Disruption of PHO13 improves ethanol production via the xylose isomerase pathway

Abstract

Xylose is the second most abundant sugar in lignocellulosic materials and can be converted to ethanol by recombinant Saccharomyces cerevisiae yeast strains expressing heterologous genes involved in xylose assimilation pathways. Recent research demonstrated that disruption of the alkaline phosphatase gene, PHO13, enhances ethanol production from xylose by a strain expressing the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes; however, the yield of ethanol is poor. In this study, PHO13 was disrupted in a recombinant strain harboring multiple copies of the xylose isomerase (XI) gene derived from Orpinomyces sp., coupled with overexpression of the endogenous xylulokinase (XK) gene and disruption of GRE3, which encodes aldose reductase. The resulting YΔGP/XK/XI strain consumed 2.08 g/L/h of xylose and produced 0.88 g/L/h of volumetric ethanol, for an 86.8 % theoretical ethanol yield, and only YΔGP/XK/XI demonstrated increase in cell concentration. Transcriptome analysis indicated that expression of genes involved in the pentose phosphate pathway (GND1, SOL3, TAL1, RKI1, and TKL1) and TCA cycle and respiratory chain (NDE1, ACO1, ACO2, SDH2, IDH1, IDH2, ATP7, ATP19, SDH4, SDH3, CMC2, and ATP15) was upregulated in the YΔGP/XK/XI strain. And the expression levels of 125 cell cycle genes were changed by deletion of PHO13.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0175-7) contains supplementary material, which is available to authorized users.

Engineering a wild-type diploid Saccharomyces cerevisiae strain for second-generation bioethanol production

“…This result implied that maintaining a moderate XI activity has a positive effect on the enhancement of xylose fermentation. The overexpression of Xks1p and four enzymes in the non-oxidative PPP is a common strategy to strengthen xylose metabolism (Bamba et al 2016; Kuyper et al 2005a; Peng et al 2012; Sharma et al 2016). Previous reports have shown that moderate, rather than extremely high, Xks1p activity is more beneficial for growth and ethanol production from xylose because this enzyme catalyzes an ATP-consuming reaction (Jin et al 2003; Peng et al 2011).…”

“…The chassis cell BSIF was diploid; therefore, to destroy two alleles of PHO13 , which would benefit xylose metabolism (Bamba et al 2016; Lee et al 2014; Shen et al 2012; Van Vleet et al 2008), two plasmids were assembled in the plasmid pUC19 as follows: pXIP1/2 (Additional file 1: Fig. S2a) containing two pairs of PHO13 -targeted recombinant arms, PHO13 - RA1 vs. PHO13 - RA2 and PHO13 - RA1 vs. PHO13 - RA3 .…”

“…To compensate for the shortfall in downstream metabolic flux in ethanol production, the xylulokinase Xks1p and the enzymes in the non-oxidative PPP are normally overexpressed in a xylose-utilizing strain (Bamba et al 2016; Peng et al 2012; Sharma et al 2016). Additionally, the inactivation of aldose reductase Gre3p, which is encoded by GRE3 , to decrease the formation of the byproduct xylitol (Bamba et al 2016; Fujitomi et al 2012; Hahn-Hägerdal et al 2007; Kuyper et al 2005a; Peng et al 2012), and the inactivation of alkaline phosphatase Pho13p, encoded by PHO13 , to reduce ATP waste (Bamba et al 2016; Lee et al 2014; Shen et al 2012; Van Vleet et al 2008), were also the considered strategies.…”

“…Additionally, the inactivation of aldose reductase Gre3p, which is encoded by GRE3 , to decrease the formation of the byproduct xylitol (Bamba et al 2016; Fujitomi et al 2012; Hahn-Hägerdal et al 2007; Kuyper et al 2005a; Peng et al 2012), and the inactivation of alkaline phosphatase Pho13p, encoded by PHO13 , to reduce ATP waste (Bamba et al 2016; Lee et al 2014; Shen et al 2012; Van Vleet et al 2008), were also the considered strategies. Normally, xylose is absorbed non-specifically and insufficiently by hexose transporters and is competitively inhibited by glucose in S. cerevisiae (Subtil and Boles 2012).…”

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

“…Intracellular metabolites were prepared by leakage‐free quenching and cold methanol extraction as previously described and analyzed by liquid chromatography–triple quadrupole mass spectrometry (LC–QqQ‐MS), using (+)‐10‐camphorsulfonic acid as an internal standard, as described by Bamba et al…”