Comparative metabolomics profiling of engineered Saccharomyces cerevisiae lead to a strategy that improving β-carotene production by acetate supplementation

Abstract

A comparative metabolomic analysis was conducted on recombinant Saccharomyces cerevisiae strain producing β-carotene and the parent strain cultivated with glucose as carbon source using gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based approach. The results showed that most of the central intermediates associated with amino acids, carbohydrates, glycolysis and TCA cycle intermediates (acetic acid, glycerol, citric acid, pyruvic acid and succinic acid), fatty acids, ergosterol and energy metabolites were produced in a lower amount in recombinant strain, as compared to the parent strain. To increase β-carotene production in recombinant strain, a strategy that exogenous addition of acetate (10 g/l) in exponential phase was developed, which could enhance most intracellular metabolites levels and result in 39.3% and 14.2% improvement of β-carotene concentration and production, respectively, which was accompanied by the enhancement of acetyl-CoA, fatty acids, ergosterol and ATP contents in cells. These results indicated that the amounts of intracellular metabolites in engineered strain are largely consumed by carotenoid formation. Therefore, maintaining intracellular metabolites pool at normal levels is essential for carotenoid biosynthesis. To relieve this limitation, rational supplementation of acetate could be a potential way because it can partially restore the levels of intracellular metabolites and improve the production of carotenoid compounds in recombinant S. cerevisiae.

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of β-carotene in Saccharomyces cerevisiae

“…For example, the mitochondrial proteins involved in TCA cycle (Sdh1p, Sdh2p Sdh3p, Lsc1p, Lsc2p, Cit2p and Cit3p) and ATP synthesis (Atp7p, Atp17p, Atp19p, Atp20p and Tim11p) were up-regulated. This indicated that the ATP generation system became abnormal due to the β-carotene accumulation, which might lead to ATP deficiency in the YBX-01 strain, as shown in our previous study [30]. The proteins related to lipid metabolism (such as Acc1p, Ino1p and Cyb5p) and ergosterol biosynthetic process (Erg1p, Erg4p, Erg6p, Erg20p, Erg24p, and Erg25p) were simultaneously enriched in YBX-01 strain (Additional file 1: Table S1).…”

“…Acetate, an abundant and renewable resource, can improve protein secretion by increasing ATP content in Schizosaccharomyces pombe [42]. As a precursor of acetyl-CoA, acetate supplementation leads to an increase in carbon flux of the TCA cycle and promotes the synthesis of ATP [30]. In this study, different concentrations of acetate (5 g/L, 10 g/L, and 15 g/L) was added after 12 h cultivation of YBX-SNQ2, and 10 g/L acetate addition showed the best performance for β-carotene production and efflux.…”

Dynamic resource allocation drives growth under nitrogen starvation in eukaryotes

“…In all organisms, arginine biosynthesis consumes ornithine and glutamine, a main nitrogen carrier, which induces an additional cost for the network, considering its high connectivity with the biosynthesis of other amino acids, pigments and nucleotides. Glutamine, specifically, has been identified as one of the most important contributors to carbon and nitrogen metabolism 40 , and a drastic decrease (e.g., 35% decrease in yeast) 6,7 in its abundance is a common response under nitrogen starvation 2,40,41 . Moreover, the consumption of ornithine fully activates the acetyl cycle, thus adding nitrogen and energy costs by consuming glutamate and ATPs.…”

“…Metabolic adaptation has been associated with changes in the production or degradation rates of biomolecules. These profiles are observed in various heterotrophic and photoautotrophic microorganisms, increasing their resistance to stress conditions and nutrients limitation 1,[3][4][5][6][7][8][9][10][11][12] . Depletion of carbon and energy sources directly reduce the synthesis of all biomass precursors since carbon is the backbone of nucleic acids, proteins, lipids, and carbohydrates and all anabolic pathways consume energy.…”

Role of phosphate limitation and pyruvate decarboxylase in rewiring of the metabolic network for increasing flux towards isoprenoid pathway in a TATA binding protein mutant of Saccharomyces cerevisiae

“…Most attempts have focused almost exclusively on the genes and regulatory controls of the isoprenoid pathway. Some efforts have also been directed towards increasing the cytosolic pools of the precursor acetyl-CoA by engineering pyruvate dehydrogenase (PDH) bypass [ 4 ], overexpression of alcohol dehydrogenase ( ADH2 ) for increasing acetaldehyde pools, a precursor for acetyl-CoA through production from ethanol [ 5 ], decreasing competition for cytosolic acetyl-CoA in the glyoxylate cycle with malate synthase ( MLS1 ) and citrate synthase ( CIT2 ) gene deletions [ 6 ], and exogenous addition of acetate in the exponential phase [ 7 ]. As increasing acetyl-CoA pools from acetate by acetyl-CoA synthases ( ACS1 , ACS2 ) requires ATP, many metabolic engineering strategies have tried to reduce overall ATP cost by decoupling acetyl-CoA supply from ATP hydrolysis [ 8 – 11 ].…”