An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus
Abstract
Rhodococcus opacus is a non-model bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in non-model organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (i.e., the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool’s utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained usingmore »
- Publication Date:
- Research Org.:
- Washington Univ., St. Louis, MO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1773341
- Grant/Contract Number:
- SC0018324
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ACS Synthetic Biology
- Additional Journal Information:
- Journal Volume: 10; Journal Issue: 4; Journal ID: ISSN 2161-5063
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; CRISPR interference; non-model organism; metabolic pathway elucidation; muconate; metabolic engineering
Citation Formats
DeLorenzo, Drew M., Diao, Jinjin, Carr, Rhiannon, Hu, Yifeng, and Moon, Tae Seok. An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus. United States: N. p., 2021.
Web. doi:10.1021/acssynbio.0c00591.
DeLorenzo, Drew M., Diao, Jinjin, Carr, Rhiannon, Hu, Yifeng, & Moon, Tae Seok. An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus. United States. https://doi.org/10.1021/acssynbio.0c00591
DeLorenzo, Drew M., Diao, Jinjin, Carr, Rhiannon, Hu, Yifeng, and Moon, Tae Seok. Wed .
"An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus". United States. https://doi.org/10.1021/acssynbio.0c00591. https://www.osti.gov/servlets/purl/1773341.
@article{osti_1773341,
title = {An Improved CRISPR Interference Tool to Engineer Rhodococcus opacus},
author = {DeLorenzo, Drew M. and Diao, Jinjin and Carr, Rhiannon and Hu, Yifeng and Moon, Tae Seok},
abstractNote = {Rhodococcus opacus is a non-model bacterium that is well suited for valorizing lignin. Despite recent advances in our systems-level understanding of its versatile metabolism, studies of its gene functions at a single gene level are still lagging. Elucidating gene functions in non-model organisms is challenging due to limited genetic engineering tools that are convenient to use. To address this issue, we developed a simple gene repression system based on CRISPR interference (CRISPRi). This gene repression system uses a T7 RNA polymerase system to express a small guide RNA, demonstrating improved repression compared to the previously demonstrated CRISPRi system (i.e., the maximum repression efficiency improved from 58% to 85%). Additionally, our cloning strategy allows for building multiple CRISPRi plasmids in parallel without any PCR step, facilitating the engineering of this GC-rich organism. Using the improved CRISPRi system, we confirmed the annotated roles of four metabolic pathway genes, which had been identified by our previous transcriptomic analysis to be related to the consumption of benzoate, vanillate, catechol, and acetate. Furthermore, we showed our tool’s utility by demonstrating the inducible accumulation of muconate that is a precursor of adipic acid, an important monomer for nylon production. While the maximum muconate yield obtained using our tool was 30% of the yield obtained using gene knockout, our tool showed its inducibility and partial repressibility. In conclusion, our CRISPRi tool will be useful to facilitate functional studies of this non-model organism and engineer this promising microbial chassis for lignin valorization.},
doi = {10.1021/acssynbio.0c00591},
journal = {ACS Synthetic Biology},
number = 4,
volume = 10,
place = {United States},
year = {Wed Mar 31 00:00:00 EDT 2021},
month = {Wed Mar 31 00:00:00 EDT 2021}
}
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