Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation
Accumulation of acetate is a limiting factor in recombinant production of ( R )-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) deletion of phosphotransacetylase ( pta ), pyruvate oxidase ( poxB ),...
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Published in | Applied microbiology and biotechnology Vol. 103; no. 14; pp. 5627 - 5639 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
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Berlin/Heidelberg
Springer Berlin Heidelberg
01.07.2019
Springer Springer Nature B.V |
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Abstract | Accumulation of acetate is a limiting factor in recombinant production of (
R
)-3-hydroxybutyrate (3HB) by
Escherichia 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 five times less acetic acid per mole 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. |
---|---|
AbstractList | Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia 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 five times less acetic acid per mole 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.sup.-1 h.sup.-1) and the highest 3HB concentration (16.3 g L.sup.-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. Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia 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 five times less acetic acid per mole 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. Accumulation of acetate is a limiting factor in recombinant production of ( R )-3-hydroxybutyrate (3HB) by Escherichia 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 five times less acetic acid per mole 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. Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia 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 five times less acetic acid per mole 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 h ) and the highest 3HB concentration (16.3 g L ) 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. 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. |
Audience | Academic |
Author | Perez-Zabaleta, Mariel Quillaguamán, Jorge van Maris, Antonius J. A. Larsson, Gen Gustavsson, Martin Guevara-Martínez, Mónica |
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Keywords | ( Acetate Nitrogen limitation Fed batch BL21 3-hydroxybutyrate (R)-3-hydroxybutyrate Escherichia coli |
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PublicationDate | 2019-07-01 |
PublicationDateYYYYMMDD | 2019-07-01 |
PublicationDate_xml | – month: 07 year: 2019 text: 2019-07-01 day: 01 |
PublicationDecade | 2010 |
PublicationPlace | Berlin/Heidelberg |
PublicationPlace_xml | – name: Berlin/Heidelberg – name: Germany – name: Heidelberg |
PublicationTitle | Applied microbiology and biotechnology |
PublicationTitleAbbrev | Appl Microbiol Biotechnol |
PublicationTitleAlternate | Appl Microbiol Biotechnol |
PublicationYear | 2019 |
Publisher | Springer Berlin Heidelberg Springer Springer Nature B.V |
Publisher_xml | – name: Springer Berlin Heidelberg – name: Springer – name: Springer Nature B.V |
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Snippet | Accumulation of acetate is a limiting factor in recombinant production of (
R
)-3-hydroxybutyrate (3HB) by
Escherichia coli
in high-cell-density processes. To... Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes. To... 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... |
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SubjectTerms | (R)-3-hydroxybutyrate acetate Acetic acid Bacteria Batch culture Biomedical and Life Sciences Bioreactors Biotechnological Products and Process Engineering Biotechnology Bioteknologi BL21 Cell density Clonal deletion Cultivation Density E coli Escherichia coli Ethylenediaminetetraacetic acid Excretion fed batch Isocitrate lyase Life Sciences Microbial Genetics and Genomics Microbiology nitrogen limitation Oxidases Pharmaceutical industry Productivity Pyruvate oxidase Pyruvic acid Screening |
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Title | Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation |
URI | https://link.springer.com/article/10.1007/s00253-019-09876-y https://www.ncbi.nlm.nih.gov/pubmed/31104101 https://www.proquest.com/docview/2226910789/abstract/ https://search.proquest.com/docview/2232071653 https://pubmed.ncbi.nlm.nih.gov/PMC6597613 https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-251046 |
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