Change search
Link to record
Permanent link

Direct link
BETA
Perez-Zabaleta, MarielORCID iD iconorcid.org/0000-0003-3873-4977
Publications (5 of 5) Show all publications
Perez-Zabaleta, M., Guevara-Martínez, M., Gustavsson, M., Quillaguamán, J., Larsson, G. & van Maris, A. J. A. (2019). Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation. Applied Microbiology and Biotechnology, 103(14), 5627-5636
Open this publication in new window or tab >>Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
Show others...
2019 (English)In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 103, no 14, p. 5627-5636Article in journal (Refereed) Accepted
Abstract [en]

Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by E. coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) Deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB) and/or the isocitrate-lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110 and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate forming background. Despite low 3HB titers in low-cell density screening, 3HB-producing BL21 produced 5 times less acetic acid per mol of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.

Place, publisher, year, edition, pages
Springer, 2019
Keywords
Escherichia coli, (R)-3-hydroxybutyrate, acetate, nitrogen limitation, fed batch, BL21.
National Category
Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-251046 (URN)10.1007/s00253-019-09876-y (DOI)000473129900012 ()2-s2.0-85066078742 (Scopus ID)
Funder
Sida - Swedish International Development Cooperation Agency, 70828
Note

QC 20190508

Available from: 2019-05-08 Created: 2019-05-08 Last updated: 2019-08-15Bibliographically approved
Guevara-Martínez, M., Perez-Zabaleta, M., Gustavsson, M., Quillaguamán, J., Larsson, G. & van Maris, A. J. A. (2019). The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli. Applied Microbiology and Biotechnology, 1-12
Open this publication in new window or tab >>The role of the acyl-CoA thioesterase YciA in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli
Show others...
2019 (English)In: Applied Microbiology and Biotechnology, p. 1-12Article in journal (Refereed) Published
Abstract [en]

Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

Place, publisher, year, edition, pages
Springer, 2019
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-249360 (URN)10.1007/s00253-019-09707-0 (DOI)000464737100008 ()2-s2.0-85062726311 (Scopus ID)
Note

QC 20190509

Available from: 2019-04-11 Created: 2019-04-11 Last updated: 2019-05-14Bibliographically approved
Perez-Zabaleta, M., Sjöberg, G., Guevara-Martínez, M., Jarmander, J., Gustavsson, M., Quillaguamán, J. & Larsson, G. (2016). Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply. Microbial Cell Factories, 15(1), Article ID 91.
Open this publication in new window or tab >>Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
Show others...
2016 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, no 1, article id 91Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Springer, 2016
Keywords
Escherichia coli, Halomonas boliviensis, (R)-3-hydroxybutyrate, Acetoacetyl-CoA reductase, NADPH, zwf overexpression, Glutamate, Nitrogen limitation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-189084 (URN)10.1186/s12934-016-0490-y (DOI)000377167900001 ()27245326 (PubMedID)2-s2.0-84971577878 (Scopus ID)
Note

QC 20160808

Available from: 2016-08-08 Created: 2016-06-27 Last updated: 2019-06-14Bibliographically approved
Perez-Zabaleta, M. (2016). (R)-3-Hydroxybutyrate production in a metabolically engineered Escherichia coli. New Biotechnology, 33, S117-S117
Open this publication in new window or tab >>(R)-3-Hydroxybutyrate production in a metabolically engineered Escherichia coli
2016 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, p. S117-S117Article in journal, Meeting abstract (Refereed) Published
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-202665 (URN)10.1016/j.nbt.2016.06.1129 (DOI)000393400600377 ()
Note

QC 20170306

Available from: 2017-03-06 Created: 2017-03-06 Last updated: 2019-06-14Bibliographically approved
Guevara-Martínez, M., Sjöberg Gällnö, K., Sjöberg, G., Jarmander, J., Perez-Zabaleta, M., Quillaguamán, J. & Larsson, G. (2015). Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation. Frontiers in Microbiology, 6, Article ID 844.
Open this publication in new window or tab >>Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
Show others...
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
Keywords
E. coil, 3-hydroxybutyrate (3HB), polyhydroxybutyrate (PHB), fed-batch, phosphate, ammonium, limitation, depletion
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-173438 (URN)10.3389/fmicb.2015.00844 (DOI)000360116800001 ()2-s2.0-84941053409 (Scopus ID)
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
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-3873-4977

Search in DiVA

Show all publications