Parallel Integration and Chromosomal Expansion of Metabolic Pathways

Hillson, Nathan J.; Goyal, Garima; Costello, Zak; Gutierrez, Jorge Alonso; Kang, Aram; Lee, Taek Soon; Martin, Hector Garcia

doi:10.1021/acssynbio.8b00243

Title: Parallel Integration and Chromosomal Expansion of Metabolic Pathways

Abstract

Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, Chemical Induced Chromosomal Expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers, while maintaining stability in the absence of antibiotic selection pressure. Here, we report Parallel Integration and Chromosomal Expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multi-gene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1, and to then generate libraries of pathway component copy-number variants to screen for improved titers. As a result, polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.

Authors:

^[1]; Goyal, Garima ^[1]; Costello, Zak ^[1]; Gutierrez, Jorge Alonso ^[2]; Kang, Aram ^[2]; Lee, Taek Soon ^[2]; Martin, Hector Garcia ^[1]

Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); DOE Agile BioFoundry, 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)

Publication Date:: Tue Oct 23 00:00:00 EDT 2018

Research Org.:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE)

Contributing Org.:: JBEI

OSTI Identifier:: 1478529

Alternate Identifier(s):: OSTI ID: 1482544

Grant/Contract Number:: AC02-05CH11231

Resource Type:: Accepted Manuscript

Journal Name:: ACS Synthetic Biology

Additional Journal Information:: Journal Volume: 7; Journal Issue: 11; Journal ID: ISSN 2161-5063

Publisher:: American Chemical Society (ACS)

Country of Publication:: United States

Language:: English

Subject:: 09 BIOMASS FUELS; metabolic pathway; chromosomal integration; stabilization; copy number; statistical modeling; machine learning; optimization; isopentenol; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats


                    Hillson, Nathan J., Goyal, Garima, Costello, Zak, Gutierrez, Jorge Alonso, Kang, Aram, Lee, Taek Soon, and Martin, Hector Garcia. Parallel Integration and Chromosomal Expansion of Metabolic Pathways.  United States: N. p., 2018. 
Web.  doi:10.1021/acssynbio.8b00243.

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                    Hillson, Nathan J., Goyal, Garima, Costello, Zak, Gutierrez, Jorge Alonso, Kang, Aram, Lee, Taek Soon, & Martin, Hector Garcia. Parallel Integration and Chromosomal Expansion of Metabolic Pathways.  United States.  https://doi.org/10.1021/acssynbio.8b00243

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                    Hillson, Nathan J., Goyal, Garima, Costello, Zak, Gutierrez, Jorge Alonso, Kang, Aram, Lee, Taek Soon, and Martin, Hector Garcia. Tue .  
"Parallel Integration and Chromosomal Expansion of Metabolic Pathways".  United States.  https://doi.org/10.1021/acssynbio.8b00243.  https://www.osti.gov/servlets/purl/1478529.

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@article{osti_1478529,

  title        = {Parallel Integration and Chromosomal Expansion of Metabolic Pathways},

  author       = {Hillson, Nathan J. and Goyal, Garima and Costello, Zak and Gutierrez, Jorge Alonso and Kang, Aram and Lee, Taek Soon and Martin, Hector Garcia},

  abstractNote = {Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, Chemical Induced Chromosomal Expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers, while maintaining stability in the absence of antibiotic selection pressure. Here, we report Parallel Integration and Chromosomal Expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multi-gene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1, and to then generate libraries of pathway component copy-number variants to screen for improved titers. As a result, polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.},

  doi          = {10.1021/acssynbio.8b00243},

  journal      = {ACS Synthetic Biology},

  number       = 11,

  volume       = 7,

  place        = {United States},

  year         = {Tue Oct 23 00:00:00 EDT 2018},

  month        = {Tue Oct 23 00:00:00 EDT 2018}

}

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https://doi.org/10.1021/acssynbio.8b00243

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Cited by: 4 works

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Figures / Tables:

Figure 1: Parallel Integration and Chromosomal Expansion (PIACE) schematic. Three gene cassettes of interest (GOI1, GOI2, and GOI3) are sequentially integrated (one copy each) at corresponding E. coli genomic loci (lacZ, intA, and fdhF) via λ-red-mediated suicide-vector homologous recombination. The introduction of recA via plasmid transformation facilitates the orthogonal copymore »

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