Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic Engineering Communications Pub Date : 2022-12-01 DOI:10.1016/j.mec.2022.e00204
Jacob A. Fenster , Allison Z. Werner , Jian Wei Tay , Matthew Gillen , Leo Schirokauer , Nicholas C. Hill , Audrey Watson , Kelsey J. Ramirez , Christopher W. Johnson , Gregg T. Beckham , Jeffrey C. Cameron , Carrie A. Eckert
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引用次数: 4

Abstract

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

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对香豆酸酯生产β-酮己二酸假单胞菌KT2440的crispr干扰工具集的动态和单细胞特性
恶臭假单胞菌KT2440是一种被充分研究的细菌,用于将木质素衍生的芳香族化合物转化为生物制品。恶臭杆菌先进遗传工具的发展减少了假设检验的周转时间,并使菌株的构建能够产生各种感兴趣的产品。在这里,我们评估了一种可诱导的crispr干扰(CRISPRi)工具集,用于荧光、必需和代谢靶标。用阿拉伯糖(8K)诱导启动子表达的核酸酶缺陷Cas9 (dCas9)在各种培养基条件下和靶向必需基因时受到严格调控。除了大量生长数据外,还进行了单细胞延时显微镜观察,结果显示了在同克隆群体中敲除率的内在异质性。在指数生长的细胞中,研究了基因组靶点的敲低动力学,揭示了诱导后普遍存在1.75±0.38 h的静止期,在观察到表型应答之前发生1.5±0.35倍。为了演示该CRISPRi工具集的应用,我们以4.39±0.5 g/L的产率和0.76±0.10 mol/mol的产率从可从草中提取的羟基肉桂酸对香豆酸中制备了性能优越的尼龙单体β-酮己二酸。这些培养指标是通过在早期指数阶段使用更高强度的IPTG (1K)诱导启动子敲除βKA通路中的pcaIJ操纵子来实现的。这使得大部分碳被分流到所需的产品中,同时消除了补充碳和能源来支持生长和维持的需要。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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