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
{"title":"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","authors":"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","doi":"10.1016/j.mec.2022.e00206","DOIUrl":null,"url":null,"abstract":"<div><p>In this study, a 14-gene edited <em>Pseudomonas putida</em> 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. <sup>13</sup>C-metabolic flux analysis (<sup>13</sup>C-MFA) revealed that the core <em>P. putida</em> 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 (Δ<em>phaAZC-IID</em>) 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 Δ<em>phaAZC-IID</em> 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.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/15/32/main.PMC9494242.pdf","citationCount":"6","resultStr":"{\"title\":\"Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production\",\"authors\":\"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\",\"doi\":\"10.1016/j.mec.2022.e00206\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>In this study, a 14-gene edited <em>Pseudomonas putida</em> 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. <sup>13</sup>C-metabolic flux analysis (<sup>13</sup>C-MFA) revealed that the core <em>P. putida</em> 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 (Δ<em>phaAZC-IID</em>) 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 Δ<em>phaAZC-IID</em> strain had a 1.5–fold and 1.8–fold increase compared to the optimized Cpf1-RBS construct and the original strain, respectively. 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Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production
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.
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.