调控酿酒酵母类异戊二烯合成途径强化芳樟醇合成

Regulation of isoprenoid pathway for enhanced production of linalool in Saccharomyces cerevisiae

摘要:

芳樟醇是一种重要单萜，广泛应用于食品、医药、日化等工业领域。然而芳樟醇在植物中含量低且难提取，限制了其大规模生产。目前通常以酿酒酵母Saccharomyces cerevisiae作为单萜生物合成宿主，其内源类异戊二烯合成途径提供合成单萜物质的前体——香叶基二磷酸 (GPP)。由于该途径代谢通量较低，导致GPP供应不足，极大地降低了异源单萜的合成效率。为了调节该途径的代谢通量，构建酿酒酵母整合表达载体pRS305-tHMG1和游离表达载体pYLIS-IDI1，并分别转入酿酒酵母CEN.PK2-1C中，获得酿酒酵母工程菌LS01和LS02。同时将载体pYLIS-IDI1转入酿酒酵母工程菌LS01中，构建酿酒酵母工程菌LS03。GC-MS检测结果显示，通过提高异戊二烯二磷酸异构酶 (IDI1) 和羟甲基戊二酸单酰辅酶A (HMG-CoA) 还原酶活性区域 (tHMG1) 的表达水平，最终使芳樟醇产量提高1.3倍至 (127.71±7.68) mg/L。结果表明，通过调控类异戊二烯合成途径，强化GPP合成前体供给，可以显著提高酿酒酵母中芳樟醇的产量。

Abstract:

Linalool is an important monoterpene, and widely used in food, pharmaceutical and cosmetic industry. The low concentration in plants and the difficulties in extraction restrict its large scale production. Saccharomyces cerevisiae can provide the monoterpene precursor, geranyl diphosphate (GPP) through its endogenous isoprenoid pathway. Therefore, it could be used as the host for monoterpene production. However, the weak metabolic flux through the isoprenoid pathway leads to the insufficient supply of GPP, and results in low monoterpene productivity. In order to increase the metabolic flux, we constructed the integrated expression plasmid pRS305-tHMG1 and free expression plasmid pYLIS-IDI1 to enhance the expression levels of isopentenyl diphosphate isomerase (IDI1) and a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene (tHMG1). The two plasmids were separately transformed into S. cerevisiae CEN.PK2-1C, resulting in strains LS01 and LS02. The plasmid pYLIS-IDI1 was further transformed into strain LS01, resulting in strain LS03. GC-MS analysis showed that the linalool concentration was increased by 1.3 times and reached (127.71±7.68) mg/L. In conclusion, enhancement of the supply of GPP precursors through the regulation of isoprenoid pathway could increase the linalool production in S. cerevisiae.