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Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
KTH, School of Biotechnology (BIO), Industrial Biotechnology. Univ Mayor de San Simon, Fac Sci & Technol, Ctr Biotechnol.ORCID iD: 0000-0001-6501-9886
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0002-7916-4731
KTH, School of Biotechnology (BIO), Industrial Biotechnology.
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2015 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 844Article in journal (Refereed) Published
Abstract [en]

The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coil strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetylCoA to 3HB also in E coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L-1 h(-1), was seen for phosphate-limitation.

Place, publisher, year, edition, pages
Frontiers Research Foundation , 2015. Vol. 6, article id 844
Keywords [en]
E. coil, 3-hydroxybutyrate (3HB), polyhydroxybutyrate (PHB), fed-batch, phosphate, ammonium, limitation, depletion
National Category
Microbiology
Identifiers
URN: urn:nbn:se:kth:diva-173438DOI: 10.3389/fmicb.2015.00844ISI: 000360116800001Scopus ID: 2-s2.0-84941053409OAI: oai:DiVA.org:kth-173438DiVA, id: diva2:855139
Funder
Swedish Research Council FormasSida - Swedish International Development Cooperation Agency
Note

QC 20150918

Available from: 2015-09-18 Created: 2015-09-11 Last updated: 2019-06-11Bibliographically approved
In thesis
1. Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
Open this publication in new window or tab >>Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Metabolic engineering and process engineering are two powerful disciplines to design and improve microbial processes for sustainable production of an extensive number of compounds ranging from chemicals to pharmaceuticals. The aim of this thesis was to synergistically combine these two disciplines to improve the production of a model chemical called (R)-3-hydroxybutyrate (3HB), which is a medium-value product with a stereocenter and two functional groups. These features make 3HB an interesting building block, especially for the pharmaceutical industry. Recombinant production of 3HB was achieved by expression of two enzymes from Halomonas boliviensis in the model microorganism Escherichia coli, which is a microbial cell factory with proven track record and abundant knowledge on its genome, metabolism and physiology.

Investigations on cultivation strategies demonstrated that nitrogen-depleted conditions had the biggest impact on 3HB yields, while nitrogen-limited cultivations predominantly increased 3HB titers and volumetric productivities. To further increase 3HB production, metabolic engineering strategies were investigated to decrease byproduct formation, enhance NADPH availability and improve the overall 3HB-pathway activity. Overexpression of glucose-6-phosphate dehydrogenase (zwf) increased cofactor availability and together with the overexpression of acyl-CoA thioesterase YciA resulted in a 2.7-fold increase of the final 3HB concentration, 52% of the theoretical product yield and a high specific productivity (0.27 g g-1 h-1). In a parallel strategy, metabolic engineering and process design resulted in an E. coli BL21 strain with the hitherto highest reported volumetric 3HB productivity (1.52 g L-1 h-1) and concentration (16.3 g L-1) using recombinant production. The concepts developed in this thesis can be applied to industrial 3HB production processes, but also advance the knowledge base to benefit design and expansion of the product range of biorefineries.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 106
Series
TRITA-CBH-FOU ; 2019:20
Keywords
Escherichia coli, (R)-3-hydroxybutyrate, nitrogen limitation, nitrogen depletion, lignocellulose, fed batch, acetate, β-ketothiolase, acetoacetyl-CoA reductase, Halomonas boliviensis.
National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-251048 (URN)978-91-7873-216-6 (ISBN)
Public defence
2019-06-05, FD5, AlbaNova, Roslagstullsbacken 21, SE-11421, Stockholm, Sweden, Stockholm, 14:00 (English)
Opponent
Supervisors
Funder
Sida - Swedish International Development Cooperation Agency, 70828
Note

QC 2019-05-08

Available from: 2019-05-08 Created: 2019-05-08 Last updated: 2019-05-09Bibliographically approved
2. 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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Guevara-Martínez, MónicaPerez-Zabaleta, MarielLarsson, Gen

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