Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae
Highlights
► Rational and evolutionary metabolic engineering were carried out in S. cerevisiae. ► Xylose fermentation performance of the evolved strain is the best reported to-date. ► High expression level of xylose isomerase is necessary for rapid xylose assimilation.
Introduction
Use of renewable biomass for the production of transport fuels is becoming increasingly important due to the decreasing fossil energy resources and growing concerns about climate change. Cost-effective production of cellulosic bio-ethanol is dependent on complete and fast utilization of lignocellulosic biomass. Although the broadly used fermentative yeast Saccharomyces cerevisiae is one of the most effective ethanol-producing organisms from hexose sugars, it does not naturally ferment pentose D-xylose, the second most abundant sugar in lignocellulosic biomass after glucose. Consequently, S. cerevisiae has been extensively engineered to incorporate an efficient D-xylose assimilation pathway (Hahn-Hägerdal et al., 2007, Maris et al., 2007, Van Vleet and Jeffries, 2009).
The conversion of xylose to xylulose can be mediated via two different pathways. Most xylose-utilizing eukaryotes convert xylose into xylulose in a two-step redox reaction, catalyzed by the predominantly NADPH-dependent xylose reductase (XR) followed by the NAD+-dependent xylitol dehydrogenase (XDH), with xylitol as the pathway intermediate (Jeffries, 1983). Recombinant S. cerevisiae strains expressing these enzymes can ferment xylose to ethanol, however, under anaerobic conditions, the different coenzyme specificities of XR and XDH generate a cofactor imbalance which results in considerable xylitol accumulation as a by-product and reduces the yield of ethanol (Bruinenberg et al., 1983, Hahn-Hägerdal et al., 2007).
In the second pathway, the isomerization of xylose to xylulose is catalyzed by the metal-ion-dependent enzyme xylose isomerase (XI). The XI pathway eliminates the cofactor imbalance and accompanying excessive production of xylitol. A number of bacterial XYLA genes coding for XIs have been heterologously expressed in S. cerevisiae, including XYLA from Escherichia coli (Sarthy et al., 1987), Clostridium thermosulfurogenes (Moes et al., 1996), Bacillus subtilis or Actinoplanes missouriensis (Amore et al., 1989), Thermus thermophilus (Walfridsson et al., 1996) and Streptomyces rubiginosus (Gardonyi and Hahn-Hägerdal, 2003). Nevertheless, all the attempts were unsuccessful until XI from the anaerobic fungus Piromyces (Kuyper et al., 2003, Kuyper et al., 2005a, Maris et al., 2007) and Orpinomyces (Madhavan et al., 2009a, Madhavan et al., 2009b) were functionally expressed at high levels in S. cerevisiae. The first bacterial XI from Clostridium phytofermentans was also expressed in S. cerevisiae, and the bacterial XI is reported to be less susceptible to inhibition by xylitol than is the enzyme from the Piromyces strain (Brat et al., 2009).
D-Xylulose is subsequently phosphorylated to D-xylulose-5-phosphate (X5P) by xylulokinase (XK), and channeled through the pentose phosphate pathway (PPP) to glycolysis. To facilitate high flux of xylose assimilation, XK including either the endogenous S. cerevisiae XKS1 (Ho et al., 1998, Johansson et al., 2001, Toivari et al., 2001) or the Pichia stipitis XYL3 (Jin et al., 2003) were also overexpressed, along with the genes of the non-oxidative PPP (RPE1, RKI1, TKL1, and TAL1) (Jin et al., 2005, Johansson and Hahn-Hägerdal, 2002, Karhumaa et al., 2005, Kuyper et al., 2005a). Still, the heterologous expression of these genes by themselves was not sufficient for efficient utilization of xylose. The problem was finally overcome by evolutionary engineering, a method that has been extensively used and proven to be very effective in obtaining S. cerevisiae strains with improved fermentation performance on xylose (Kuyper et al., 2005b, Pitkänen et al., 2005, Sauer, 2001, Sonderegger and Sauer, 2003), arabinose (Becker and Boles, 2003) or co-utilization of D-xylose and L-arabinose (Karhumaa et al., 2006, Rosa et al., 2010, Wiedemann and Boles, 2008). Furthermore, successful genetic analysis of an evolved strain can identify useful genotypes aiding in further rational engineering (Nevoigt, 2008).
In the present paper, we report on a metabolic engineering study to improve xylose fermentation by a S. cerevisiae strain. The key genes for the xylose metabolic pathway, Piromyces XYLA and P. stipitis XYL3, were introduced to enable xylose utilization. In addition, the genes for PPP were overexpressed to overcome other potential limitations of xylose assimilation. Sequential batch cultivation followed by cultivation in a xylose-limited chemostat with increasing dilution rate was finally used to select for strains with improved growth and xylose consumption rate. The fermentation performance of the constructed and evolved yeast strains was evaluated yielding one isolate, H131-A3SB-3, with very high rates of growth, xylose assimilation and ethanol production. An inversed metabolic engineering based on functional complementation was deployed to discover the dominant genotype of the rapid xylose-fermenting strain.
Section snippets
Strains and maintenance
Yeast strains used in this study are summarized in Table 2. Yeast and bacterial strains were stored in 15% glycerol at −80 °C. E. coli and grown in Luria–Bertani medium. Ampicillin (50 mg/L) was added to the medium when required. Yeast strains were routinely cultivated at 30 °C in YNB (6.7 g/L yeast nitrogen base without amino acids; Becton, Dickinson and Company, MD, USA), plus 20 g/L of glucose (YNBG) or xylose (YNBX) and a mixture of appropriate nucleotides and amino acids. For growth on plates,
Construction of xylose-utilizing strains
Several plasmids were constructed to integrate the genes of the non-oxidative PPP. In order to express the endogenous RPE1, RKI1, TKL1 and P. stipitis TAL1 genes under the strong constitutive glyceraldehydes-3-phosphate dehydrogenase promoter (TDH3p), the genes were amplified from S. cerevisiae or P. stipitis genomic DNA using appropriate primers (Table 3) and integrated to the genome of BF264-15Dau (Table 2), resulting in the pentose phosphate pathway overexpression strain H131 (Table 2. MATa,
Discussion
We have applied evolutionary engineering to a metabolically engineered S. cerevisiae strain to improve growth and fermentation on xylose. Both the anaerobic growth rate and the xylose consumption rate were significantly elevated in the ultimately evolved strain H131-A3-ALCS. Ethanol yields remained close to the theoretical maximum in most strains.
Evolved strains exhibiting high xylose assimilation rates harbor a large number of XYLA copies, which is consistent with the high transcription level
Acknowledgments
This project was financially supported by the MIT Energy Initiative and the Low Carbon Emissions University Alliance (LCEUA). We thank Paula Grisafi, Anna Drinnenberg for sharing the expertise on PFGE and chromosome blot.
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