Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.ORCID-id: 0000-0003-3873-4977
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.ORCID-id: 0000-0002-7916-4731
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. Universidad Mayor de San Simón, Bolivia.ORCID-id: 0000-0001-6501-9886
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi. (Industrial Biotechnology)
Vise andre og tillknytning
2016 (engelsk)Inngår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, nr 1, artikkel-id 91Artikkel i tidsskrift (Fagfellevurdert) Published
Resurstyp
Text
Abstract [en]

Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.

sted, utgiver, år, opplag, sider
Springer, 2016. Vol. 15, nr 1, artikkel-id 91
Emneord [en]
Escherichia coli, Halomonas boliviensis, (R)-3-hydroxybutyrate, Acetoacetyl-CoA reductase, NADPH, zwf overexpression, Glutamate, Nitrogen limitation
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-189084DOI: 10.1186/s12934-016-0490-yISI: 000377167900001PubMedID: 27245326Scopus ID: 2-s2.0-84971577878OAI: oai:DiVA.org:kth-189084DiVA, id: diva2:951344
Merknad

QC 20160808

Tilgjengelig fra: 2016-08-08 Laget: 2016-06-27 Sist oppdatert: 2019-06-14bibliografisk kontrollert
Inngår i avhandling
1. Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
Åpne denne publikasjonen i ny fane eller vindu >>Metabolic engineering and cultivation strategies for recombinant production of (R)-3-hydroxybutyrate
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
KTH Royal Institute of Technology, 2019. s. 106
Serie
TRITA-CBH-FOU ; 2019:20
Emneord
Escherichia coli, (R)-3-hydroxybutyrate, nitrogen limitation, nitrogen depletion, lignocellulose, fed batch, acetate, β-ketothiolase, acetoacetyl-CoA reductase, Halomonas boliviensis.
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-251048 (URN)978-91-7873-216-6 (ISBN)
Disputas
2019-06-05, FD5, AlbaNova, Roslagstullsbacken 21, SE-11421, Stockholm, Sweden, Stockholm, 14:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Sida - Swedish International Development Cooperation Agency, 70828
Merknad

QC 2019-05-08

Tilgjengelig fra: 2019-05-08 Laget: 2019-05-08 Sist oppdatert: 2019-05-09bibliografisk kontrollert
2. Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
Åpne denne publikasjonen i ny fane eller vindu >>Strain- and bioprocess-design strategies to increase production of (R)-3-hydroxybutyrate by Escherichia coli
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2019. s. 100
Serie
TRITA-CBH-FOU ; 2019:25
Emneord
E. coli, (R)-3-hydroxybutyrate, metabolic engineering, bioprocess design, NADPH, acetic acid, thioesterase, BAC
HSV kategori
Forskningsprogram
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-251096 (URN)978-91-7873-205-0 (ISBN)
Disputas
2019-06-03, F3, Lindstedtsvägen 26, Sing-Sing, våningsplan 2, KTH Campus, Stockholm, 14:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Sida - Swedish International Development Cooperation AgencySwedish Research Council Formas
Merknad

QC 2019-05-09

Tilgjengelig fra: 2019-05-09 Laget: 2019-05-09 Sist oppdatert: 2019-05-09bibliografisk kontrollert

Open Access i DiVA

Fulltekst mangler i DiVA

Andre lenker

Forlagets fulltekstPubMedScopus

Personposter BETA

Perez-Zabaleta, MarielGuevara-Martínez, MónicaGustavsson, Martin

Søk i DiVA

Av forfatter/redaktør
Perez-Zabaleta, MarielSjöberg, GustavGuevara-Martínez, MónicaJarmander, JohanGustavsson, MartinLarsson, Gen
Av organisasjonen
I samme tidsskrift
Microbial Cell Factories

Søk utenfor DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric

doi
pubmed
urn-nbn
Totalt: 118 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf