Bacterial Production of Pinene by a Laboratory-Evolved Pinene-Synthase
Bacterial Production of Pinene by a Laboratory-Evolved Pinene-Synthase
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DOI：
10.1021/acssynbio.6b00140
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发表时间：
2016-09-01
影响因子：
4.7
通讯作者：
Umeno, Daisuke
中科院分区：
文献类型：
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作者：
Tashiro, Miki;Kiyota, Hiroshi;Umeno, Daisuke
成功地将底物香叶酰焦磷酸(GPP)提供给单萜烯合成酶是微生物高效生产单萜烯的关键。GPP合酶的过度表达、从GPP合酶到萜烯合酶的代谢通道以及内源竞争对手的下调已成功地用于增加单萜的产量。然而，单萜类化合物的产量仍然大大低于半/倍半萜类化合物。我们测试了它是否有效地提高了单萜合成酶的细胞活性。为此，我们开发了一种高通量筛查系统来监测GPP消耗的增加。通过单轮诱变和筛选，我们在大肠杆菌和蓝藻中分离到了一个在多种环境中表现优于野生型(亲本)酶的Pinene合成酶变体。纯化的突变体对金属的依赖性发生了很大的变化，使其能够在缺乏锰的胞浆中保持活性。在摇瓶培养中，该变异体与甲戊酸途径酶、异戊烯焦磷酸异构酶和GPP合成酶共表达，产量为140 mg/L。
Successful feeding of the substrate geranylpyrophosphate (GPP) to monoterpene synthase is critical to the efficient microbial production of monoterpenes. Overexpression of GPP synthases, metabolic channeling from GPP synthase to terpene synthases, and down-tuning of endogenous competitors have been successfully used to increase the production of monoterpene. Nevertheless, the production of monoterpenes has remained considerably lower than that of hemi-/sesqui-terpenoids. We tested whether it is effective to improve the cellular activity of monoterpene synthases. To this end, we developed a high-throughput screening system to monitor for elevated GPP consumption. Through a single round of mutagenesis and screening, we isolated a pinene synthase variant that outperformed the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibited drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenylpyrophosphate isomerase, and GPP synthase yielded 140 mg/L pinene in a flask culture.