Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation

Guevara-Martínez, Mónica; Gällnö, Karin Sjöberg; Sjöberg, Gustav; Jarmander, Johan; Pérez-Zabaleta, Mariel; Quillaguamán, Jorge; Larsson, Gen

doi:10.3389/fmicb.2015.00844

Cited by 22 publications

(21 citation statements)

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“…4 , it is evident that both zwf overexpression and the use of a glutamate-supplemented medium led to increased HAc production. In the case of zwf overexpression, the specific production rate of HAc (q HAc ) correlates well with the specific production rate (q 3HB ), which is in line with previous ammonium-depleted cultivations [ 15 ]. However, in the case of glutamate excess, HAc production seems not directly coupled to 3HB production and here we also find the highest HAc production.…”

Section: Resultssupporting

confidence: 86%

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

et al. 2016

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BackgroundIn 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.ResultsThe production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the Vmax 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.ConclusionsIncreased 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0490-y) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 86%

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

et al. 2016

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“…In E. coli, acetate is often produced concomitantly with R3HBA (Gao et al, 2002;Tseng et al, 2009;Guevara-Martínez et al, 2015), decreasing product yield and lowering the growth rate at concentrations as low as 0.5 g L −1 ; thus, difficulting the use of high-cell-density cultures with high volumetric productivities (Nakano et al, 1997;Roe et al, 2002). Perez-Zabaleta et al (2019) studied the production of R3HBA using the native E. coli acyl-CoA thioesterases (fadM, tesA, tesB, ybgC, ydiI, and yciA).…”

Section: Production Of 3has In Recombinant Ecoli Strainsmentioning

confidence: 99%

Beyond Intracellular Accumulation of Polyhydroxyalkanoates: Chiral Hydroxyalkanoic Acids and Polymer Secretion

Yáñez

Conejeros

Vergara-Fernández

et al. 2020

Front. Bioeng. Biotechnol.

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Polyhydroxyalkanoates (PHAs) are ubiquitous prokaryotic storage compounds of carbon and energy, acting as sinks for reducing power during periods of surplus of carbon source relative to other nutrients. With close to 150 different hydroxyalkanoate monomers identified, the structure and properties of these polyesters can be adjusted to serve applications ranging from food packaging to biomedical uses. Despite its versatility and the intensive research in the area over the last three decades, the market share of PHAs is still low. While considerable rich literature has accumulated concerning biochemical, physiological, and genetic aspects of PHAs intracellular accumulation, the costs of substrates and processing costs, including the extraction of the polymer accumulated in intracellular granules, still hampers a more widespread use of this family of polymers. This review presents a comprehensive survey and critical analysis of the process engineering and metabolic engineering strategies reported in literature aimed at the production of chiral (R)-hydroxycarboxylic acids (RHAs), either from the accumulated polymer or by bypassing the accumulation of PHAs using metabolically engineered bacteria, and the strategies developed to recover the accumulated polymer without using conventional downstream separations processes. Each of these topics, that have received less attention compared to PHAs accumulation, could potentially improve the economy of PHAs production and use. (R)-hydroxycarboxylic acids can be used as chiral precursors, thanks to its easily modifiable functional groups, and can be either produced de-novo or be obtained from recycled PHA products. On the other hand, efficient mechanisms of PHAs release from bacterial cells, including controlled cell lysis and PHA excretion, could reduce downstream costs and simplify the polymer recovery process.

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“…Poly(3-hydroxybutyrate) (PHB) has been considered to be good candidate for completely biodegradable polymers due to the mechanical properties similar to petroleum-derived polymers and complete biodegradability. PHB, the most common PHA, is synthesized by numerous prokaryotes as Cupriavidus necator (Ralstonia eutropha) [1], in response to limitation of nitrogen [2] in presence of high carbon sources. Several strategies are used to produce P(3HB), one-stage batch [3], two-stage batch [4,5] or high-cell-density fed-batch cultures [6,7].…”

Section: Introductionmentioning

confidence: 99%

PHB Production in Biofermentors Assisted through Biosensor Applications

Poltronieri¹,

Mezzolla²,

D’Urso³

2016

Proceedings of the 3rd International Electronic Conference on Sensors and Applications, 15–30 November 2016; Availabl

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Poly-hydroxy-alcanoates (PHAs) are biodegradable and biocompatible polymers synthesized and accumulated in intracellular compartments in several bacterial species. Polyhydroxyalcanoates (PHAs) are synthesized by numerous prokaryotes, such as Cupriavidus necator (Ralstonia eutropha), Pseudomonas spp., Comamonas spp., in response to stress conditions, i.e., under high carbon and low nitrogen (24:1 ratio). PHA can be synthesized using recombinant microorganisms (provided with the operon phbA/phbB/phbC), escaping the constrains of nutrient request, except addition of high amount of sugar (glucose, lactose, fructose). Recombinant E. coli systems were studied to produce PHB using metabolic engineering. In biofermentors, the critical points are the excess of fermentable sugars and the ratio of nutrients versus cell optical density. In order to allow production in biofermentors in automated system, sensors are envisaged to evaluate critical parameters such as sugar consumption, bacteria concentration and level of synthesis of PHA. The need of fermentors and operation control has compelled for application of three biosensing units, one linked to a Nanodrop to evaluate OD, one linked to an enzymatic reaction chamber to measure sugars consumed by enzyme linked sugar biosensing tools, and one for sampling the bacteria, Nile Blue staining, and fluorescence intensity reads. These detectors will make possible to exploit the full potential of bioreactors optimizing the time of use and maximizing the number of bacteria synthesizing PHA.

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Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation

Cited by 22 publications

References 22 publications

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

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

Beyond Intracellular Accumulation of Polyhydroxyalkanoates: Chiral Hydroxyalkanoic Acids and Polymer Secretion

PHB Production in Biofermentors Assisted through Biosensor Applications

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