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α-Humulene is a natural sesquiterpenoid with multiple pharmacological activities including anti-inflammatory, antibacterial and antitumor effects, and also serves as the key precursor for zerumbone biosynthesis. In this study, Saccharomyces cerevisiae was used as the chassis for heterologous production of α-humulene. One novel α-humulene synthase gene, AsTPS8, was mined from the transcriptome of Aquilaria sinensis. Compared with the reported ZSS1 from Zingiber zerumbet, AsTPS8 exhibited higher catalytic efficiency. Furthermore, fusion expression of ERG20 with AsTPS8 via a (GGGS)2 linker combined with multi-copy integration at the TY1 locus increased the α-humulene titer to 1.41 g·L-1 in shake flasks. On this basis, the bypass phosphatase gene LPP1 was knocked out using CRISPR/Cas9, and the transcriptional regulators IME4 and UPC2-1 were simultaneously introduced, further elevating the titer to 1.80 g·L-1. Finally, fed-batch high-density fermentation in a 5 L bioreactor achieved an α-humulene titer of 22.02 g·L-1 at 144 h, representing the highest reported level to date. This study provides an efficient chassis strain and technical support for the industrial production of α-humulene and downstream zerumbone.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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