Project description:With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints-in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints.We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate.By combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoA derived fuels and chemicals.

Project description:The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment by SAGE analysis. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unabl e to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g., an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modeling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors. SAGE analysis of C. neoformans cells isolated from macrophages The murine macrophage-like cell line J774A.1 was maintained at 37M-BM-0C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 M-BM-5g mL-1 penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against capsule (1 M-BM-5g mL-1), and macrophages were treated with recombinant mouse gamma interferon (IFN-gamma) (50 U mL-1) and lipopolysaccharide (LPS) (0.3 M-BM-5g mL-1) prior to co-incubation at a multiplicity of infection (MOI) of 1:1. Macrophages were inoculated with H99 cells and washed after 1 h of inoculation to remove unattached, extracellular fungal cells. After 6 h of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells (4 x 107) were harvested by centrifugation. H99 control cells were prepared by growth under the same condition but without macrophages, and 108 cell s were harvested. SAGE library construction, sequencing and analysis SAGE library construction, sequencing and analysis were as previously described (Hu et al., 2007; Hu et al., 2008; Steen et al., 2002; Steen et al., 2003). Briefly, RNA was isolated from lyophilized cells by vortexing with glass beads (3.0 mm, acid-washed and RNase-free) for 15 min in 15 mL of TRIZOL extraction buffer (Invitrogen, Carlsbad, CA, USA). The mixture was incubated for 15 min at room temperature and total RNA was isolated according to the manufacturer's instructions. Total RNA was used directly for SAGE library construction as described by Velculescu et al. (1995) using the I-Long SAGE kit (Invitrogen). The tagging enzyme for cDNA digestion was NlaIII and 29 PCR cycles were performed to amplify the ditags during library construction. Colonies were screened by PCR (M13F and M13R primers) to assess the average clone insert size and the percentage of recombinants. Clones from the libraries were sequenced by BigDye primer cycle sequencing on an ABI PRISM 3700 DNA analy zer. Sequence chromatograms were processed using PHRED (Ewing et al., 1998), and vector sequence was detected using Cross_match (Gordon et al., 1998). Fourteen-base-pair tags were extracted from the vector-clipped sequence, and an overall quality score for each tag was derived based on the cumulative PHRED score. Duplicate ditags and linker sequences were removed as described previously (Steen et al., 2002). Only tags with a predicted accuracy of 99% were used, and statistical differences between tag abundance in different libraries were determined as described (Audic and Claverie, 1997). The libraries yielded 23,904 tags from cells grown in media without macrophages for 6hr and 38,5444 tags from cells grown in media with macrophages for 6hr. An overview of the abundance classes for both SAGE libraries is presented in Table S1 in the supplementary material, with both the number of different tag sequences and the total number of tags present in each class for the cells from th e 6h infection and the control. All libraries were normalized to 23,904 to allow direct comparisons, and the tags that appeared less than once in any given library were removed. The EST database available for strain H99 at the University of Oklahoma's Advanced Center for Genome Technology http://www.genome.ou.edu/cneo.html) was used for the preliminary assignment of tags to genes. When an EST sequence could not be identified for a particular tag, the genomic sequence for H99 at the Duke University Center for Genome Technology (http://cgt.genetics.duke.edu/data/index.html) and the Broad Institute (http://cneo.genetics.duke.edu/) was used to identify contigs with unambiguous tag assignments. Note that a limitation of the SAGE approach is that some transcripts are not detected because of low abundance and/or the absence of an NlaIII site for transcript processing. The control (fungal cells alone) and cryptococcal-macrophage interaction SAGE datasets were deposited to the NCBI under accession number GSM986206 and GSM986207 respectively.

Project description:The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment by SAGE analysis. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unabl e to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g., an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modeling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors. SAGE analysis of C. neoformans cells isolated from macrophages The murine macrophage-like cell line J774A.1 was maintained at 37°C in 10% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FBS), 1% nonessential amino acids, 100 µg mL-1 penicillin-streptomycin, and 4 mM L-glutamine (Invitrogen). Cryptococcus cells were opsonized with monoclonal antibody 18B7 against capsule (1 µg mL-1), and macrophages were treated with recombinant mouse gamma interferon (IFN-gamma) (50 U mL-1) and lipopolysaccharide (LPS) (0.3 µg mL-1) prior to co-incubation at a multiplicity of infection (MOI) of 1:1. Macrophages were inoculated with H99 cells and washed after 1 h of inoculation to remove unattached, extracellular fungal cells. After 6 h of incubation, sterile, ice-cold distilled H2O was applied to each well to lyse the macrophages, and the fungal cells (4 x 107) were harvested by centrifugation. H99 control cells were prepared by growth under the same condition but without macrophages, and 108 cell s were harvested.

Project description:Aspergillus luchuensis mut. kawachii (A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpA ΔyhmA strain, we constructed an S-tagged ctpA (ctpA-S) conditional expression strain in the ΔyhmA background using the Tet-On promoter system. Knockdown of ctpA-S in ΔyhmA resulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis in A. kawachiiIMPORTANCE Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from Aspergillus luchuensis mut. kawachii Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

Project description:Background:Bio-based production of organic acids promises to be an attractive alternative for the chemicals industry to substitute petrochemicals as building-block chemicals. In recent years, itaconic acid (IA, methylenesuccinic acid) has been established as a sustainable building-block chemical for the manufacture of various products such as synthetic resins, coatings, and biofuels. The natural IA producer Aspergillus terreus is currently used for industrial IA production; however, the filamentous fungus Aspergillus niger has been suggested to be a more suitable host for this purpose. In our previous report, we communicated the overexpression of a putative cytosolic citrate synthase citB in an A. niger strain carrying the full IA biosynthesis gene cluster from A. terreus, which resulted in the highest final titer reported for A. niger (26.2 g/L IA). In this research, we have attempted to improve this pathway by increasing the cytosolic acetyl-CoA pool. Additionally, we have also performed fermentation optimization by varying the nitrogen source and concentration. Results:To increase the cytosolic acetyl-CoA pool, we have overexpressed genes acl1 and acl2 that together encode for ATP-citrate lyase (ACL). Metabolic engineering of ACL resulted in improved IA production through an apparent increase in glycolytic flux. Strains that overexpress acl12 show an increased yield, titer and productivity in comparison with parental strain CitB#99. Furthermore, IA fermentation conditions were improved by nitrogen supplementation, which resulted in alkalization of the medium and thereby reducing IA-induced weak-acid stress. In turn, the alkalizing effect of nitrogen supplementation enabled an elongated idiophase and allowed final titers up to 42.7 g/L to be reached at a productivity of 0.18 g/L/h and yield of 0.26 g/g in 10-L bioreactors. Conclusion:Ultimately, this study shows that metabolic engineering of ACL in our rewired IA biosynthesis pathway leads to improved IA production in A. niger due to an increase in glycolytic flux. Furthermore, IA fermentation conditions were improved by nitrogen supplementation that alleviates IA induced weak-acid stress and extends the idiophase.

Dataset Information

ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in Aspergillus nidulans.

Publications

ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in Aspergillus nidulans.

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