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Because it is an outstanding antioxidant with wide applications, biotechnological production of astaxanthin has attracted increasing research interest. However, the astaxanthin titer achieved to date is still rather low, attributed to the poor efficiency of β-carotene ketolation and hydroxylation, as well as the adverse effect of astaxanthin accumulation on cell growth.To address these problems, we constructed an efficient astaxanthin-producing Saccharomyces cerevisiae strain by combining protein engineering and dynamic metabolic regulation. First, superior mutants of β-carotene ketolase and β-carotene hydroxylase were obtained by directed coevolution to accelerate the conversion of β-carotene to astaxanthin. Subsequently, the Gal4M9-based temperature-responsive regulation system was introduced to separate astaxanthin production from cell growth. Finally, 235 mg/L of (3S,3′S)-astaxanthin was produced by two-stage, high-density fermentation. This study demonstrates the power of combining directed coevolution and temperature-responsive regulation in astaxanthin biosynthesis and may provide methodological reference for biotechnological production of other value-added chemicals.

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Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growthproduction conflict and intra-pathway competition were simultaneously resolved.Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.

The vitamin E component δ-tocotrienol has shown impressive activities in radioprotection, neuroprotection, and cholesterol reduction. Its production is limited by the low content in plants and difficulty in separation from other tocotrienols. Fermentative production using a microbial cell factory that exclusively produces and secretes δ-tocotrienol is a promising alternative approach. Assembly of the δ-tocotrienol synthetic pathway in Saccharomyces cerevisiae followed by comprehensive pathway engineering led to the production of 73.45 mg/L δ-tocotrienol. Subsequent addition of 2-hydroxypropyl-β-cyclodextrin (CD) and overexpression of the transcription factor PDR1 significantly elevated δ-tocotrienol titer to 241.7 mg/L (63.65 mg/g dry cell weight) in shake flasks, with 30.4% secreted. By properly adding CD and the in situ extractant olive oil, 181.12 mg/L of δ-tocotrienol was collected as an extracellular product, accounting for 85.6% of the total δ-tocotrienol production. This process provides not only a promising δ-tocotrienol cell factory but also insights into yeast engineering toward secretory production of other terpenoids.