Parallel Integration and Chromosomal Expansion of Metabolic Pathways
[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)
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.
- Research Organization:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Energy Efficiency and Renewable Energy (EERE)
- Contributing Organization:
- JBEI
- Grant/Contract Number:
- AC02-05CH11231
- OSTI ID:
- 1478529
- Alternate ID(s):
- OSTI ID: 1482544
- Journal Information:
- ACS Synthetic Biology, Vol. 7, Issue 11; ISSN 2161-5063
- Publisher:
- American Chemical Society (ACS)Copyright Statement
- Country of Publication:
- United States
- Language:
- English
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