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

Abstract

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 b-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. INTRODUCTIONLipids are dynamically bioactive molecules that contribute to the regulation of several complex systems of living cells. Besides the medical perspective, the remarkable growth of the lipid field is undoubtedly driven by the demand for feedstock for the oleochemical industry. In addition to the production of n-3 and n-6 polyunsaturated fatty acids that are beneficial to human health, extensive attention is being directed to biodiesel production from micro-organisms to replace non-sustainable petroleum (Liu & Zhao, 2007; Vicente et al., 2010). Certain strains, such as Rhodosporidiun toruloides, Lipomyces starkeyi, Yarrowia lipolytica and Mucor circinelloides, are known to accumulate substantial amounts of lipids, accounting for more than 20 % of their biomass, and are thus called oleaginous strains (Ageitos et al., 2011;Beopoulos et al., 2009;Meng et al. 2009;Ratledge, 2004). Due to their short cultivation time, their high level of intracellular lipids that are predominantly triacylglycerol (TAG) and their utilization of various substrates, oleaginous yeasts and fungi have become important model systems for alternatives to traditional sources of lipids derived from fossil, animal and plant origins (Beopoulos et al., 2009;Ratledge, 2004). Therefore, an understanding of lipid physiology is required to improve the efficient production of lipids of commercial interest. An integrated approach has been implemented using recent developments for studying the lipid metabolism of these promising micro-organisms. It has been reported that the lipid production of these oleaginous species is enhanced by controlling cultivation or nutritional conditions (Certik et al., 1999;Ruenwai et al., 2010). Based on the bioc...

Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

“…Specifically, we contrasted the effects of leucine and isoleucine supplementation on lipogenesis and demonstrated that only leucine acts to promote lipogenesis. As Y. lipolytica possesses degradation pathways for both leucine and isoleucine to acetyl-CoA fatty-acid precursors [30][31][32] , leucine must act as an intracellular signal to permit lipid production, rather than as a carbon source. This is further established by the leucine-dependent TOR-inhibition phenotype of the PO1f leucine À uracil þ and PO1f leucine þ uracil þ strains (Supplementary Fig.…”

“…1a). Leucine biosynthetic capacity has also been implicated as an effector of lipogenic ability in oleaginous organisms 25,29 , and a putative acetyl-CoA-generating leucine degradation pathway was recently identified through a comparative genomics analysis of non-oleaginous and oleaginous yeast strains 30 . In this regard, Y. lipolytica also possesses homologues for every enzyme and enzymatic subunit necessary for acetyl-CoA generation through isoleucine degradation (445% similarity to Homo sapiens enzymes) 31,32 .…”

Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum

“…It has been reported that Leu metabolism is involved in the generation of the acetyl-CoA required for fatty acid biosynthesis in the oleaginous fungus Mucor circinelloides (Vorapreeda et al, 2012;Rodríguez-Frómeta et al, 2013). In addition, catabolism of amino acids such as Ala and Gln could provide carbon skeletons directed to fatty acid elongation (Schwender et al, 2006;Junker et al, 2007;Hockin et al, 2012).…”

Oleaginous Yeasts form Uruguay and Antarctica as Renewable Raw Material for Biodiesel Production

“…After 5 days of incubation, Sudan Black-stained smears from each culture were observed under optical microscopy looking for the presence of intracellular blue oil globules which are characteristic of oleaginous yeasts. Non oleaginous yeast (Saccharomyces cerevisiae) was used as a negative control [8]. For the oleaginous isolates, intracellular content of saponifiable lipids was quantified as described by Burja et al [9] with modifications.…”

“…Intracellular saponifiable lipids were evaluated daily from the second to the sixth day. Ammonia was determined using the commercial kit UREA/BUN-COLOR (Biosystems COD 11536) and nitrate was analyzed by HPLC with a IC-Pack Anion column (Waters) maintained at 40 °C, using phosphate buffer (0,01 M, pH 6,8) as mobile phase at a flux of 1,2 mL min -1 and a UV detector (Shimadzu SPD-10AV UV-VIS Detector) at 210 nm. Glycerol was determined by HPLC using an Aminex HPX-87C (Bio-Rad), 250 x 4 mm column, at 67 °C with water as mobile phase and a RID-10A Shimadzu detector.…”