Enhanced β-carotene production in Yarrowia lipolytica through the metabolic and fermentation engineering

Abstract

β-Carotene is a kind of high-value tetraterpene compound, which shows various applications in medical, agricultural, and industrial areas owing to its antioxidant, antitumor, and anti-inflammatory activities. In this study, Yarrowia lipolytica was successfully metabolically modified through the construction and optimization of β-carotene biosynthetic pathway for β-carotene production. The β-carotene titer in the engineered strain Yli-C with the introduction of the carotenogenesis genes crtI, crtE, and crtYB can reach 34.5 mg/L. With the overexpression of key gene in the mevalonate pathway and the enhanced expression of the fatty acid synthesis pathway, the β-carotene titer of the engineered strain Yli-CAH reached 87 mg/L, which was 152% higher than that of the strain Yli-C. Through the further expression of the rate-limiting enzyme tHMGR and the copy number of β-carotene synthesis related genes, the β-carotene production of Yli-C2AH2 strain reached 117.5 mg/L. The final strain Yli-C2AH2 produced 2.7 g/L β-carotene titer by fed-batch fermentation in a 5.0-L fermenter. This research will greatly speed up the process of developing microbial cell factories for the commercial production of β-carotene.

One-sentence summary: In this study, the β-carotene synthesis pathway in engineered Yarrowia lipolytica was enhanced, and the fermentation conditions were optimized for high β-carotene production.

Keywords: Yarrowia lipolytica; Fatty acid pathway; Fermentation engineering; MVA pathway; Metabolic engineering; β-Carotene.

Graphical Abstract

Synthesis pathways of β-carotene and fatty acids in the engineered Yarrowia lipolytica.

Fig. 1.

β-Carotene biosynthetic pathway. Black arrows indicate β-carotene synthesis pathways. Yellow arrows indicate the lipid biosynthesis pathway. Red is the enzyme that is overexpressed in the cell.

Fig. 2.

β-Carotene production of strain Yli-C and exploration of its fermentation conditions. (a) and (b) β-carotene production of po1f and Yli-C. (c) β-Carotene titer of strain Yli-C when glucose or glycerol was used as carbon source. (d) β-Carotene titer of strain Yli-C at different temperatures.

Fig. 3.

β-Carotene yield of engineered strains after optimization of mevalonate and fatty acid pathway. (a) β-carotene titer of strain Yli-CH. (b) β-carotene titer of strain Yli-CA. (c) β-Carotene titer of strain Yli-CAH.

Fig. 4.

β-Carotene production in engineered strains with multi-copy expression of genes and construction of genetically engineered strains. (a) β-Carotene titer of strain Yli-C2AH. (b) β-Carotene titer of strain Y li-C2AH2. (c) Genetic modification of β-Carotene producing strains.

Fig. 5.

Optimization of fermentation conditions for Yli-C2AH2. (a) β-Carotene titer of strain Yli-C2AH2 at different concentrations of sodium citrate. (b) β-Carotene titer of strain Yli-C2AH2 at different concentrations of H₂O₂.

Fig. 6.

Production of β-carotene by strain Yli-C2AH2 through the fed-batch fermentation in a 5.0 L bioreactor. (a) Single cell morphology under optical microscope and fermentation broth status in a 5.0 L bioreactor. (b) Dynamic parameters during 168 hr fermentation in a 5.0 L bioreactor.