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.

Fig 1. Biosynthetic pathway of β-carotene in recombinant S. cerevisiae.

The dashed lines indicate multiple step reactions. HMG1: HMG-CoA reductase, ERG9: squalene synthase. Carotenoids pathway: CrtE: GGPP synthase, BTS1: S. cerevisiae GGPP synthase, CrtYB: Lycopene cyclase, CrtI: Phytoene desaturase. IPP: isopentenyl pyrophosphate; DMAP: dimethylallyl pyrophosphate; GPP: geranyl pyrophosphate; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate.

Fig 2

Cell growth (A) and glucose consumption (B) of recombinant S. cerevisiae T73-63 and parent strain T73-4 and β-carotene concentration and production (C) of recombinant S. cerevisiae T73-63. Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test).

Fig 3. Time courses of intracellular amino acids concentration (μmol/g) of recombinant S. cerevisiae T73-63 (marked grey) and parent strain T73-4 (marked white).

Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test).

Fig 4. Time courses of relative concentration of metabolites in recombinant S. cerevisiae T73-63 (marked grey) and parent strain T73-4 (marked white).

Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test).

Fig 5. Cell growth of recombinant S. cerevisiae T73-63 in mixed carbon source cultures and YPD medium containing 2% and 5% glucose, respectively.

Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test). Abbreviations: Glu: glucose, Gly: glycerol, Gala: galactose, AceNa: sodium acetate, CitrNa: sodium citrate.

Fig 6

β-Carotene concentration (A) and production (B) of recombinant S. cerevisiae T73-63 in mixed carbon source cultures and YPD medium containing 2% and 5% glucose, respectively at 12, 16 and 20 h. Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test). Abbreviations: Glu: glucose, Gly: glycerol, Gala: galactose, AceNa: sodium acetate.

Fig 7. Cell growth of recombinant S. cerevisiae T73-63 with different acetate supplementation modes.

Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test). Abbreviations: Glu: glucose, AceNa: sodium acetate.

Fig 8

The β-carotene concentration (A) and production (B) of recombinant S. cerevisiae T73-63 with different acetate supplementation modes. Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test). Abbreviations: Glu: glucose, AceNa: sodium acetate.

Fig 9. Relative concentration of metabolites in recombinant S. cerevisiae T73-63 supplemented with 10 g/l acetate at 10 h (marked grey) and T73-63 (marked white) at 12 and 16 h.

Values represent the average of three independent cultures, and error bars correspond to the standard deviation (P< 0.05, Student’s t test).

Fig 10. The conversion pathway of acetate into β-carotene in recombinant S. cerevisiae.

Acetate addition are shown in red, central metabolite (acetyl-CoA) are shown in rose. The dashed lines represent multiple step reactions, double arrows indicate the enhancement of metabolic flow. ACS1/ACS2: Acetyl-CoA synthetase, FPP: farnesyl pyrophosphate, MVA pathway: mevalonate pathway.