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

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.

Computer-Aided Rational Design of Efficient NADPH Production System by Escherichia coli pgi Mutant Using a Mixture of Glucose and Xylose

“…Since the lignocellulosic biomass can be hydrolyzed into two major sugars of glucose and xylose (Gawand et al, 2013), the strategy for the efficient consumption of both sugars is highly desirable for the production of useful chemicals with low cost. In the production of industrially important valueadded products such as 3-hydroxybutyrate (3HB), methyl 3hydoxybutyrate (MHB), amino acids, fatty acids, and isoprenoids (Siedler et al, 2011;Perez-Zabaleta et al, 2016;Yanase et al, 2016;Niu et al, 2017;Li et al, 2018), NADPH is the essential molecule, and thus its availability remains a major hurdle for the efficient production of useful chemicals and fuels (Spaans et al, 2015). Several metabolic engineering strategies have, therefore, been considered, and tested in practice.…”

“…Therefore, the cell growth rate of the pgi mutant becomes significantly lower (by about 80 %) than that of the wild-type strain when glucose was used as a carbon source (Charusanti et al, 2010). In order to avoid the lower cell growth rate inherent in the pgi-knockout mutant, several attempts have been made by partially increasing the Pgi activity (Usui et al, 2012), by reducing the expression level of its gene via replacement of its start codon ATG with GTG without completely removing Pgi (Park et al, 2014;Kim et al, 2015), and by overexpressing the genes of the OPP pathway (Lim et al, 2002;Perez-Zabaleta et al, 2016).…”

Anaerobic poly-3-d-hydroxybutyrate production from xylose in recombinant Saccharomyces cerevisiae using a NADH-dependent acetoacetyl-CoA reductase

“…All three enzymes required for PHB synthesis in bacteria are located in the cytoplasm of the cell where PHB granule accumulation takes place [5, 6]. Most characterized PHB-producers carry an NADPH-dependent AAR gene, whereas AAR with putative promiscuous cofactor utilization are only reported in few cases: for the halotolerant bacterium Allochromatium vinosum, formerly known as Chromatium vinosum [6], the anaerobic syntrophic bacterium Syntrophomonas wolfei [7] and the recently described AAR from the halophilic bacterium Halomonas boliviensis [8]. …”

Increasing Ascomycin Yield in Streptomyces Hygroscopicus var. Ascomyceticus by Using Polyhydroxybutyrate as an Intracellular Carbon Supply Station

“…In the most PHB-producing bacteria, the synthesis of PHB monomer is catalyzed by the NADPHdependent acetoacetyl-CoA reductase (encoding by phaB), and the accumulation of PHB mainly depended on the supply of NADPH [50]. However, NADPH is mainly responsible for the reduction in anabolism, so the biosynthesis of PHB is often limited due to the lack of NADPH.…”