganda, which evaluated a novel orally delivered lipid nanocrystal (LNC) amphotericin B in combination with flucytosine for the treatment of cryptococcal meningitis. When flucytosine (5FC) is used as monotherapy in cryptococcosis, 5FC can induce resistant Cryptococcus mutants. Oral amphotericin B uses a novel drug delivery mechanism, and we assessed whether resistance to 5FC develops during oral LNC-amphotericin B therapy. Methods: We enrolled Ugandans with HIV diagnosed with cryptococcal meningitis and who were randomized to receive 5FC and either standard intravenous (IV) amphotericin B or oral LNC-amphotericin B. We used broth microdilution to measure the minimum inhibitory concentration (MIC) of the first and last cryptococcal isolates in each participant. Breakpoints are inferred from 5FC in Candida albicans. We measured cerebral spinal fluid (CSF) 5FC concentrations by liquid chromatography and tandem mass spectrometry. Results: Cryptococcus 5FC MIC50 was 4 µg/mL, and MIC90 was 8 µg/mL. After 2 weeks of therapy, there was no evidence of 5FC resistance developing, defined as a >4-fold change in susceptibility in any Cryptococcus isolate tested. The median CSF 5FC concentration to MIC ratio (interquartile range) was 3.0 (1.7-5.5) µg/mL. There was no association between 5FC/MIC ratio and early fungicidal activity of the quantitative rate of CSF yeast clearance (R2 = 0.004; P = .63). Conclusions: There is no evidence of baseline resistance to 5FC or incident resistance during combination therapy with oral or IV amphotericin B in Uganda. Oral amphotericin B can safely be used in combination with 5FC.

Lack of Association between Fluconazole Susceptibility and ERG11 Nucleotide Polymorphisms in Cryptococcus neoformans Clinical Isolates from Uganda.

Fluconazole is the drug of choice for cryptococcal meningitis (CM) monoprophylaxis in resource-limited settings such as Uganda. Emerging fluconazole resistance linked to mutations in the Cryptococcus neoformansERG11 gene (CYP51) has been observed in clinical isolates. Currently, the single nucleotide polymorphisms [SNPs] in the Cryptococcus spp. ERG11 gene that could be responsible for fluconazole resistance are poorly characterized within Ugandan C. neoformans clinical isolates. If available, this information would be useful in the management of cryptococcosis among HIV patients. This cross-sectional study investigates the SNPs present in the coding region of the C. neoformansERG11 gene to determine the relationship between the SNPs identified and fluconazole susceptibility of the clinical isolates. 310 C. neoformans isolates recovered from the Cerebrospinal Fluid (CSF) of patients with HIV and cryptococcal meningitis were examined. The fluconazole half-maximal inhibitory concentrations (IC50 range: 0.25−32 µg/mL) was determined using the microbroth dilution method. A total of 56.1% of the isolates had low IC50 values of <8 µg/mL while 43.9% had high IC50 values ≥ 8 µg/mL. We amplified and sequenced 600 bp of the ERG11 coding sequence from 40 of the clinical isolates. Novel synonymous and 2 missense mutations, S460T and A457V, were identified in the ERG11 gene. The identified SNPs were not associated with differences in fluconazole IC50 values in vitro (p = 0.179).

Co-feeding glucose with either gluconate or galacturonate during clostridial fermentations provides metabolic fine-tuning capabilities.

Clostridium acetobutylicum ATCC 824 effectively utilizes a wide range of substrates to produce commodity chemicals. When grown on substrates of different oxidation states, the organism exhibits different recycling needs of reduced intracellular electron carrying co-factors. Ratios of substrates with different oxidation states were used to modulate the need to balance electron carriers and demonstrate fine-tuned control of metabolic output. Three different oxidized substrates were first fed singularly, then in different ratios to three different strains of Clostridium sp. Growth was most robust when fed glucose in exclusive fermentations. However, the use of the other two more oxidized substrates was strain-dependent in exclusive feeds. In glucose-galacturonate mixed fermentation, the main products (acetate and butyrate) were dependant on the ratios of the substrates. Exclusive fermentation on galacturonate was nearly homoacetic. Co-utilization of galacturonate and glucose was observed from the onset of fermentation in growth conditions using both substrates combined, with the proportion of galacturonate present dictating the amount of acetate produced. For all three strains, increasing galacturonate content (%) in a mixture of galacturonate and glucose from 0 to 50, and 100, resulted in a corresponding increase in the amount of acetate produced. For example, C. acetobutylicum increased from ~ 10 mM to ~ 17 mM, and then ~ 23 mM. No co-utilization was observed when galacturonate was replaced with gluconate in the two substrate co-feed.

ATI-2307 Exhibits Equivalent Antifungal Activity in Cryptococcus neoformans Clinical Isolates With High and Low Fluconazole IC50.

Half maximal inhibitory concentrations (IC50) to the experimental drug ATI-2307 and complete inhibition (IC90) of the common clinically used antifungal drug amphotericin B were determined by microbroth dilution assay for a collection of 69 clinical isolates of Cryptococcus neoformans from Uganda that had high fluconazole IC50 values. The majority of the clinical isolates tested had fluconazole IC50 at or above 8 µg/mL, but were susceptible to both amphotericin B (IC90 ≤1 µg/mL) and ATI-2307 (IC50 ≤0.0312 µg/mL). No correlation between increased fluconazole minimum inhibitory concentration (MIC) and ATI-2307 or amphotericin B MIC was observed, suggesting that the cellular changes impacting fluconazole susceptibility did not impact the effectiveness of ATI-2307. Our results suggest that ATI-2307 is a promising new antifungal drug for use in the context of high fluconazole or other antifungal drug MICs and/or in combination drug therapy regimens.

Arabinose-Induced Catabolite Repression as a Mechanism for Pentose Hierarchy Control in Clostridium acetobutylicum ATCC 824.

Bacterial fermentation of carbohydrates from sustainable lignocellulosic biomass into commodity chemicals by the anaerobic bacterium Clostridium acetobutylicum is a promising alternative source to fossil fuel-derived chemicals. Recently, it was demonstrated that xylose is not appreciably fermented in the presence of arabinose, revealing a hierarchy of pentose utilization in this organism (L. Aristilde, I. A. Lewis, J. O. Park, and J. D. Rabinowitz, Appl Environ Microbiol 81:1452-1462, 2015, https://doi.org/10.1128/AEM.03199-14). The goal of the current study is to characterize the transcriptional regulation that occurs and perhaps drives this pentose hierarchy. Carbohydrate consumption rates showed that arabinose, like glucose, actively represses xylose utilization in cultures fermenting xylose. Further, arabinose addition to xylose cultures led to increased acetate-to-butyrate ratios, which indicated a transition of pentose catabolism from the pentose phosphate pathway to the phosphoketolase pathway. Transcriptome sequencing (RNA-Seq) confirmed that arabinose addition to cells actively growing on xylose resulted in increased phosphoketolase (CA_C1343) mRNA levels, providing additional evidence that arabinose induces this metabolic switch. A significant overlap in differentially regulated genes after addition of arabinose or glucose suggested a common regulation mechanism. A putative open reading frame (ORF) encoding a potential catabolite repression phosphocarrier histidine protein (Crh) was identified that likely participates in the observed transcriptional regulation. These results substantiate the claim that arabinose is utilized preferentially over xylose in C. acetobutylicum and suggest that arabinose can activate carbon catabolite repression via Crh. Furthermore, they provide valuable insights into potential mechanisms for altering pentose utilization to modulate fermentation products for chemical production. IMPORTANCE Clostridium acetobutylicum can ferment a wide variety of carbohydrates to the commodity chemicals acetone, butanol, and ethanol. Recent advances in genetic engineering have expanded the chemical production repertoire of C. acetobutylicum using synthetic biology. Due to its natural properties and genetic engineering potential, this organism is a promising candidate for converting biomass-derived feedstocks containing carbohydrate mixtures to commodity chemicals via natural or engineered pathways. Understanding how this organism regulates its metabolism during growth on carbohydrate mixtures is imperative to enable control of synthetic gene circuits in order to optimize chemical production. The work presented here unveils a novel mechanism via transcriptional regulation by a predicted Crh that controls the hierarchy of carbohydrate utilization and is essential for guiding robust genetic engineering strategies for chemical production.

Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105.

Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Šresolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two ß-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Šand contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR.