Characterization of oil-producing yeast Lipomyces starkeyi on glycerol carbon source based on metabolomics and 13C-labeling

Abstract

Lipomyces starkeyi is an oil-producing yeast that can produce triacylglycerol (TAG) from glycerol as a carbon source. The TAG was mainly produced after nitrogen depletion alongside reduced cell proliferation. To obtain clues for enhancing the TAG production, cell metabolism during the TAG-producing phase was characterized by metabolomics with C labeling. The turnover analysis showed that the time constants of intermediates from glycerol to pyruvate (Pyr) were large, whereas those of tricarboxylic acid (TCA) cycle intermediates were much smaller than that of Pyr. Surprisingly, the time constants of intermediates in gluconeogenesis and the pentose phosphate (PP) pathway were large, suggesting that a large amount of the uptaken glycerol was metabolized via the PP pathway. To synthesize fatty acids that make up TAG from acetyl-CoA (AcCoA), 14 molecules of nicotinamide adenine dinucleotide phosphate (NADPH) per C16 fatty acid molecule are required. Because the oxidative PP pathway generates NADPH, this pathway would contribute to supply NADPH for fatty acid synthesis. To confirm that the oxidative PP pathway can supply the NADPH required for TAG production, flux analysis was conducted based on the measured specific rates and mass balances. Flux analysis revealed that the NADPH necessary for TAG production was supplied by metabolizing 48.2% of the uptaken glycerol through gluconeogenesis and the PP pathway. This result was consistent with the result of theC-labeling experiment. Furthermore, comparison of the actual flux distribution with the ideal flux distribution for TAG production suggested that it is necessary to flow more dihydroxyacetonephosphate (DHAP) through gluconeogenesis to improve TAG yield.

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions

“…However, only a small amount of glycerol (only ~1.2% of the 13% of consumed glycerol), ended up as the backbone for the lipid fractions, with more being funnelled into de novo acyl chain synthesis, the majority of which ended up on the membrane lipids (Table 1). This contrasts with previous literature, in which the addition of glycerol was shown to induce TAG accumulation significantly in the diatom P. tricornutum (Villanova et al, 2017) and the oil‐producing yeast Lipomyces starkeyi (Maruyama et al, 2018) by increasing intracellular carbon intermediates of the Calvin–Benson cycle.…”

“…However, the assimilation pathways of glycerol into the microalgal lipidome are not yet fully understood. Previously, it has been shown that supplying exogenous glycerol can augment TAG production in diatoms (Villanova et al, 2017), yeast (Maruyama et al, 2018), and plant seeds (Pollard, Delamarter, Martin, & Shachar-Hill, 2015). TAG is composed of three fatty acyl chains linked to a glycerol backbone (glycerol-3-phosphate, G3P) via ester linkages.…”

A metabolic model of Lipomyces starkeyi for predicting lipogenesis potential from diverse low-cost substrates

“…The PPP was the unique supplier of NADPH. Indeed, the NADPH required for FA synthesis by L. starkeyi was majorly provided by PPP according to the C 13 metabolic flux analysis using C 13 -labeled glycerol as substrate [ 14 ]. The NADH generated during the metabolic process from glucose to acetyl-CoA was excessive and could not be used for FA synthesis according to the flux distribution analysis.…”

“…A site-directed gene knockout strategy has been reported in L. starkeyi NRRL Y-11558 [ 11 ]. The development of synthetic biology approaches, coupled with the omics technologies [ 12 – 14 ], has continuously deepened the understanding of lipid metabolism of L. starkeyi [ 3 ].…”

¹³C-metabolic flux analysis in glycerol-assimilating strains of <i>Saccharomyces cerevisiae</i>

“…Metabolic flux analysis with 13 C-labeled glycerol has been reported in previous studies (Alagesan et al, 2018;Maruyama et al, 2018;Tomàs-Gamisans et al, 2019;Toya et al, 2018;Yao et al, 2019), but, until now, has not been reported for S. cerevisiae. In the present study, to understand intracellular metabolism of S. cerevisiae obtained by ALE and the RIM15 disruptant grown on glycerol as a main carbon source, we performed metabolic flux analysis of evolved S. cerevisiae grown on a mixture of naturally-labeled and 13 C-labeled glycerol.…”