Cloning and Expression of Xylitol Dehydrogenase Enzyme from Spathaspora passalidarum in Saccharomyces cerevisiae

Mamoori, Yaseen I.; Al-Jelawi, Majed H.; Yahya, Abdul Ghani I.

doi:10.22401/jnus.15.1.17

Cited by 2 publications

(2 citation statements)

References 21 publications

(22 reference statements)

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“…Yeasts that have NAD(P)Hdependent XR are able to ferment xylose to ethanol under oxygen limitation (Bruinenberg et al, 1984). The best xylose fermenters belong in the Scheffersomyces-Spathaspora clade, and have NAD(P)H-dependent XR encoded by XYL1.2 (Table 1) (Mamoori et al, 2013). XYL1.2 originated from a tandem duplication of XYL1, in a common ancestor of Spathaspora and Scheffersomyces (Figures S7 and S8).…”

Section: Resultsmentioning

confidence: 99%

Flux balance analysis predicts NADP phosphatase and NADH kinase are critical to balancing redox during xylose fermentation inScheffersomyces stipitis

Correia

Khusnutdinova

et al. 2018

Preprint

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Xylose is the second most abundant sugar in lignocellulose and can be used as a feedstock for nextgeneration biofuels by industry. Saccharomyces cerevisiae, one of the main workhorses in biotechnology, is unable to metabolize xylose natively but has been engineered to ferment xylose to ethanol with the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Scheffersoymces stipitis. In the scientific literature, the yield and volumetric productivity of xylose fermentation to ethanol in engineered S. cerevisiae still lags S. stipitis, despite expressing of the same XR-XDH genes. These contrasting phenotypes can be due to differences in S. cerevisiae's redox metabolism that hinders xylose fermentation, differences in S. stipitis' redox metabolism that promotes xylose fermentation, or both. To help elucidate how S. stipitis ferments xylose, we used flux balance analysis to test various redox balancing mechanisms, reviewed published omics datasets, and studied the phylogeny of key genes in xylose fermentation. In vivo and in silico xylose fermentation cannot be reconciled without NADP phosphatase (NADPase) and NADH kinase. We identified eight candidate genes for NADPase. PHO3.2 was the sole candidate showing evidence of expression during xylose fermentation. Pho3.2p and Pho3p, a recent paralog, were purified and characterized for their substrate preferences. Only Pho3.2p was found to have NADPase activity. Both NADPase and NAD(P)H-dependent XR emerged from recent duplications in a common ancestor of Scheffersoymces and Spathaspora to enable efficient xylose fermentation to ethanol. This study demonstrates the advantages of using metabolic simulations, omics data, bioinformatics, and enzymology to reverse engineer metabolism. METHODSGenome-scale network reconstruction and analysis. The S. stipitis GENRE was obtained from a panfungal GENRE that combined the GENRE's of S. stipitis (Balagurunathan et al., 2012;Caspeta et al., 2012;Li, 2012;Liu et al., 2012), Schizosaccharomyces pombe (Sohn et al., 2012), Aspergillus niger (Andersen et al., 2008), Yarrowia lipolytica (Pan and Hua, 2012; Loira et al., 2012), Komagataella phaffii (Caspeta 3/28 et al., 2012), Kluyveromyces lactis (Dias et al., 2014), and S. cerevisiae (Heavner et al., 2013). Model simulations were carried out using COnstraints Based Reconstruction and Analysis for Python version 0.9.1 (COBRApy) (Ebrahim et al., 2013). The xylose uptake rate was set to 10 mmol · gDCW -1 · h -1 .The growth associated maintenance (GAM) and non-growth associated maintenance (NGAM) were set to 60 mmol · gDCW -1 and 0 mmol · gDCW -1 · h -1 , respectively. Flux variability analysis (FVA) was used to evaluate alternative optimum solutions (Mahadevan and Schilling, 2003). The exchange bounds for erythritol, ribitol, arabitol, sorbitol, and glycerol were all set to zero to simplify the solution space for polyols. The cofactor selectivities of NADH and NADPH were varied in a single XR reaction (Balagurunathan et al., 2012). XR solely driven by NADH or NADPH were blocked. The e...

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Section: Resultsmentioning

confidence: 99%

Flux balance analysis predicts NADP phosphatase and NADH kinase are critical to balancing redox during xylose fermentation inScheffersomyces stipitis

Correia

Khusnutdinova

et al. 2018

Preprint

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“…The known genome of Sp. passalidarum [21] has two genes encoding xylose reductases, SpXYL1.1 and SpXYL1.2, and recent data show that the SpXYL1.2 gene encodes for a xylose reductase with higher activity with NADH, allowing efficient anaerobic xylose consumption and fermentation by recombinant S. cerevisiae strains [19,22]. Regarding xylitol dehydrogenase activity, all the Spathaspora yeasts analyzed showed a NAD + -dependent activity, and, again, the Sp.…”

Section: Introductionmentioning

confidence: 87%

Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae

et al. 2020

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In recent years, many novel xylose-fermenting yeasts belonging to the new genus Spathaspora have been isolated from the gut of wood-feeding insects and/or wood-decaying substrates. We have cloned and expressed, in Saccharomyces cerevisiae, a Spathaspora arborariae xylose reductase gene (SaXYL1) that accepts both NADH and NADPH as co-substrates, as well as a Spathaspora passalidarum NADPH-dependent xylose reductase (SpXYL1.1 gene) and the SpXYL2.2 gene encoding for a NAD+-dependent xylitol dehydrogenase. These enzymes were co-expressed in a S. cerevisiae strain over-expressing the native XKS1 gene encoding xylulokinase, as well as being deleted in the alkaline phosphatase encoded by the PHO13 gene. The S. cerevisiae strains expressing the Spathaspora enzymes consumed xylose, and xylitol was the major fermentation product. Higher specific growth rates, xylose consumption and xylitol volumetric productivities were obtained by the co-expression of the SaXYL1 and SpXYL2.2 genes, when compared with the co-expression of the NADPH-dependent SpXYL1.1 xylose reductase. During glucose-xylose co-fermentation by the strain with co-expression of the SaXYL1 and SpXYL2.2 genes, both ethanol and xylitol were produced efficiently. Our results open up the possibility of using the advantageous Saccharomyces yeasts for xylitol production, a commodity with wide commercial applications in pharmaceuticals, nutraceuticals, food and beverage industries.

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Cloning and Expression of Xylitol Dehydrogenase Enzyme from Spathaspora passalidarum in Saccharomyces cerevisiae

Cited by 2 publications

References 21 publications

Flux balance analysis predicts NADP phosphatase and NADH kinase are critical to balancing redox during xylose fermentation inScheffersomyces stipitis

Flux balance analysis predicts NADP phosphatase and NADH kinase are critical to balancing redox during xylose fermentation inScheffersomyces stipitis

Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae

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