Acceleration of target production in co‐culture by enhancing intermediate consumption through adaptive laboratory evolution
Acceleration of target production in co‐culture by enhancing intermediate consumption through adaptive laboratory evolution
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DOI：
10.1002/bit.28007
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发表时间：
2021-12
影响因子：
3.8
通讯作者：
Ryutaro Kawai;Yoshihiro Toya;K. Miyoshi;Manami Murakami;Teppei Niide;Takaaki Horinouchi;Tomoya Maeda;Atsushi Shibai;C. Furusawa;H. Shimizu
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作者：
Ryutaro Kawai;Yoshihiro Toya;K. Miyoshi;Manami Murakami;Teppei Niide;Takaaki Horinouchi;Tomoya Maeda;Atsushi Shibai;C. Furusawa;H. Shimizu
共培养是通过将代谢途径分成几个模块并与多个菌株共享转化过程来减轻代谢负担的一种有前途的方法。由于中间体通过细胞外环境从供体传递到受体，因此不可避免地被稀释。因此，提高中间消耗率对于提高目标生产率非常重要。在本研究中，我们证明了通过适应性实验室进化提高大肠杆菌中甲羟戊酸的消耗，并将进化的菌株应用于E. coli（上游：葡萄糖至甲羟戊酸）-E.大肠杆菌（下游：甲羟戊酸转化为异戊二烯醇）共培养。将工程化的甲羟戊酸营养缺陷型菌株在补充有甲羟戊酸的合成培养基中重复传代培养，其中甲羟戊酸浓度从100 μ M逐步降低至20 μM。在五个平行进化实验中，所有生长速率逐渐增加，导致五个进化菌株。全基因组重新测序和反向工程确定了三个参与增强甲羟戊酸消耗的突变。在引入用于生产异戊二烯醇的nudF基因后，将异戊二烯醇生产亲本和进化菌株分别与甲羟戊酸生产菌株共培养。在1：3的接种比例（上游：下游），异戊烯醇生产使用的进化菌株是3.3倍，高于使用亲本菌株。
Co‐culture is a promising way to alleviate metabolic burden by dividing the metabolic pathways into several modules and sharing the conversion processes with multiple strains. Since an intermediate is passed from the donor to the recipient via the extracellular environment, it is inevitably diluted. Therefore, enhancing the intermediate consumption rate is important for increasing target productivity. In the present study, we demonstrated the enhancement of mevalonate consumption in Escherichia coli by adaptive laboratory evolution and applied the evolved strain to isoprenol production in an E. coli (upstream: glucose to mevalonate)–E. coli (downstream: mevalonate to isoprenol) co‐culture. An engineered mevalonate auxotroph strain was repeatedly sub‐cultured in a synthetic medium supplemented with mevalonate, where the mevalonate concentration was decreased stepwise from 100 to 20 µM. In five parallel evolution experiments, all growth rates gradually increased, resulting in five evolved strains. Whole‐genome re‐sequencing and reverse engineering identified three mutations involved in enhancing mevalonate consumption. After introducing nudF gene for producing isoprenol, the isoprenol‐producing parental and evolved strains were respectively co‐cultured with a mevalonate‐producing strain. At an inoculation ratio of 1:3 (upstream:downstream), isoprenol production using the evolved strain was 3.3 times higher than that using the parental strain.