Abstract
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.
Highlights
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
Cofactor specificity of the H. boliviensis acetoacetyl‐coenzyme A (CoA) reductase In order to determine the cofactor specificity of the reductase, the enzyme was produced in E. coli and purified using metal ion chromatography
Several factors influence the production of metabolites from transplantation of new pathways into microorganisms and one central point is the balancing of cofactors
Summary
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. For the production of PHB three enzymes are needed and H. boliviensis possesses seven alleles of the β-ketothiolase, one allele of the acetoacetyl-CoA reductase and three alleles of the PHB synthase. These alleles are scattered over the chromosome and are not organized into operons, as in e.g. Cupriavidus necator [9]. From earlier experiments it was seen that E. coli most likely expresses the enzyme thioesterase II (tesB), which is capable of hydrolysis of (R)-3-hydroxybutyrateCoA (3HB-CoA), which in turn leads to excretion of 3HB to the medium [10]. This is an added benefit with expression in E. coli since its results in a primary purification of the product
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