Engineering of Yarrowia lipolytica for production of astaxanthin

Abstract

Astaxanthin is a red-colored carotenoid, used as food and feed additive. Astaxanthin is mainly produced by chemical synthesis, however, the process is expensive and synthetic astaxanthin is not approved for human consumption. In this study, we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation. First, we screened 12 different Y. lipolytica isolates for β-carotene production by introducing two genes for β-carotene biosynthesis: bi-functional phytoene synthase/lycopene cyclase (crtYB) and phytoene desaturase (crtI) from the red yeast Xanthophyllomyces dendrorhous. The best strain produced 31.1 ± 0.5 mg/L β-carotene. Next, we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG1) and geranylgeranyl diphosphate synthase (GGS1/crtE) in the best producing strain and obtained 453.9 ± 20.2 mg/L β-carotene. Additional downregulation of the competing squalene synthase SQS1 increased the β-carotene titer to 797.1 ± 57.2 mg/L. Then we introduced β-carotene ketolase (crtW) from Paracoccus sp. N81106 and hydroxylase (crtZ) from Pantoea ananatis to convert β-carotene into astaxanthin. The constructed strain accumulated 10.4 ± 0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates, 5.7 ± 0.5 mg/L canthaxanthin, and 35.3 ± 1.8 mg/L echinenone. Finally, we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW (54.6 mg/L) of astaxanthin in a microtiter plate cultivation. Our study for the first time reports engineering of Y. lipolytica for the production of astaxanthin. The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y. lipolytica-based fermentation process for astaxanthin production.

Keywords: Astaxanthin; Isoprenoids; Metabolic engineering; Oleaginous yeast; Yarrowia lipolytica; β-carotene.

Fig. 1

Strategies for optimization of β-carotene production in Y. lipolytica. The engineered steps are highlighted. Abbreviations: DMAPP, dimethylallyl pyrophosphate; IPP, isopenthenyl pyrophosphate; GPP, geranyl pyrophosphate, FPP, farnesyl pyrophosphate, GGPP, geranylgeranyl pyrophosphate; HMG1 and tHMG1, full-length and truncated alleles of 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Y. lipolytica, respectively; crtE and GGS1, GGPP synthase-encoding genes from X. dendrorhous and Y. lipolytica, respectively; crtYB, phytoene synthase and lycopene cyclase genes from X. dendrorhous; crtI, phytoene desaturase-encoding gene from X. dendrorhous; SQS1, squalene synthase gene from Y. lipolytica; P_ERG1, squalene epoxidase promoter; P_ERG11, Lanosterol 14-alpha demethylase promoter; P_SQS1, squalene synthase promoter.

Fig. 2

Production of β-carotene in engineered Y. lipolytica strains. A. The effect of HMG-CoA reductase and GGPP synthase overexpression on β-carotene production. B. The effect of downregulation of squalene synthase on β-carotene production. All strains were cultivated in YP+8%glucose in 24-deep-well plates for 72 h. The error bars represent standard deviations calculated from triplicate experiments.

Fig. 3

Astaxanthin production in Y. lipolytica. A. Astaxanthin biosynthesis pathways. The black thick arrow indicates the reactions catalyzed by the CrtW and CrtZ enzymes. The gray dashed arrow indicates the reaction catalyzed by the CrtS enzyme. To obtain a functional expression of crtS, crtR must be co-expressed. B. Engineered strains cultivated on YPD agar plates for 72 h. Strain abbreviations: (A) ST3683, Wild type; (B) ST5204, β-carotene-producing non-optimized strain; (C) ST6899, β-carotene-producing optimized strain; (D) ST6074, built by expressing crtS and crtR in ST5204; (E) ST6075, built by expressing crtW and crtZ in ST5204; (F) ST7022, built by expressing crtS and crtR in ST6899; and (G) ST7023, built by expressing crtW and crtZ in ST6899. C. HPLC analysis of carotenoids. ST3683, wild type; β-caro_op, β-carotene producer with precursor optimization [ST6899]; Asta_op (crtS-crtR), astaxanthin producer carrying crtS and crtR (precursor optimized) [ST7022]; Asta_op (crtW-crtZ), astaxanthin producer carrying crtW and crtZ (precursor optimized) [ST7023]; Ι, β-carotene; II, echinenone; III, canthaxanthin; IV, astaxanthin. All strains were cultivated in YP+8%glucose in 24-deep-well plates for 72 h.

Fig. 4

The effect of gene copy number on astaxanthin production. A. Scheme of the strain construction process for increasing the copy number of astaxanthin biosynthetic genes. B. HPLC analysis of carotenoids. Strain abbreviations: crtW-crtZ, astaxanthin producer carrying crtW and crtZ (precursor optimized) [ST7023]; crtW-crtZ + crtW↑↑↑, built by multiple integrations of crtW in ST7023 [ST7399]; crtW-crtZ + crtZ↑↑↑, built by multiple integrations of crtZ in ST7023 [ST7400]; crtW-crtZ + crtW↑↑↑-crtZ↑↑↑, built by multiple integrations of crtW-crtZ in ST7023 [ST7402]. Ι, β-carotene; II, echinenone; III, canthaxanthin; IV, astaxanthin. C. The box plot represents the effect of gene copy number adjustment on the production of astaxanthin and its intermediates. Different combination of additional copies of either crtW, crtZ or both genes under the control of P_TEFintron were introduced in the astaxanthin platform strain ST7023. Strain abbreviations: Ref, reference strain ST7023; 1, ST7399; 2, ST7400; 3, ST7401; 4, ST7402; 5, ST7403; 6, ST7404; 7, ST7405; 8, ST7406. Ten individual isolates from each construct were cultivated in YP+8%glucose in 96 deep-well-plates for 72 h.