Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway
Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway
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DOI：
10.1002/bit.26530
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发表时间：
2018-04-01
影响因子：
3.8
通讯作者：
Lee, Taek S.
中科院分区：
文献类型：
--
作者：
Alonso-Gutierrez, Jorge;Koma, Daisuke;Lee, Taek S.
大肠杆菌已成为通过改造天然和异源途径生产不同化学品的首选生物体。在本研究中，我们同时解决了与大肠杆菌作为类异戊二烯工业平台相关的一些主要问题，包括无法在蔗糖上生长、缺乏对有毒甲羟戊酸（MVA）途径中间体的内源控制以及对染色体的有限途径工程。作为概念证明，我们生成了一种大肠杆菌 DH1 菌株，该菌株能够通过将 cscAKB 操纵子整合到染色体中并在应激反应控制下表达异源 MVA 途径，从蔗糖中生产类异戊二烯红没药烯。当整个途径整合到染色体中时，产量水平相对于质粒介导的表达急剧下降。为了优化染色体整合的MVA途径，我们建立了CRISPR-Cas9系统来快速、系统地替换启动子序列。这一策略导致了更高的途径表达和红没药烯产量的五倍提高。更有趣的是，我们分析了蛋白质组学数据集，以了解和解决与染色体整合途径代谢工程相关的一些挑战。该报告表明，将质粒优化的操纵子整合到基因组中并使其发挥最佳作用并不是一项简单的任务，染色体上任何不良的工程选择都可能导致细胞死亡，而不仅仅是导致低效价。基于这些结果，我们还提出了染色体代谢工程的方向。
Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.