Selective biosynthesis of retinol in S. cerevisiae

Abstract

The vitamin A component retinol has become an increasingly sought-after cosmetic ingredient. In previous efforts for microbial biosynthesis of vitamin A, a mixture of retinoids was produced. In order to efficiently produce retinol at high purity, the precursor and NADPH supply was first enhanced to improve retinoids accumulation in the S. cerevisiae strain constructed from a β-carotene producer by introducing β-carotene 15,15'-dioxygenase, following by screening of heterologous and endogenous oxidoreductases for retinal reduction. Env9 was found as an endogenous retinal reductase and its activity was verified in vitro. By co-expressing Env9 with the E. coli ybbO, as much as 443.43 mg/L of retinol was produced at 98.76% purity in bi-phasic shake-flask culture when the antioxidant butylated hydroxytoluene was added to prevent retinoids degradation. The retinol titer reached 2479.34 mg/L in fed-batch fermentation. The success in selective biosynthesis of retinol would lay a solid foundation for its biotechnological production.

Keywords: Biosynthesis; Metabolic engineering; Retinal reductase; Retinol; Vitamin A.

Fig. 1

Vitamin A biosynthesis. a Biosynthesis of retinal and its conversion to other retinoids. BCDO, β-carotene 15,15ʹ-dioxygenase; BCMO, β-carotene 15,15′-monooxygenase; ACO, apo-carotenoid 15,15′-oxygenase; ALDH, aldehyde dehydrogenase; SDR, short-chain dehydrogenase/reductase; AKR, aldo-keto reductase; ADH, alcohol dehydrogenase. b Vitamin A biosynthetic pathway in S. cerevisiae. The biosynthetic pathway starts from the endogenous mevalonic acid (MVA) pathway, extended by a β-carotene synthetic pathway module consisting of CrtE03M, CrtYB and CrtI from Xanthophyllomyces dendrorhous and the vitamin A formation module containing the Halobacterium sp. β-carotene dioxygenase gene BLH and the retinal reductase genes (ybbO from E. coli and ENV9 from S. cerevisiae). The major competing branches for the key precursor farnesyl pyrophosphate (FPP) include the ergosterol synthetic pathway and the phosphatase-catalyzed formation of farnesol. Diacylglycerol pyrophosphate phosphatase gene DPP1 and lipid phosphate phosphatase gene LPP1 were knocked out and used as integration sites. The exogenous enzymes introduced are shown in green, the endogenous enzymes are depicted in orange and those deleted are depicted in gray. HMG-CoA, 3-hydroxy-3-methyl-glutaryl-CoA; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl diphosphate; tHmg1, truncated 3-hydroxy-3-methyl-glutaryl reductase; CrtE, geranylgeranyl diphosphate synthase; CrtI, phytoene desaturase; CrtYB, phytoene synthase/ lycopene cyclase; Blh, bacterirhodopsin-related-protein-like homolog protein (later identified as β-carotene dioxygenase); ybbO, aldehyde reductase; tPos5, truncated NADH kinase

Fig. 2

Retinoids biosynthesis in the engineered yeast strain Y03. a HPLC chromatograms of strain Y03, together with the retinal and retinol standards. b Effect of extractant type on bi-phasic fermentation of retinoids. c Effect of dodecane overlay volume on retinoids production. d Distribution of retinoids in mono- and bi-phasic fermentation. e Effect of Fe²⁺ addition on retinoids production. f Intracellular accumulation of carotenoids in mono- and bi-phasic fermentation. Statistical significance was evaluated using Student’s t test (*, P < 0.05; **, P < 0.01)

Fig. 3

Effect of CrtE03M overexpression, ERG9 downregulation, and deletion of ROX1, MOT3 or YPL062W on retinoids titer (a), carotenoids titer (b), and dry cell weight (c). The error bars represent standard deviations calculated from triplicate experiments, and statistical significance of the different retinoids levels in comparison with the control was evaluated using Student’s t test (*, P < 0.05; **, P < 0.01)

Fig. 4

Effect of enhancing NADPH supply on retinoids production. a Retinoids titer and b dry cell weight of each strain are indicated. The error bars represent standard deviations calculated from triplicate experiments, and statistical significance of the different retinoids levels in comparison with the control was evaluated using Student’s t test (*, P < 0.05; **, P < 0.01)

Fig. 5

Enhancement of retinol production through mining and evaluation of endogenous and exogenous alcohol dehydrogenases and investigation of the role of antioxidant BHT. a Effect of overexpressing endogenous or exogenous dehydrogenases on retinol formation. b Retinoids production by the engineered yeast strains in the absence and presence of BHT. The error bars represent standard deviations calculated from triplicate experiments and statistical significance of the different retinol levels in comparison with the control was evaluated using Student’s t test (*, P < 0.05; **, P < 0.01). ‘+’ represents the introduction of exogenous genes or overexpression of endogenous genes, while its number represents the copy number of the respective gene in the yeast strain

Fig. 6

Retinol production of strain Y03-43(+) in fed-batch fermentation. a Profile of glucose, ethanol, biomass and retinol accumulation. b Accumulation of precursors. The arrows and asterisks indicate the time of adding dodecane and Fe²⁺, respectively