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

Abstract

BackgroundPoly-3-d-hydroxybutyrate (PHB) that is a promising precursor for bioplastic with similar physical properties as polypropylene, is naturally produced by several bacterial species. The bacterial pathway is comprised of the three enzymes β-ketothiolase, acetoacetyl-CoA reductase (AAR) and PHB synthase, which all together convert acetyl-CoA into PHB. Heterologous expression of the pathway genes from Cupriavidus necator has enabled PHB production in the yeast Saccharomyces cerevisiae from glucose as well as from xylose, after introduction of the fungal xylose utilization pathway from Scheffersomyces stipitis including xylose reductase (XR) and xylitol dehydrogenase (XDH). However PHB titers are still low.ResultsIn this study the acetoacetyl-CoA reductase gene from C. necator (CnAAR), a NADPH-dependent enzyme, was replaced by the NADH-dependent AAR gene from Allochromatium vinosum (AvAAR) in recombinant xylose-utilizing S. cerevisiae and PHB production was compared. A. vinosum AAR was found to be active in S. cerevisiae and able to use both NADH and NADPH as cofactors. This resulted in improved PHB titers in S. cerevisiae when xylose was used as sole carbon source (5-fold in aerobic conditions and 8.4-fold under oxygen limited conditions) and PHB yields (4-fold in aerobic conditions and up to 5.6-fold under oxygen limited conditions). Moreover, the best strain was able to accumulate up to 14% of PHB per cell dry weight under fully anaerobic conditions.ConclusionsThis study reports a novel approach for boosting PHB accumulation in S. cerevisiae by replacement of the commonly used AAR from C. necator with the NADH-dependent alternative from A. vinosum. Additionally, to the best of our knowledge, it is the first demonstration of anaerobic PHB synthesis from xylose.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0598-0) contains supplementary material, which is available to authorized users.

Engineering NADH/NAD+ ratio in Halomonas bluephagenesis for enhanced production of polyhydroxyalkanoates (PHA)

“…Based on the fact that PHB accumulation by H. bluephagenesis TD01 is mainly a fermentation process consuming NADH under oxygen limitation condition, NADH/NAD + ratio becomes very important to channel the acetyl-CoA flux to PHB synthesis (Anderson and Dawes, 1990;de las Heras et al, 2016). The NADH/NAD + ratio varies from species to species, it is usually less than 1, with the only exception of Pseudomonas aeruginosa (Farhana et al, 2010;Wimpenny and Firth, 1972), which contains phenazine as the electron receptor under hypoxic condition, this is similar to the acetoacetyl-CoA reduction during PHB synthesis by H. bluephagenesis TD01 (Farhana et al, 2010).…”

“…The limitations on essential nutrients (such as N, P, S, Fe and Mg) and presence of sufficient carbon source are critical for most known NADPH dependent species to accumulate PHA (Steinbüchel, 1991). While oxygen limitation has been proven functional for inducing PHA synthesis only in A. beijerinckii and recombinant Saccharomyces cerevisiae harboring phaCAB genes from A. vinosum, as both contain NADH consumable PhaB (Portugal-Nunes et al, 2017;de las Heras et al, 2016;Jackson and Dawes, 1976;Senior and Dawes, 1971;Senior and Dawes, 1973).…”

“…Whereas oxygen limitation is the key factor for the NADH dependent fermentation process, NADH/NAD + ratio increases significantly under oxygen limitation. Citrate synthase has been reported to be inhibited by NADH, as a result acetyl-CoA can no longer enter the TCA cycle, instead, it is channeled to the NADH consuming PHB synthesis pathway (Anderson and Dawes, 1990;de las Heras et al, 2016;Farhana et al, 2010;Jackson and Dawes, 1976;Lee, 1996;Steinbüchel, 1991).…”

“…Compared with other reported PHA producing strains, the aeration or agitation during growth of H. bluephagenesis TD01 is much more approachable, the maximal agitations of 800 rpm (revolutions per minute), 280 rpm, 170 rpm, and a constant aeration rate of 1 vvm (air volume/culture volume/min), 1 vvm, 0.85 vvm for 7.5 L, 1 m 3 and 5 m 3 bioreactors, can satisfy the high cell density growth, respectively (Ye et al, 2018b;Kim Beom et al, 2004;Lee, 1996;Steinbüchel, 1991). The product formations under oxygen limitation allow lower agitation rate, less aeration and thus less energy consumption, increasing the competitiveness of the next generation industrial biotechnology (NGIB) based on halophiles (Chen and Jiang, 2018;de las Heras et al, 2016).…”

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

“…For the biosynthesis of PHB, the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is an important step, which catalyzed by the NADH-dependent 3-hydroxybutyryl-CoA dehydrogenase (encoding by hcd) or the NAD(P)H-dependent acetoacetyl-CoA reductase (encoding by phaB) (Figure 5a). The NAD(P)H pool and the ratio of NAD(P)H/NAD(P) + could signi cantly in uence the content of PHB [40,41]. The sequence alignment showed that there is a copy of hcd gene in the genome of S. hygroscopicus var.…”