Engineering of Saccharomyces cerevisiae for the production of poly-3-d-hydroxybutyrate from xylose

Sandström, Anders; Heras, Alejandro Muñoz de las; Portugal-Nunes, Diogo; Gorwa-Grauslund, Marie-Françoise

doi:10.1186/s13568-015-0100-0

Cited by 33 publications

(29 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The AAR from C. necator uses NADPH as a cofactor [24], which competes with biomass synthesis in recombinant S. cerevisiae and could explain the low PHB titers observed on glucose and xylose [15, 16]. This led us to look for NADH-dependent alternatives in reported studies.…”

Section: Resultsmentioning

confidence: 99%

“…The coding sequence was designed to be under the control of the constitutive promoter-terminator pair of the gene TPI1 flanked by the restriction sites Sac I and Spe I. The custom-synthesized coding sequence was cloned on the integrative plasmid YIpAGS2 [16] generating YIpAGS3 (Additional file 1). …”

Section: Methodsmentioning

confidence: 99%

“…The custom-synthesized coding sequence was cloned on the integrative plasmid YIpAGS2 [16] generating YIpAGS3 (Additional file 1). …”

Section: Methodsmentioning

confidence: 99%

“…In addition the pool of cytosolic acetyl-CoA was increased in S. cerevisiae by overexpressing the alcohol dehydrogenase ( ADH2 ), the acetaldehyde dehydrogenase ( ALD6 ), the acetyl-CoA acetyltransferase ( ERG10 ) and the Salmonella enterica acetyl-CoA synthetase variant ( acs L641P ) and it resulted in improved PHB productivity from glucose [15]. Finally since a major fraction of sugars from lignocellulosic biomass may consist of xylose, PHB pathway has been introduced in S. cerevisiae engineered for xylose utilization, leading to PHB synthesis from xylose [16]. In the present study we demonstrate that PHB can be produced anaerobically, and in combination with ethanol, from xylose by cofactor shift through the introduction of the NADH-dependent AAR alternative from A. vinosum in recombinant S. cerevisiae .…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The custom-synthesized coding sequence was cloned on the integrative plasmid YIpAGS2 [16] generating YIpAGS3 (Additional file 1). …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Xylose catabolism is a key component of sustainable processes that produce useful secondary products from lignocellulosic biomass (2)(3)(4). Xylose catabolism is also of commercial interest because xylose conversion to useful secondary chemicals such as bioethanol and biodegradable plastics can reduce losses associated with lignocellulose bioprocessing (5,6). Industrially important by-products of xylose metabolism include xylitol, which is used as a natural sweetener in the food and confectionary industries (7).…”

mentioning

confidence: 99%

Activation of an Otherwise Silent Xylose Metabolic Pathway in Shewanella oneidensis

Sekar

Shin

DiChristina

2016

Appl Environ Microbiol

View full text Add to dashboard Cite

Shewanella oneidensis is unable to metabolize the sugar xylose as a carbon and energy source. In the present study, an otherwise silent xylose catabolic pathway was activated in S. oneidensis by following an adaptive evolution strategy. Genome-wide scans indicated that the S. oneidensis genome encoded two proteins similar to the xylose oxido-reductase pathway enzymes xylose reductase (SO_0900) and xylulokinase (SO_4230), and purified SO_0900 and SO_4230 displayed xylose reductase and xylulokinase activities, respectively. The S. oneidensis genome was missing, however, an Escherichia coli XylE-like xylose transporter. After 12 monthly transfers in minimal growth medium containing successively higher xylose concentrations, an S. oneidensis mutant (termed strain XM1) was isolated for the acquired ability to grow aerobically on xylose as a carbon and energy source. Wholegenome sequencing indicated that strain XM1 contained a mutation in an unknown membrane protein (SO_1396) resulting in a glutamine-to-histidine conversion at amino acid position 207. Homology modeling demonstrated that the Q207H mutation in SO_1396 was located at the homologous xylose docking site in XylE. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis. Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass. IMPORTANCEThe activation of an otherwise silent xylose metabolic system in Shewanella oneidensis is a powerful example of how accidental mutations allow microorganisms to adaptively evolve. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis. Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass. Xylose is one of the primary products of lignocellulose degradation and, after glucose, is the second most abundant carbohydrate in nature. The xylose polymer xylan is the primary constituent of hemicellulose, which comprises approximately 17% of the dry weight of hardwoods and up to 31% of plants (1). Xylose catabolism is a key component of sustainable processes that produce useful secondary products from lignocellulosic biomass (2-4). Xylose catabolism is also of commercial interest because xylose conversion to useful secondary chemicals such as bioethanol and biodegradable plastics can reduce losses associated with lignocellulose bioprocessing (5, 6). Industrially important by-products of xylose metabolism include xylitol, which is used as a natural sweetener in the food and confectionary industries (7).Xylose metabolic pathways include the oxido-reductase, isomerase, and Weimberg-Dahms pathways (Fig. 1) (1, 8). Extracellular xylose is transported inside the cell via the ATP-bindi...

show abstract

Glucose assimilation rate determines the partition of flux at pyruvate between lactic acid and ethanol in Saccharomyces cerevisiae

Lane

Turner

Jin

2023

Biotechnology Journal

View full text Add to dashboard Cite

Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endogenous PDCs will eliminate ethanol production, but the resulting strain suffers from C2 auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product without the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae expressing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L−1 from the 12 g L−1 observed during sequential consumption of sugars. This work elucidates the carbon source‐dependent effects on product selection in engineered yeast.

show abstract

Engineering of Saccharomyces cerevisiae for the production of poly-3-d-hydroxybutyrate from xylose

Cited by 33 publications

References 30 publications

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

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

Activation of an Otherwise Silent Xylose Metabolic Pathway in Shewanella oneidensis

Glucose assimilation rate determines the partition of flux at pyruvate between lactic acid and ethanol in Saccharomyces cerevisiae

Contact Info

Product

Resources

About