Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Jeffrey J. Czajka , Deepanwita Banerjee , Thomas Eng , Javier Menasalvas , Chunsheng Yan , Nathalie Munoz Munoz , Brenton C. Poirier , Young-Mo Kim , Scott E. Baker , Yinjie J. Tang , Aindrila Mukhopadhyay
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引用次数: 6

Abstract

In this study, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using three distinct omic datasets. Transcriptomic data indicated that CRISPR/dCpf1-interference (CRISPRi) mediated multiplex repression caused global gene expression changes, implying potential undesirable changes in metabolic flux. 13C-metabolic flux analysis (13C-MFA) revealed that the core P. putida flux network after CRISPRi repression was conserved, with moderate reduction of TCA cycle and pyruvate shunt activity along with glyoxylate shunt activation during glucose catabolism. Metabolomic results identified a change in intracellular TCA metabolites and extracellular metabolite secretion profiles (sugars and succinate overflow) in the engineered strains. These omic analyses guided further strain engineering, with a random mutagenesis screen first identifying an optimal ribosome binding site (RBS) for Cpf1 that enabled stronger product-substrate pairing (1.6–fold increase). Then, deletion strains were constructed with excision of the PHA operon (ΔphaAZC-IID) resulting in a 2.2–fold increase in indigoidine titer over the optimized Cpf1-RBS construct at the end of the growth phase (∼6 h). The maximum indigoidine titer (at 72 h) in the ΔphaAZC-IID strain had a 1.5–fold and 1.8–fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. Overall, this study demonstrated that integration of omic data types is essential for understanding responses to complex metabolic engineering designs and directly quantified the effect of such modifications on central metabolism.

调整一个高性能的多重crispri恶臭假单胞菌菌株,进一步提高靛蓝苷的产量
在这项研究中,使用三个不同的组学数据集对一株14个基因编辑的恶臭假单胞菌KT2440菌株进行了异种靛蓝素生产的检测。转录组学数据表明,CRISPR/ dcpf1干扰(CRISPRi)介导的多重抑制导致了全球基因表达的变化,这意味着代谢通量可能发生不良变化。13c -代谢通量分析(13C-MFA)显示,CRISPRi抑制后的核心P. putida通量网络是保守的,在葡萄糖分解代谢过程中,TCA循环和丙酮酸分流活性以及glyoxylate分流活性适度降低。代谢组学结果确定了工程菌株细胞内TCA代谢物和细胞外代谢物分泌谱(糖和琥珀酸溢出)的变化。这些组学分析指导了进一步的菌株工程,随机突变筛选首先确定Cpf1的最佳核糖体结合位点(RBS),使产物-底物配对更强(增加1.6倍)。然后,通过去除PHA操纵子(ΔphaAZC-IID)构建缺失菌株,在生长阶段结束时(~ 6 h),其靛蓝素滴度比优化的Cpf1-RBS结构提高了2.2倍。与优化的Cpf1-RBS结构和原始菌株相比,ΔphaAZC-IID菌株的最大靛蓝素滴度(72 h)分别提高了1.5倍和1.8倍。总体而言,本研究表明,整合组学数据类型对于理解对复杂代谢工程设计的反应至关重要,并可直接量化此类修饰对中枢代谢的影响。
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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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