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Microb Cell Fact. 2016 Jun 1;15:91. doi: 10.1186/s12934-016-0490-y.

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

Author information

1
Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.
2
Center of Biotechnology, Faculty of Science and Technology, Universidad Mayor de San Simón, Cochabamba, Bolivia.
3
Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden. gen@kth.se.

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 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.

KEYWORDS:

(R)-3-hydroxybutyrate; Acetoacetyl-CoA reductase; Escherichia coli; Glutamate; Halomonas boliviensis; NADPH; Nitrogen limitation; zwf overexpression

PMID:
27245326
PMCID:
PMC4888404
DOI:
10.1186/s12934-016-0490-y
[Indexed for MEDLINE]
Free PMC Article

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