Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden.
Metab Eng. 2012 Mar;14(2):91-103. doi: 10.1016/j.ymben.2012.01.007. Epub 2012 Feb 8.
Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.
通过合理设计的代谢工程方法，首次构建了能够高效生产植物倍半萜烯的酿酒酵母菌株，这使得这些化合物的可持续和规模化生产成为可能，这些化合物可用于香水和制药等领域。这里，首次构建了一种能够高效生产α-檀香烯（一种具有商业价值的化合物前体）的酿酒酵母菌株，该菌株通过合理设计的代谢工程方法实现。通过调节甲羟戊酸（MVA）途径中关键代谢步骤之一——鲨烯合酶（Erg9）的表达，实现了最优的倍半萜烯生产。为了将 ERG9 的表达与葡萄糖浓度偶联，用 HXT1 启动子替换了其启动子。在第二种方法中，使用 HXT2 启动子表达 ERG9 反义构建体。使用 HXT1 启动子控制 ERG9 的表达，有可能将碳通量从固醇合成转向α-檀香烯，从而将生产力提高 3.4 倍。将这种方法与 3-羟甲基戊二酰辅酶 A 还原酶（HMGR）截断形式的过表达以及 LPP1 编码的脂质磷酸酶的缺失相结合，导致产生了一种生产力为 0.18mg/gDCWh 的菌株。通过删除编码第二种消耗 FPP 的焦磷酸磷酸酶的 DPP1，进一步提高了滴度，最终生产力和α-檀香烯的滴度分别为 0.21mg/gDCWh 和 92mg/L。
