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Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0002-7916-4731
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0001-6501-9886
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0002-3314-6060
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0001-5319-7511
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3 hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization. 

Keywords [en]
3-hydroxybutyrate, Chromosomal expression, Pathway optimization, F plasmid, Bacterial artificial chromosome, Metabolic engineering
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-251090OAI: oai:DiVA.org:kth-251090DiVA, id: diva2:1314516
Note

QC 20190619

Available from: 2019-05-08 Created: 2019-05-08 Last updated: 2019-06-19Bibliographically approved
In thesis
1. Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
Open this publication in new window or tab >>Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microbial bio-based processes have emerged as an alternative to replace fossil-based processes for the production of fuels and chemicals. (R)-3-hydroxybutyrate (3HB) is a medium-value chemical that has gained special attention as a precursor of antibiotics and vitamins, as a monomer for the synthesis of tailor-made polyesters and as a nutritional source for eukaryotic cells. By integrating strain and bioprocess-design strategies the work of this thesis has aimed to improve microbial 3HB production by the well-studied platform organism Escherichia coli (strain AF1000) expressing a thiolase and a reductase from Halomonas boliviensis.

Uncoupling growth and product formation by NH4+- or PO43-- limited fed-batch cultivations allowed for 3HB titers of 4.1 and 6.8 g L-1 (Paper I). Increasing the NADPH supply by overexpression of glucose-6-phosphate dehydrogenase (zwf) resulted in 1.7 times higher 3HB yield compared to not overexpressing zwf in NH4depleted conditions (Paper II). To increase 3HB production in high-cell density cultures, strain BL21 was selected as a low acetate-forming, 3HB-producing platform. BL21 grown in NH4limited fed-batch cultivations resulted in 2.3 times higher 3HB titer (16.3 g L-1) compared to strain AF1000 (Paper III). Overexpression of the native E. coli thioesterase “yciA”, identified as the largest contributor in 3HB-CoA hydrolysis, resulted in 2.6 times higher 3HB yield compared to AF1000 not overexpressing yciA. Overexpressing zwf and yciA in NH4depleted fed-batch experiments resulted in 2 times higher total 3HB yield (0.210 g g-1) compared to AF1000 only overexpressing zwf (Paper IV)Additionally, using 3HB as a model product, the bacterial artificial chromosome was presented as a simple platform for performing pathway design and optimization in E. coli (Paper V)While directly relevant for 3HB production, these findings also contribute to the knowledge on how to improve the production of a chemical for the development of robust and scalable processes.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. p. 100
Series
TRITA-CBH-FOU ; 2019:25
Keywords
E. coli, (R)-3-hydroxybutyrate, metabolic engineering, bioprocess design, NADPH, acetic acid, thioesterase, BAC
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-251096 (URN)978-91-7873-205-0 (ISBN)
Public defence
2019-06-03, F3, Lindstedtsvägen 26, Sing-Sing, våningsplan 2, KTH Campus, Stockholm, 14:00 (English)
Opponent
Supervisors
Funder
Sida - Swedish International Development Cooperation AgencySwedish Research Council Formas
Note

QC 2019-05-09

Available from: 2019-05-09 Created: 2019-05-09 Last updated: 2019-05-09Bibliographically approved

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Sjöberg, GustavGustavsson, Martinvan Maris, Antonius J. A.

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