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Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply

Perez-Zabaleta, Mariel LU orcid ; Sjöberg, Gustav ; Guevara-Martínez, Mónica ; Jarmander, Johan ; Gustavsson, Martin ; Quillaguamán, Jorge LU and Larsson, Gen (2016) In Microbial Cell Factories 15(1).
Abstract

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... (More)

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 (q3HB = 0.12 g g-1 h-1), while nitrogen depletion/zwf overexpression gave the highest yield (Y3HB/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.

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author
; ; ; ; ; and
publishing date
type
Contribution to journal
publication status
published
keywords
(R)-3-hydroxybutyrate, Acetoacetyl-CoA reductase, Escherichia coli, Glutamate, Halomonas boliviensis, NADPH, Nitrogen limitation, zwf overexpression
in
Microbial Cell Factories
volume
15
issue
1
article number
91
publisher
BioMed Central (BMC)
external identifiers
  • pmid:27245326
  • scopus:84971577878
ISSN
1475-2859
DOI
10.1186/s12934-016-0490-y
language
English
LU publication?
no
additional info
Publisher Copyright: © 2016 The Author(s).
id
6d2c60a5-de66-4a96-bc2d-05c845aad560
date added to LUP
2026-02-26 11:42:12
date last changed
2026-04-23 23:02:57
@article{6d2c60a5-de66-4a96-bc2d-05c845aad560,
  abstract     = {{<p>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 <sub> max </sub> 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<sub>3HB</sub> = 0.12 g g<sup>-1</sup> h<sup>-1</sup>), while nitrogen depletion/zwf overexpression gave the highest yield (Y<sub>3HB/CDW</sub> = 0.53 g g<sup>-1</sup>) and a 3HB concentration of 1 g L<sup>-1</sup>, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L<sup>-1</sup> and a productivity of 0.42 g L<sup>-1</sup> h<sup>-1</sup>, 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.</p>}},
  author       = {{Perez-Zabaleta, Mariel and Sjöberg, Gustav and Guevara-Martínez, Mónica and Jarmander, Johan and Gustavsson, Martin and Quillaguamán, Jorge and Larsson, Gen}},
  issn         = {{1475-2859}},
  keywords     = {{(R)-3-hydroxybutyrate; Acetoacetyl-CoA reductase; Escherichia coli; Glutamate; Halomonas boliviensis; NADPH; Nitrogen limitation; zwf overexpression}},
  language     = {{eng}},
  month        = {{06}},
  number       = {{1}},
  publisher    = {{BioMed Central (BMC)}},
  series       = {{Microbial Cell Factories}},
  title        = {{Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply}},
  url          = {{http://dx.doi.org/10.1186/s12934-016-0490-y}},
  doi          = {{10.1186/s12934-016-0490-y}},
  volume       = {{15}},
  year         = {{2016}},
}