Kobkul Laoteng

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.

Mechanisms controlling lipid accumulation and polyunsaturated fatty acid synthesis in oleaginous fungi

Polyunsaturated fatty acids (PUFAs) are functional lipids that have been widely incorporated into several industrial sectors. Apart from animal- and plant-derived origins, oleaginous fungi belonging to Mucorales have been identified as promising alternatives for production of n-3 and n-6 PUFAs. It was found, in Mucorales fungi, that ATP:citrate lyase, acetyl-CoA carboxylase and malic enzyme trigger lipid overproduction, and biosynthesis of PUFA requires membrane-bound desaturases with fatty acyl substrate specificities. Accumulation of PUFAs in the cells is associated not only with the desaturation system, but it is also tightly bound with acyltransferases that facilitate the distribution of newly synthesized PUFA to individual lipid structures. Several physical parameters, such as temperature, aeration, and nutrient regimes, greatly affect either the lipid content or fatty acid composition among different Mucorales species. Conclusive evidence showed that the PUFA production yield of the fungi depends on the environmental control of “oleaginous” enzymes, and on the transcriptional expression of the desaturase genes. These valuable studies provide perspectives with biological rationale for microbial production of economically important lipids.

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.

Gamma-linolenic acid production of Mucor rouxii by solid-state fermentation using agricultural by-products

Aims:  This study aims to maximize the yield of gamma‐linolenic acid by a filamentous fungus, Mucor rouxii, using low cost production by solid‐state fermentation.

Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi

Microbial lipids are becoming an attractive option for the industrial production of foods and oleochemicals. To investigate the lipid physiology of the oleaginous microorganisms, at the system level, genome-scale metabolic networks of Mortierella alpina and Mucor circinelloides were constructed using bioinformatics and systems biology. As scaffolds for integrated data analysis focusing on lipid production, consensus metabolic routes governing fatty acid synthesis, and lipid storage and mobilisation were identified by comparative analysis of developed metabolic networks. Unique metabolic features were identified in individual fungi, particularly in NADPH metabolism and sterol biosynthesis, which might be related to differences in fungal lipid phenotypes. The frameworks detailing the metabolic relationship between M. alpina and M. circinelloides generated in this study is useful for further elucidation of the microbial oleaginicity, which might lead to the production improvement of microbial oils as alternative feedstocks for oleochemical industry.

Substrate specificity and preference of Δ6-desaturase of Mucor rouxii

The Δ6‐fatty acid desaturase is a key enzyme in the synthesis of an important fatty acid, γ‐linolenic acid. We have characterized, by heterologous expression in Saccharomyces cerevisiae, substrate specificity and preference of Δ6‐desaturase of Mucor rouxii. Fatty acid supplementation was carried out based on the predicted enzyme topology, fatty acid phenotype and the corresponding metabolic pathway in M. rouxii. The enzyme has a broad substrate specificity as based on C15–C18. The result also supported classification of the M. rouxii Δ6‐desaturase into a front‐end desaturase. Interestingly, a relatively rare activity based on odd acyl chains and not described previously in other eukaryotic Δ6‐desaturases was also observed.

Evaluation of inoculum performance for enhancing gamma‐linolenic acid production from Mucor rouxii

Aims:  To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii.

Microbial production of γ-linolenic acid: Submerged versus solid-state fermentations

One of the major interests of lipid biotechnology is targeted on natural manufacturing of healthy oils containing polyunsaturated fatty acids. Of them, γ-linolenic acid (C18:3 n-6; GLA) as the key intermediate in the n-6 fatty acid family is involved to maintain the proper mammalian cell functions. Insufficient supply of GLA from agricultural and animal sources resulted in ‘hunting’ for appropriate microorganisms suitable to produce this essential fatty acid in high yield. Extensive studies on oleaginous lower filamentous fungi have led to development of two basic fermentation techniques for GLA production: submerged and solid-state fermentations. Each of the processes provide specific advantages in various applications depending on the GLA product form (GLA-rich oil, whole cells, and fermented mass) and might bring new prospects to fill marketing claims in food, feed, pharmaceutical, and veterinary fields.