Highly efficient biosynthesis of astaxanthin in Saccharomyces cerevisiae by integration and tuning of algal crtZ and bkt

Abstract

Astaxanthin is a highly valued carotenoid with strong antioxidant activity and has wide applications in aquaculture, food, cosmetic, and pharmaceutical industries. The market demand for natural astaxanthin promotes research in metabolic engineering of heterologous hosts for astaxanthin production. In this study, an astaxanthin-producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis β-carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed β-carotene hyperproducer. Further integration of strategies including codon optimization, gene copy number adjustment, and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in S. cerevisiae reported to date. Besides, the substrate specificity of H. pluvialis CrtZ and BKT and the probable formation route of astaxanthin from β-carotene in S. cerevisiae were figured out by expressing the genes separately and in combination. The yeast strains engineered in this work provide a basis for further improving biotechnological production of astaxanthin and might offer a useful general approach to the construction of heterologous biosynthetic pathways for other natural products.

Development of a temperature‐responsive yeast cell factory using engineered Gal4 as a protein switch

“…Carotenoids were extracted from the HCl‐heat‐treated cells with 4 ml acetone as previously described (Zhou, Ye, Xie, Lv, & Yu, ) and measured by HPLC (SHIMADZU LC‐20 AT) equipped with an Amethyst C18‐H column (4.6 × 150 mm, 5 µm, Sepax Technologies, Inc., Newark, DE) and a UV/VIS detector at 470 nm. Samples were eluted with 50% acetonitrile (HPLC grade) and 50% methyl alcoholisopropyl alcohol (3:2, v/v, HPLC grade) at a flow rate of 1.0 ml/ min at 40 °C.…”

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae

“…Saccharomyces cerevisiae has been reported as a safe (Generally Recognized as Safe, GRAS) and robust host cell to produce heterologous carotenoids, including lycopene [19], β-carotene [20] and astaxanthin [21]. Thus, in our study, crocetin was successfully synthesized in S. cerevisiae through incorporating heterologous CrtZ and CCD in an existing β-carotene producing strain SyBE_Sc0014CY06 (with β-carotene titer of 220 mg/L) (Table 1).…”

A Review of Pigments Derived from Marine Natural Products

“…For example, an astaxanthin producing Saccharomyces cerevisiae strain was created by successively introducing the Haematococcus pluvialis β‐carotenoid hydroxylase (crtZ) and ketolase (bkt) genes into a previously constructed β‐carotene hyperproducer. Through codon optimization, gene copy number adjustment and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake‐flask cultures which is the highest astaxanthin content in Saccharomyces cerevisiae reported to date . Recently, the production of astaxanthin is mainly based on microbial fermentation, including yeast and algae.…”

“…Through codon optimization, gene copy number adjustment and iron cofactor supplementation led to significant increase in the astaxanthin production, reaching up to 4.7 mg/g DCW in the shake-flask cultures which is the highest astaxanthin content in Saccharomyces cerevisiae reported to date. [108] Recently, the production of astaxanthin is mainly based on microbial fermentation, including yeast and algae. Interestingly, astaxanthin from yeast is 100 % right-handed (3R-3'R) with partial antioxidant activity, while astaxanthin from algae is 100 % left-handed (3S-3'S) with the strongest biological activity.…”