Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning.
Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning.
复制标题
通过 T7 启动子调整，将工程化三酮化合物和四酮化合物合酶的滴度提高至创纪录水平。
DOI：
10.1016/j.ymben.2023.05.008
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发表时间：
2023
影响因子：
8.4
通讯作者：
Keatinge-Clay,AdrianT
中科院分区：
文献类型：
--
作者：
Zhang,Jie;Bista,Ramesh;Miyazawa,Takeshi;Keatinge-Clay,AdrianT
模块化聚酮合成酶（PKS）是设计聚酮和商品化学品的合理工程平台，但其低生产率是这些化合物实际生物合成的障碍。之前，我们使用最近更新的模块定义设计了三酮内酯合成酶，如Pik167，并显示它们每升大肠杆菌207-3摇瓶培养物产生数百毫克的产物。由于Pik167的两个多肽的摩尔比是高度偏斜的，我们试图减弱T7启动子控制更小、表达更好的多肽的产生的强度，从而在未优化的T7启动子的控制下增加第一个多肽的产生。通过这种策略，获得了1.8倍的提高滴度。通过将培养基中的丙酸浓度从20 mM增加到80 mM，进一步提高1.5倍后，达到了791 mg L−1（分离的627 mg L−1）的创纪录滴度，总体提高了2.6倍。在这一结果的刺激下，对四肽合成酶Pik1567进行了工程设计，并将T7启动子衰减策略应用于其第二和第三个基因。四肽产物的滴度从20 mg L−1提高到100 mg L−1，提高了5倍。
Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities are a barrier to the practical biosynthesis of these compounds. Previously, we engineered triketide lactone synthases such as Pik167 using the recently updated module definition and showed they generate hundreds of milligrams of product per liter ofEscherichia coliK207-3 shake flask culture. As the molar ratio between the 2 polypeptides of Pik167 is highly skewed, we sought to attenuate the strength of the T7 promoter controlling the production of the smaller, better-expressing polypeptide and thereby increase production of the first polypeptide under the control of an unoptimized T7 promoter. Through this strategy, a 1.8-fold boost in titer was obtained. After a further 1.5-fold boost obtained by increasing the propionate concentration in the media from 20 to 80 mM, a record titer of 791 mg L−1(627 mg L−1isolated) was achieved, a 2.6-fold increase overall. Spurred on by this result, the tetraketide synthase Pik1567 was engineered and the T7 promoter attenuation strategy was applied to its second and third genes. A 5-fold boost, from 20 mg L−1to 100 mg L−1, in the titer of its tetraketide product was achieved.