High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae

Kim, Soo Rin; Ha, Suk Jin; Kong, In Iok; Jin, Yong Su

doi:10.1016/j.ymben.2012.04.001

Cited by 68 publications

(44 citation statements)

References 44 publications

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“…4). Interestingly, the rate of xylose fermentation by the engineered S. boulardii strain (SB-X123) was higher than that of S. cerevisiae strains from previous studies (37,38). Our results suggest that S. boulardii can be employed as a potential host for producing cellulosic biofuels as well as a probiotic yeast strain.…”

Section: Resultsmentioning

confidence: 50%

“…Similarly, we observed that S. boulardii is also incapable of naturally fermenting xylose. Thus, we introduced an integrating expression cassette (pSR6-X123) (37,38) expressing the XYL1, XYL2, and XYL3 genes under the control of constitutive promoters of S. cerevisiae into the ura3 locus of the S. boulardii uracil auxotroph. The resulting engineered S. boulardii strain (SB-X123) showed decent xylose consumption and produced ethanol, suggesting that the xylose metabolic pathways are operational in S. boulardii (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Metabolic Engineering of Probiotic Saccharomyces boulardii

Liu

Kong

Zhang

et al. 2016

Appl Environ Microbiol

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Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae. Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants (leu2, ura3, his3, and trp1) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae. We demonstrated the overexpression of a heterologous gene (lacZ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii. We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii. Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii.

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Section: Resultsmentioning

confidence: 50%

Section: Resultsmentioning

confidence: 99%

Metabolic Engineering of Probiotic Saccharomyces boulardii

Liu

Kong

Zhang

et al. 2016

Appl Environ Microbiol

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“…Xylitol accumulation, which results in wasted carbons and a reduced target product yield, is a long-term and yet unsolved problem for xylose utilization by engineered yeast possessing the heterologous xylose reductase (XR)/xylitol dehydrogenase (XDH) pathways (Kim et al 2012;Kötter and Ciriacy 1993). Xylitol accumulation has been resolved in engineered yeast which utilize the xylose isomerase (XI) pathway (Kuyper et al 2005).…”

Section: Discussionmentioning

confidence: 99%

Lactic acid production from xylose by engineered Saccharomyces cerevisiae without PDC or ADH deletion

Turner

Zhang

Kim

et al. 2015

Appl Microbiol Biotechnol

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Production of lactic acid from renewable sugars has received growing attention as lactic acid can be used for making renewable and bio-based plastics. However, most prior studies have focused on production of lactic acid from glucose despite that cellulosic hydrolysates contain xylose as well as glucose. Microbial strains capable of fermenting both glucose and xylose into lactic acid are needed for sustainable and economic lactic acid production. In this study, we introduced a lactic acid-producing pathway into an engineered Saccharomyces cerevisiae capable of fermenting xylose. Specifically, ldhA from the fungi Rhizopus oryzae was overexpressed under the control of the PGK1 promoter through integration of the expression cassette in the chromosome. The resulting strain exhibited a high lactate dehydrogenase activity and produced lactic acid from glucose or xylose. Interestingly, we observed that the engineered strain exhibited substrate-dependent product formation. When the engineered yeast was cultured on glucose, the major fermentation product was ethanol while lactic acid was a minor product. In contrast, the engineered yeast produced lactic acid almost exclusively when cultured on xylose under oxygen-limited conditions. The yields of ethanol and lactic acid from glucose were 0.31 g ethanol/g glucose and 0.22 g lactic acid/g glucose, respectively. On xylose, the yields of ethanol and lactic acid were <0.01 g ethanol/g xylose and 0.69 g lactic acid/g xylose, respectively. These results demonstrate that lactic acid can be produced from xylose with a high yield by S. cerevisiae without deleting pyruvate decarboxylase, and the formation patterns of fermentations can be altered by substrates.

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“…3 Saccharomyces cerevisiae is currently the most employed microbial catalyst in the biotechnology industry, but it is limited in its range of substrates for producing fuel ethanol, although genetic engineering has improved its utilization of some of the constituent sugars of lignocellulosic materials. [4][5][6][7][8][9][10][11][12][13] Recently, increasing attention has been directed toward developing microbial catalysts for ethanol production at elevated temperatures. [14][15][16][17][18] Fermentation processes conducted at elevated temperatures will significantly reduce cooling costs, improve efficiency of simultaneous saccharification and fermentation, allow continuous ethanol removal by evaporation under reduced pressure, and reduce risk of contamination, all of which would improve profitability of fuel ethanol production from biomass.…”

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confidence: 99%