Temperature-dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli.
Temperature-dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli.
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DOI：
10.1002/bit.26446
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发表时间：
2018-01
影响因子：
3.8
通讯作者：
Klamt S
中科院分区：
文献类型：
--
作者：
Harder BJ;Bettenbrock K;Klamt S
基于最近构建的大肠杆菌衣康酸生产菌株ita 23，我们旨在通过应用生物质和衣康酸解耦生产的两阶段工艺策略来提高生产率。我们构建了菌株ita 32（MG 1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs），与ita 23相反，它具有活性三羧酸（TCA）循环和在37°C下0.52 hr−1的快速生长速率，因此代表了第一阶段（生长期）的理想表型。随后，我们实施了一种合成遗传控制，允许下调TCA循环，从而在第二阶段从生长切换到衣康酸生产。异柠檬酸脱氢酶的启动子被λ启动子（PR）取代，其表达由温度敏感性阻遏物CI 857控制，该阻遏物在较低温度（30°C）下有活性。以葡萄糖为底物，相应菌株ita 36 A在37°C下生长速度快，并在28°C下转向生产衣康酸。为了研究工艺策略对生产率的影响，我们进行了一阶段和两阶段生物反应器培养。与一阶段工艺相比，两阶段工艺能够快速形成生物质，从而提高峰值生产率0.86 g/L/hr（+48%）和体积生产率0.39 g/L/hr（+22%）。利用我们的动态生产菌株，我们还解决了ita 23的谷氨酸营养缺陷型，并将衣康酸滴度提高到47 g/L。通过λ启动子（p R/p L）的基因表达的温度依赖性激活经常用于改善蛋白质或在少数情况下改善两阶段过程中的代谢物产生。在这里，我们证明了该系统也可以用于相反的方向，选择性地敲低E.大肠杆菌设计一个两阶段的过程，以提高体积生产率。通过温度控制避免了昂贵的诱导剂，并且具有普遍用于改善细胞工厂性能的潜力。
Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two‐stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr−1 at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage. The promoter of the isocitrate dehydrogenase was replaced by the Lambda promoter (p R) and its expression was controlled by the temperature‐sensitive repressor CI857 which is active at lower temperatures (30°C). With glucose as substrate, the respective strain ita36A grew with a fast growth rate at 37°C and switched to production of itaconic acid at 28°C. To study the impact of the process strategy on productivity, we performed one‐stage and two‐stage bioreactor cultivations. The two‐stage process enabled fast formation of biomass resulting in improved peak productivity of 0.86 g/L/hr (+48%) and volumetric productivity of 0.39 g/L/hr (+22%) in comparison to the one‐stage process. With our dynamic production strain, we also resolved the glutamate auxotrophy of ita23 and increased the itaconic acid titer to 47 g/L. The temperature‐dependent activation of gene expression by the Lambda promoters (p R/p L) has been frequently used to improve protein or, in a few cases, metabolite production in two‐stage processes. Here we demonstrate that the system can be as well used in the opposite direction to selectively knock‐down an essential gene (icd) in E. coli to design a two‐stage process for improved volumetric productivity. The control by temperature avoids expensive inducers and has the potential to be generally used to improve cell factory performance.
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影响因子：
3.7
作者：
Steinsiek S;Stagge S;Bettenbrock K
通讯作者：
Bettenbrock K
影响因子：
9.9
作者：
Orth, Jeffrey D.;Conrad, Tom M.;Na, Jessica;Lerman, Joshua A.;Nam, Hojung;Feist, Adam M.;Palsson, Bernhard O.
通讯作者：
Palsson, Bernhard O.
影响因子：
6.4
作者：
Valdez-Cruz NA;Caspeta L;Pérez NO;Ramírez OT;Trujillo-Roldán MA
通讯作者：
Trujillo-Roldán MA
影响因子：
8.4
作者：
Zhou, Li;Niu, Dan-Dan;Wang, Zheng-Xiang
通讯作者：
Wang, Zheng-Xiang
影响因子：
8.4
作者：
Harder, Bjoern-Johannes;Bettenbrock, Katja;Klamt, Steffen
通讯作者：
Klamt, Steffen