View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Universidade do Minho: RepositoriUM AUTOPHAGIC PUNCTUM ARTICLE ADDENDUM Bioengineered Bugs 1:6, 1-6; November/December 2010; © 2010 Landes Bioscience Metabolic engineering for improved microbial pentose fermentation Sara Fernandes1 and Patrick Murray2,* 1IBB-Institute for Biotechnology and Bioengineering; Centre of Biological Engineering; Universidade do Minho; Braga Portugal; 2Shannon Applied Biotechnology Centre; Limerick Institute Technology; Limerick Ireland lobal concern over the depletion of yeast species such as Pachysolen sp. and Gfossil fuel reserves, and the detri- Pichia sp., S. cerevisiae does not metabo- mental impact that combustion of these lise the pentose sugars, xylose and arabi- materials has on the environment, is nose, and it was not until the late 1970s focusing attention on initiatives to cre- that the first steps were taken to develop ate sustainable approaches for the pro- methods to engineer pentose metabolism duction and use of biofuels from various in this yeast. The ability of S. cerevisiae in biomass substrates. The development of fermenting lignocellulose hydrolysates has a low-cost, safe and eco-friendly process been demonstrated repeatedly.1 S. cerevi- for the utilisation of renewable resources siae produces ethanol with stoichiometric to generate value-added products with yields from hexose sugars and tolerates a biotechnological potential as well as wide spectrum of inhibitors and elevated robust microorganisms capable of effi- osmotic pressure. For these reasons, it has cient fermentation of all types of sugars been recognised that genetic engineering are essential to underpin the economic of naturally fermenting microorganisms production of biofuels from biomass such as S. cerevisiae is required for trans- feedstocks. Saccharomyces cerevisiae, the port and efficient bioconversion of pentose most established fermentation yeast used sugars to bioethanol. Pathways for pentose in large scale bioconversion strategies, sugar metabolism are essential for microor- does not however metabolise the pentose ganisms living on decaying plant material sugars, xylose and arabinose and bioen- and are of prime interest in biotechnology gineering is required for introduction when low-cost plant hydrolysates are to be of efficient pentose metabolic pathways fermented to ethanol efficiently. and pentose sugar transport proteins for Key words: pentose fermentation, cofac- bioconversion of these substrates. Our tor imbalance, metabolic engineering Pentose Metabolism approach provided a basis for future Submitted: 05/04/10 experiments that may ultimately lead to A common step in the catabolism of both Revised: 05/24/10 the development of industrial S. cerevi- xylose and arabinose in all microorgan- siae strains engineered to express pentose isms is that both sugars are converted to Accepted: 05/26/10 metabolising proteins from thermophilic D-xylulose-5-phosphate. However, the Previously published online: fungi living on decaying plant material pathways to convert L-arabinose and This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly. the issue is complete and page numbers have Once to printing. has been published online, prior This manuscript www.landesbioscience.com/journals/ and here we expand our original article D-xylose to D-xylulose-5-phosphate are biobugs/article/12724 and discuss the strategies implemented distinctly different in bacteria and fungi *Correspondence to: Patrick G. Murray; to improve pentose fermentation. (Fig. 1). In bacteria, D-xylose is converted Email: [email protected] to D-xylulose by an isomerase (EC 5.3.1.5) Introduction and then phosphorylated by xylulokinase Addendum to: Fernandes S, Tuohy MG, Murray (EC 2.1.7.53) while L-arabinose is first PG. Xylose reductase from the thermophilic The baker’s yeast, Saccharomyces cerevisiae converted to L-ribulose by an isomerase fungus Talaromyces emersonii: cloning and het- erologous expression of the native gene (Texr) is the most well established fermentation (EC 5.3.1.3), and then phosphorylated by and a double mutant (TexrK271R+N273D) with altered yeast for large scale ethanolic fermentation ribulokinase (EC 2.1.7.47). L-Ribulose-5- coenzyme specificity. J Biosci 2009; 34:881–90; of the hexose sugars glucose, mannose and phosphate is then converted to D-xylulose- PMID: 20093741. galactose. However, unlike some other 5-phosphate by an epimerase (EC 5.3.1.3). www.landesbioscience.com Bioengineered Bugs 1 organisms (yeasts or bacteria) that can produce ethanol efficiently from bio- mass-derived hydrolysates. To date, no studies have been conducted to engi- neer filamentous fungi for ethanolic fer- mentation even though some species of anaerobic filamentous fungi were shown to produce ethanol and also ferment pentose sugars.1 For commercial fermen- tation, yeasts present a number of advan- tages over bacteria. Yeasts have superior resistance to hydrolysate inhibitors, bet- ter growth at low pH and less stringent nutritional requirements. Saccharomyces sp. have been traditionally used in indus- try for fermentation of sugar-based materials. However, the best hexose fer- mentor to produce ethanol, S. cerevisiae, is unable to metabolise the pentose sugar components of biomass. Metabolic engi- neering has been used to improve the fermentative capability of S. cerevisiae. Expression of a xylose reductase (XYL1) and a xylitol dehydrogenase (XYL2) from Pichia stipitis in S. cerevisiae resulted in growth on xylose but low levels of etha- nol production.3 In addition, the over- expression of the endogenous xylulose Figure 1. Bacterial (A) and fungal (B) pentose utilisation pathways. Enzyme designations are: kinase (XYL3) together with XYL1 and XI-xylose isomerase; XK-xylulose kinase; AI-arabinose isomerase; RI-ribulose kinase; XR-xylose XYL2 was undertaken4 and the effect reductase; LAD-L-arabitol dehydrogenase; LXR-L-xylulose reductase; XDH-xylitol dehydrogenase. and optimization of expression levels of these genes has been studied.5 The use of In fungi, both pentose sugars go engineered for pentose metabolism but the NAD(P)H-dependent XR and NAD(+)- through oxidation and reduction reactions fermentation requires careful aeration oth- dependent XDH from P. stipitis creates before they are phosphorylated by xylulo- erwise the fermentation product is mainly a cofactor imbalance resulting in xylitol kinase. D-xylose is reduced to xylitol by biomass or xylitol and CO2. Pentoses accumulation. The effect of replacing a reduced nicotinamide adenine dinucleo- are therefore not efficiently fermented to the native P. stipitis Xr with a K270M- tide phosphate (NADPH)-consuming ethanol because of the imbalance of these mutated Xr6 which has reduced affinity reaction and xylitol is then oxidised by redox cofactors. Since NADPH is regener- for NADPH was investigated, resulting an NAD+-consuming reaction to form ated mainly in the oxidative phase of the in enhanced ethanol yields and decreased + D-xylulose. L-Arabinose goes through four PPP, where the reduction of NADP is xylitol formation. However, when the Km + redox reactions; two are NAD -dependent coupled to the generation of CO2, it has an for NADPH was enhanced, the Km for oxidation reactions and two reductions are effect on the redox balance. When extra xylose also increased concomitantly. linked to NADPH consumption. All of the CO2 is produced in this pathway, the pen- NADH-specific xylose reductase enzymes in the fungal D-xylose pathway tose fermentation to ethanol and CO2 is enzymes would enable efficient recycling can also be used in the L-arabinose path- no longer redox neutral. To remove excess of the co-enzyme in the next step in xylose way. D-xylulose then enters the Pentose NADPH, either xylitol is produced or and arabinose metabolism, which involves Phosphate Pathway (PPP) after phosphor- aeration is required, which leads to further conversion of xylitol to xylulose and arabi- + ylation to D-xylulose-5-phosphate. The unwanted CO2 production or a combina- tol to L-xylulose by NAD -specific dehy- conversion of L-arabinose and D-xylose to tion of both processes.2 drogenases. Furthermore NADH is more D-xylulose is redox neutral, but different stable and intracellular concentrations are redox cofactors are used, which affects cel- Metabolic Engineering naturally much higher than NADPH. lular demands for oxygen. Fermentation and the Redox Metabolism Recombinant S. cerevisiae strain carrying of D-xylose and L-arabinose to equimolar a single copy of the Candida tenuis xylose 274 276 amounts of ethanol and CO2 under anaer- Several metabolic engineering strate- reductase K R - N D double mutant obic conditions is possible in S. cerevisiae gies have been developed to generate which was shown to have undergone 2 Bioengineered Bugs Volume 1 Issue 6 Figure 2. Superimposition using SWISS-MODEL of 2–319 amino acid backbone atoms (1,260) between the template structure 1mi3A (pink) TeXR (black) model (A) Cα trace, (B) ribbon trace and (C) amino acids relevant to catalysis, substrate binding and co-enzyme (blue) interaction. almost complete reversal of co-enzyme imbalance showing that T. emersonii may engineered with the P. stipitis PsXrK270R + preference from NADPH to NADH and be a novel and highly efficient ‘toolbox’ for N272D double mutant.12 displayed improved fermentative capabili- biotechnological conversion