Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway
Abstract
Abstract 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 notmore »
- Authors:
-
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Osaka Municipal Technical Research Inst. (Japan)
- Univ. of Queensland, St. Lucia, QLD (Australia)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technical Univ. of Denmark, Horsholm (Denmark); Univ. of California, Berkeley, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1530349
- Alternate Identifier(s):
- OSTI ID: 1419808
- Grant/Contract Number:
- AC02-05CH11231; DE‐AC02‐05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Biotechnology and Bioengineering
- Additional Journal Information:
- Journal Volume: 115; Journal Issue: 4; Journal ID: ISSN 0006-3592
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
Alonso‐Gutierrez, Jorge, Koma, Daisuke, Hu, Qijun, Yang, Yuchen, Chan, Leanne J. G., Petzold, Christopher J., Adams, Paul D., Vickers, Claudia E., Nielsen, Lars K., Keasling, Jay D., and Lee, Taek S. Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway. United States: N. p., 2017.
Web. doi:10.1002/bit.26530.
Alonso‐Gutierrez, Jorge, Koma, Daisuke, Hu, Qijun, Yang, Yuchen, Chan, Leanne J. G., Petzold, Christopher J., Adams, Paul D., Vickers, Claudia E., Nielsen, Lars K., Keasling, Jay D., & Lee, Taek S. Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway. United States. https://doi.org/10.1002/bit.26530
Alonso‐Gutierrez, Jorge, Koma, Daisuke, Hu, Qijun, Yang, Yuchen, Chan, Leanne J. G., Petzold, Christopher J., Adams, Paul D., Vickers, Claudia E., Nielsen, Lars K., Keasling, Jay D., and Lee, Taek S. Tue .
"Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway". United States. https://doi.org/10.1002/bit.26530. https://www.osti.gov/servlets/purl/1530349.
@article{osti_1530349,
title = {Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR-Cas9 based optimization of a chromosomally integrated mevalonate pathway},
author = {Alonso‐Gutierrez, Jorge and Koma, Daisuke and Hu, Qijun and Yang, Yuchen and Chan, Leanne J. G. and Petzold, Christopher J. and Adams, Paul D. and Vickers, Claudia E. and Nielsen, Lars K. and Keasling, Jay D. and Lee, Taek S.},
abstractNote = {Abstract 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.},
doi = {10.1002/bit.26530},
journal = {Biotechnology and Bioengineering},
number = 4,
volume = 115,
place = {United States},
year = {Tue Dec 26 00:00:00 EST 2017},
month = {Tue Dec 26 00:00:00 EST 2017}
}
Web of Science
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