Supapon Cheevadhanarak

The genome-scale metabolic model iIN800 of Saccharomyces cerevisiae and its validation: A scaffold to query lipid metabolism

BackgroundUp to now, there have been three published versions of a yeast genome-scale metabolic model: iFF708, iND750 and iLL672. All three models, however, lack a detailed description of lipid metabolism and thus are unable to be used as integrated scaffolds for gaining insights into lipid metabolism from multilevel omic measurement technologies (e.g. genome-wide mRNA levels). To overcome this limitation, we reconstructed a new version of the Saccharomyces cerevisiae genome-scale model, iIN800 that includes a more rigorous and detailed description of lipid metabolism.ResultsThe reconstructed metabolic model comprises 1446 reactions and 1013 metabolites. Beyond incorporating new reactions involved in lipid metabolism, we also present new biomass equations that improve the predictive power of flux balance analysis simulations. Predictions of both growth capability and large scale in silico single gene deletions by iIN800 were consistent with experimental data. In addition, 13C-labeling experiments validated the new biomass equations and calculated intracellular fluxes. To demonstrate the applicability of iIN800, we show that the model can be used as a scaffold to reveal the regulatory importance of lipid metabolism precursors and intermediates that would have been missed in previous models from transcriptome datasets.ConclusionPerforming integrated analyses using iIN800 as a network scaffold is shown to be a valuable tool for elucidating the behavior of complex metabolic networks, particularly for identifying regulatory targets in lipid metabolism that can be used for industrial applications or for understanding lipid disease states.

Alternative routes of acetyl-CoA synthesis identified by comparative genomic analysis: involvement in the lipid production of oleaginous yeast and fungi

For a bio-based economy, microbial lipids offer a potential solution as alternative feedstocks in the oleochemical industry. The existing genome data for the promising strains, oleaginous yeasts and fungi, allowed us to investigate candidate orthologous sequences that participate in their oleaginicity. Comparative genome analysis of the non-oleaginous (Saccharomyces cerevisiae, Candida albicans and Ashbya gossypii) and oleaginous strains (Yarrowia lipolytica, Rhizopus oryzae, Aspergillus oryzae and Mucor circinelloides) showed that 209 orthologous protein sequences of the oleaginous microbes were distributed over several processes of the cells. Based on the 41 sequences categorized by metabolism, putative routes potentially involved in the generation of precursors for fatty acid and lipid synthesis, particularly acetyl-CoA, were then identified that were not present in the non-oleaginous strains. We found a set of the orthologous oleaginous proteins that was responsible for the biosynthesis of this key two-carbon metabolite through citrate catabolism, fatty acid β-oxidation, leucine metabolism and lysine degradation. Our findings suggest a relationship between carbohydrate, lipid and amino acid metabolism in the biosynthesis of acetyl-CoA, which contributes to the lipid production of oleaginous microbes.

Overexpression of acetyl-CoA carboxylase gene of Mucor rouxii enhanced fatty acid content in Hansenula polymorpha.

Malonyl-CoA is an essential precursor for fatty acid biosynthesis that is generated from the carboxylation of acetyl-CoA. In this work, a gene coding for acetyl-CoA carboxylase (ACC) was isolated from an oleaginous fungus, Mucor rouxii. According to the amino acid sequence homology and the conserved structural organization of the biotin carboxylase, biotin carboxyl carrier protein, and carboxyl transferase domains, the cloned gene was characterized as a multi-domain ACC1 protein. Interestingly, a 40% increase in the total fatty acid content of the non-oleaginous yeast Hansenula polymorpha was achieved by overexpressing the M. rouxii ACC1. This result demonstrated a significant improvement in the production of fatty acids through genetic modification in this yeast strain.

Proteome analysis at the subcellular level of the cyanobacterium Spirulina platensis in response to low‐temperature stress conditions

The present study addresses the differential expression of Spirulina platensis proteins detected during cold-induced stress, analyzed at the subcellular level. In performing differential expression analysis, the results revealed upregulated proteins in every subcellular fraction, including two-component response systems, DNA repair, molecular chaperones, stress-induced proteins and proteins involved in other biological processes such as secretion systems and nitrogen assimilation. The chlorophyll biosynthetic proteins, protochlorophyllide oxidoreductase and ChlI, had unique expression patterns as detected in the thylakoid membrane; the levels of these proteins immediately decreased during the first 45 min of low-temperature exposure. In contrast, their expression levels significantly increased after low-temperature exposure, indicating the relevance of the chlorophyll biosynthesis in Spirulina in response to low-temperature stress in the light condition. In addition, this is the first report in which genome-based protein identification in S. platensis by peptide mass fingerprinting was performed using the database derived from the unpublished Spirulina genome sequence.

Identification of the nonribosomal peptide synthetase gene responsible for bassianolide synthesis in wood-decaying fungus Xylaria sp. BCC1067.

Intensive study of gene diversity of bioactive compounds in a wood-rot fungus, Xylaria sp. BCC1067, has made it possible to identify polyketides and nonribosomal peptides (NRPs) unaccounted for by conventional chemical screening methods. Here we report the complete nonribosomal peptide synthetase (NRPS) gene responsible for the biosynthesis of an NRP, bassianolide, using a genetic approach. Isolation of the bassianolide biosynthetic gene, nrpsxy, was achieved using degenerate primers specific to the adenylation domain of NRPS. The complete ORF of nrpsxy is 10.6 kb in length. Based on comparisons with other known NRPSs, the domain arrangement of NRPSXY is most likely to be C-A-T-C-A-M-T-T-C-R. The other ORF found upstream of nrpsxy, designated efxy, is 1.8 kb in length and shows high similarity to members of the major facilitator superfamily of transporters. Functional analysis of the nrpsxy gene was conducted by gene disruption, and the missing metabolite in the mutant was identified. Chemical analysis revealed the structure of the metabolite to be a cyclooctadepsipeptide, bassianolide, which has been found in other fungi. A bioassay of bassianolide revealed a wide range of biological activities other than insecticidal uses, which have been previously reported, thus making bassianolide an interesting candidate for future structural modification. This study is the first evidence for a gene involved in the biosynthesis of bassianolide.

Mutation study of conserved amino acid residues of Spirulina Δ6-acyl-lipid desaturase showing involvement of histidine 313 in the regioselectivity of the enzyme

In the cyanobacterium Spirulina platensis, the desaturation process is carried out by three desaturases: the Δ9, Δ12 and Δ6 desaturases, encoded by desC, desA and desD, respectively. The Δ6 desaturase is responsible for the catalysis of linoleic acid, yielding γ-linolenic acid (18:3Δ9,12,6), the end-product of the process. In this study, the desD gene was expressed in Escherichia coli using a pTrcHisA expression system. In order to identify the amino acid residues involved in the enzymatic activity, a sequence comparison was performed using various organisms. The alignment revealed three conserved histidine clusters, a number of conserved residues among all listed organisms and a few conserved residues among cyanobacterial species possibly involved in the desaturation activity. A series of site-directed mutations were generated in the desD gene to evaluate the role of these residues vis-à-vis the enzyme function. This approach revealed that: (1) H313 is involved in the regioselectivity of the enzyme, (2) the three histidine clusters together with H313, H315, D138 and E140 are required for enzymatic activity, most likely as providers of the catalytic Fe center and (3) W294 is also essential for the activity of Δ6 desaturase, possibly by forming part of the substrate-binding pocket.

Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis

The present study examined the changes in protein expression in Spirulina platensis upon exposure to high temperature, with the changes in expression analyzed at the subcellular level. In addition, the transcriptional expression level of some differentially expressed proteins, the expression pattern clustering, and the protein-protein interaction network were analyzed. The results obtained from differential expression analysis revealed up-regulation of proteins involved in two-component response systems, DNA damage and repair systems, molecular chaperones, known stress-related proteins, and proteins involved in other biological processes, such as capsule formation and unsaturated fatty acid biosynthesis. The clustering of all differentially expressed proteins in the three cellular compartments showed: (i) the majority of the proteins in all fractions were sustained tolerance proteins, suggesting the roles of these proteins in the tolerance to high temperature stress, (ii) the level of resistance proteins in the photosynthetic membrane was 2-fold higher than the level in two other fractions, correlating with the rapid inactivation of the photosynthetic system in response to high temperature. Subcellular communication among the three cellular compartments via protein-protein interactions was clearly shown by the PPI network analysis. Furthermore, this analysis also showed a connection between temperature stress and nitrogen and ammonia assimilation.

Heterologous production of polyunsaturated fatty acids in Saccharomyces cerevisiae causes a global transcriptional response resulting in reduced proteasomal activity and increased oxidative stress

Due to their health benefits there is much interest in developing microbial processes for efficient production of polyunsaturated fatty acids (PUFAs). In this study we co-expressed Mucor rouxii Δ(12) - and Δ(6) -desaturase genes in Saccharomyces cerevisiae, which resulted in a yeast strain that accumulated linoleic acid and γ-linolenic acid in the different lipid species. Additionally, the strain contained higher levels of phospholipids and lower levels of ergosterol than the reference strain. Integrated analysis of the transcriptome revealed decreased expression of genes involved in ergosterol biosynthesis, but more unexpectedly it also pointed towards attenuated activity of the ubiquitin-proteasome system and a reduced oxidative stress response. In vitro and in vivo measurements showed reduced levels of all three proteasomal activities and also increased levels of reactive oxidative species in the PUFA-producing strain. Overall our results clearly show that PUFAs in yeast can be detrimental for several key cellular pathways, such as the oxidative stress response and proteasomal activity, suggesting that the membrane composition is of vital importance for these processes.

Differential expression of desaturases and changes in fatty acid composition during sporangiospore germination and development in Mucor rouxii.

Polyunsaturated fatty acids (PUFAs), namely, oleic (C18:1), linoleic (C18:2), and gamma-linolenic acid (C18:3), constituted the majority in the total fatty acid content (44%) of sporangiospores of Mucor rouxii. At 30 degrees C, the germination begins within 1h at which time spore swelling occurs, followed by germ tube emergence within 3-4h. Throughout germination, an increase in gamma-linolenic acid (GLA) was observed and its content was highest at germ tube emergence. It took longer for sporangiospores of M. rouxii to germinate at sub-optimal temperatures (15 and 35 degrees C). However, the content of GLA was higher at the germ tube initiation than at the mycelial stage at all temperatures, suggesting the association of GLA and germination of sporangiospores. This finding was substantially confirmed by differential expression of delta9-, delta12-, and delta6-desaturase genes measured during spore germination. The expression of three desaturase genes parallels the pattern of GLA synthesis. By using RT-PCR techniques to follow gene expression, we found that mRNA of delta12- and delta6-desaturase genes were translated as soon as the spores were introduced into a fresh medium while the mRNA of delta9-desaturase gene could not be detected until 2h after introduction. A sharp increase in mRNA of delta6-desaturase genes correlated well with an increase in GLA content at germ tube emergence (4h). These results demonstrated that changes in fatty acid composition of sporangiospore of M. rouxii and differential expression of desaturase genes occurred during germination, and that extensive changes in GLA synthesis associated with some events in germination process.

Draft genome sequence of Arthrospira platensis C1 (PCC9438)

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.