Metabolic Engineering for Improved Microbial Pentose Fermentation

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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 heterologous 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 biomass-derived hydrolysates. To date, no studies have been conducted to engineer filamentous fungi for ethanolic fermentation even though some species of anaerobic filamentous fungi were shown to produce ethanol and also ferment pentose sugars.1 For commercial fermentation, yeasts present a number of advan- tages over bacteria. Yeasts have superior resistance to hydrolysate inhibitors, better 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 engineering 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 ethanol production.3 In addition, the overexpression 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 of lignocellu- Other strategies to introduce xylose ties in terms of ethanol when compared to lose to bioethanol. Comparable changes metabolic pathways into S. cerevisiae S. cerevisiae harbouring the wild type C. in altered coenzyme preference from in an attempt to decrease xylitol for- tenuis xylose reductase.7 NADPH to NADH were obtained with a mation included the expression of the The gene encoding Xylose reductase CtXrK274R+N276D double mutant10 PsXrK270R Thermus thermophilus xylose (glucose) (TeXR ) was also isolated from the ther- +N272D double mutant11,12 and a PsXrK270S+ isomerase14 and the XYLA gene encod- mophilic fungus Talaromyces emersonii,8 S271G+N272P+R276F quadruple mutant.11 The ing the Piromyces sp. xylose isomerase.15 a fungus known by its thermostable lig- next logical steps in our approach would However, the latter enzyme is strongly nocellulolytic enzyme systems.9 Amino be to perform a detailed investigation of inhibited by xylitol and because S. cerevi- acid residues identified inC. tenuis xylose TeXrK271R + N273D double mutant effects in siae produces an aldose reductase (Gre3) reductase critical in substrate recognition engineered strains in order to improve capable of reducing xylose to xylitol, a and co-factor preference were conserved xylose fermentation rates and establish a major by-product in the process was xyli- in TeXr (Fig. 2). The coenzyme selectiv- comparison between other xylose reduc- tol. Therefore GRE3 deletion strains are ity of TeXr was altered by site-directed tase mutants. The S. cerevisiae strain engi- essential for xylose fermentation when a mutagenesis and the ability of TeXrK271R + neered with the C. tenuis double mutant xylose isomerase is introduced into S. cere- N273D double mutant to use NADH prefer- showed 42% enhanced ethanol yield and visiae.16 Comparison of xylose-fermenting entially to NADPH as a coenzyme in the decreased xylitol and glycerol production ability by S. cerevisiae starins engineered first step of pentose metabolism could have compared to the reference strain harbour- with xylose reductase and xylitol dehydro- dramatic effects on improving the xylose ing wild-type XR.13 Similar tendencies genase with strains engineered with xylose conversion process by reducing the redox were observed with S. cerevisiae strains isomerase revealed that the Xr-Xdh xylose

www.landesbioscience.com Bioengineered Bugs 3 and overexpression of NADH-dependent GDH2 or GS-GOGAT complex was shown to improve cofactor utilisation in xylose-fermenting S. cerevisiae and improved the ethanol yield.21 In further metabolic engineering of xylose metabolism, the xylose transport step should receive special attention. The

rather high Km values of xylose reductase and xylose isomerise enzymes implies that achieving high rates of xylose fermentation may require the introduction of heterologous high-affinity xylose transporters that catalyses xylose uptake. S. cerevisiae also lacks an efficient transport system for pentose sugars, although the transport of these sugars occurs through hexose transporters with very low affinity and competition with glucose restricts xylose assimilation. Simultaneous and effec- Figure 3. Ideal simultaneous transport and fermentation of hexose and pentose sugars. tive transport of both hexose and pentose sugars by pentose utilising strains of S. utilization pathway is much better than Insertion of a NADP+-dependent cerevisiae would therefore be a significant the xylose isomerase pathway due to the D-glyceraldehyde-3-phosphate dehy- improvement for bioconversion of biomass insufficient in vivo activity of xylose isom- drogenase (NADP-GAPDH) from feedstocks to bioethanol (Fig. 3). A gene erase. This low xylose isomerase activity Kluyveromyces lactis facilitated NADPH encoding a glucose/xylose facilitated dif-

limits xylose utilization in recombinant regeneration with no production of CO2 fusion transporter and a gene encoding a S. cerevisiae strains and therefore it would in S. cerevisiae strains with a ZWF1 dele- glucose/xylose symporter from C. inter- be useful to exploit further the Xr-Xdh tion.17 In S. cerevisiae strains exibiting a media36 were successfully expressed in pathway using T. emersonii XrK271R + N273D range of productions levels of G6PDH, S. cerevisiae. However the C. intermedia double mutant to potentially construct a overexpression of a transhydrogenase symporter was not able to support vigor- new pathway to reduce the redox imbal- from Azotobacter vinelandii, reduced the ous growth of the recombinant S. cerevi- ance achieving more effective ethanol xylitol yield but enhanced the yield of siae strain on xylose or glucose when used production from xylose by recombinant S. glycerol during xylose fermentation.20 as sole carbon sources. Among filamen- cerevisiae. A different approach to modifying tous fungi relatively few sugar transport- NADPH is mainly regenerated in the redox metabolism would require ers have been identified and characterised. the oxidative part of PPP and is coupled modification of ammonia assimilation in T. emersonii, the thermophilic fun-

to the generation of CO2. In this path- recombinant S. cerevisiae. For ammonia gus used in our studies, inhabits the soil, way, D-glucose-6-phosphate dehydroge- incorporation, ATP-dependent synthesis decaying masses of plant material piles nase (G6PDH), encoded by ZWF1 and of glutamine from glutamate and ammo- of agricultural and forestry products, 6-phosphogluconate dehydrogenase, nia is catalysed by glutamine synthetase, and other accumulations of organic mat- encoded by GND1 and GND2 are respon- while a NADPH-dependent glutamate ter wherein the warm, humid and aero- sible for the oxidation of D-glucose- dehydrogenase, encoded by GDH1 is bic environment with accessible carbon 6-phosphate and release of 2 moles of responsible for 2-ketoglutarate amina- sources provides the basic conditions for 23 NADPH and 1 mole of CO2 per mole of tion. Another glutamate dehydrogenase, survival. T. emersonii also grows rapidly D-glucose-6-phosphate.17 Ethanol yield encoded by GDH2 is NADH-dependent on hexoses and pentoses as a sole carbon from xylose can be increased by lower- and converts glutamate to 2-ketoglu- sources and thermostable enzyme systems ing the oxidative PPP flux. Modifications tarate and ammonium. Virtually all required for hemicellulose degradation in the pathway such as deletion of ZWF1 microorganisms that fix atmospheric have been described and characterised pre- and GND1 were proven to block PPP nitrogen assimilate ammonia through the viously.24,25 So we therefore anticipated the giving an ethanol yield of 0.41 g g-1 and GS-GOGAT complex which converts presence of high affinity sugar transporter a xylitol yield of only 0.05 g g-1,18 and a ammonia to glutamate using NADH and genes in T. emersonii genome. reduced phosphoglucose isomerase activ- ATP and consists of two enzymes: glu- We searched the T. emersonii chro- ity, an enzyme that converts glucose- tamate synthetase encoded by GLT1 and mosomal DNA for genes with strong 6-phosphate into fructose-6-phosphate, glutamine synthetase encoded by GLN1. sequence and predicted structural simi- was shown to increase ethanol yield.19 Deletion of NADPH-dependent GDH1 larities with transporter genes from the

4 Bioengineered Bugs Volume 1 Issue 6 8. Fernandes S, Tuohy MG, Murray PG. Xylose reduc- microorganisms whose function has been and the simultaneous co-fermentation of tase from the thermophilic fungus Talaromyces emer- demonstrated and therefore the isolation hexose and pentose sugars still constitutes sonii: cloning and heterologous expression of the native gene (Texr) and a double mutant (Texr K271R + of two putative transporters from the a major strain engineering challenge. N273D) with altered coenzyme specificity. Journal of filamentous fungusT. emersonii, TeHTX There is an increasing need to iden- Biosciences 2009; 34:881-90. (GenBank FJ985745) and TeXYLT tify new sources of more stable biomass 9. Moloney AP, McCrae SI, Wood TM, Coughlan MP. Isolation and characterization of the endoglucanases (GenBank Accession No.: FJ985746) converting enzymes and more efficient of T. emersonii. Biochem J 1985; 225:365-74. has been achieved. The transporters were systems to utilise all carbohydrate com- 10. Petschacher B, Leitgeb S, Kavanagh KL, Wilson DK, shown to have 12 transmembrane domains ponents of lignocellulose. Also better Nidetzky B. The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site- which is a characteristic feature of the understanding of the sugar and oxygen directed mutagenesis and X-ray crystallography. Major Facilitator Superfamily. Detailed regulatory system, sugar transport and Biochem J 2005; 385:75-83. 11. Liang L, Zhang J, Lin Z. Altering coenzyme speci- sequence analysis of both transporters the development of further transforma- ficity of Pichia stipitis xylose reductase by the semi- suggests that TeHXT is potentially a hex- tion and expression systems with genes rational approach CASTing. Microb Cell Fact 2007; ose transporter similarly to Gal2 from S. capable to overcome the cofactor imbal- 6:1-11. 12. Watanabe S, Saleh AA, Pack SP, Annaluru N, Kodaki cerevisiae while TeXYLT has the potential ance will enable the construction of T, Makino K. Ethanol production from xylose by to be a glucose/xylose transporter (unpub- robust industrial yeast strains for pentose recombinant Saccharomyces cerevisiae expressing pro- tein-engineered NADH-preferring xylose reductase lished data). fermentation. The thermophilic fungus T. from Pichia stipitis. Microbiology 2007; 153:3044- Despite the high similarity of TeHXT emersonii proved to be a potential source 54. to fungal high-affinity transporters and organism for extracellular and intracel- 13. Petschacher B, Nidetzky B. Altering the coenzyme preference of xylose reductase to favor utilization of the similarity of TeXYLT to other trans- lular proteins with applications in bio- NADH enhances ethanol yield from xylose in a meta- porters previously shown to transport mass bioconversion strategies and future bolically engineered strain of Saccharomyces cerevisiae. xylose, Talaromyces genes did not restore experiments using T. emersonii genes may Microb Cell Fact 2008; 7:1-12. 14. Walfridsson M, Bao X, Anderlund M, Lilius G, growth on different sugars of an engi- further improve the desired traits or fer- Bulow L, Hahn-Hagerdal B. Ethanolic fermentation neered S. cerevisiae strain, in which all hex- mentative properties of the industrial of xylose with S. cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active ose transporters were deleted (unpublished organisms leading to a more efficient bio- xylose (glucose) isomerase. Appl Environ Microbiol data). We hypothesized that T. emersonii technological conversion of lignocellulose 1996; 62:4648-51. transporter proteins may not have been to bioethanol. 15. Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MS, de Laat WT, et al. High-level functional correctly directed to the plasma mem- expression of a fungal xylose isomerase: the key to brane, were folded incorrectly or a differ- Acknowledgements efficient ethanolic fermentation of xylose by S. cerevi- ent composition of phospholipid sterols This work was supported by an Irish siae. FEMS Yeast Res 2003; 4:69-78. 16. Träff KL, Cordero Otero RR, van Zyl WH, Hahn- in S. cerevisiae may have caused changes government Department of Agriculture, Hägerdal B. Deletion of the GRE3 aldose reductase in T. emersonii transporter conformation. Food and Fisheries award (DAFF RSF-05- gene and its influence on xylose metabolism in recombinant strains of S. cerevisiae expressing the Further expansion of these results of our 225) under the Research stimulus Fund xylA and XKS1 genes. Appl Environ Microbiol 2001; research can open an alternative route to Programme 2007–13. 67:5668-74. the development of industrial xylose-uti- 17. Verho R, Londesborough J, Penttilä M, Richard P. References Engineering redox cofactor regeneration for improved lizing strains of S. cerevisiae. pentose fermentation in S. cerevisiae. Appl Environ 1. Hahn-Hägerdal B, Jeppsson H, Olsson L, Microbiol 2003; 69:5892-7. Mohagheghi A. An inter-laboratory comparison of 18. Jeppsson M, Johansson B, Hahn-Hägerdal B, Gorwa- Conclusion the performance of ethanol-producing microorgan- Grauslund MF. Reduced oxidative pentose phosphate isms in a xylose rich acid hydrolysate. Applied pathway flux in recombinant xylose-utilizing S. cere- More than a decade of research has been Microbial Biotechnology 1994; 41:62-72. visiae strains improves the ethanol yield from xylose. 2. Hahn-Hägerdal B, Karhumaa K, Fonseca C, Appl Environ Microbiol 2002; 68:1604-9. devoted to the development of strains for Spencer-Martins I, Gorwa-Grauslund MF. Towards 19. Eliasson A, Boles E, Johansson B, Österberg M, efficient pentose fermentation. The major- industrial pentose-fermenting yeast strains. Appl Thevelein JM, Spencer-Martins I, et al. Xylulose Microbiol Biotechnol 2007; 74:937-53. ity of the studies conducted used metabolic fermentation by mutant and wild-type strains of 3. Kotter P, Ciriacy M. Xylose fermentation by S. cerevi- Zygosaccharomyces and Saccharomyces cerevisiae. Appl. engineering through a rational selection of siae. App Microb Biotechnol 1993; 38:776-83. Microbiol. Biotechnol 2000; 53:376-82. genes to be manipulated for the develop- 4. Ho NW, Chen Z, Brainard AP. Genetically engi- 20. Jeppsson M, Johansson B, Jensen PR, Hahn- neered Saccharomyces yeast capable of effective Hägerdal B, Gorwa-Grauslund MF. The level of ment of novel pentose-fermenting strains cofermentation of glucose and xylose. Appl Environ glucose-6-phosphate dehydrogenase activity strongly of S. cerevisiae with varying levels of suc- Microbiol 1998; 64:1852-9. influences xylose fermentation and inhibitor sensitiv- cess. The increased knowledge about pen- 5. Bao X, Gao D, Qu Y, Wang Z, Walfridssion M, ity in recombinant S. cerevisiae strains. Yeast 2003; Hahn-Hägerbal B. Effect on product formation in 20:1263-72. tose metabolism, substrate binding and recombinant S. cerevisiae strains expressing different 21. Roca C, Nielsen J, Olsson L. Metabolic engineering cofactor specificity of pentose assimilating levels of xylose metabolic genes. 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www.landesbioscience.com Bioengineered Bugs 5 24. Tuohy MG, Puls J, Claeyssens M, Vrsanská M, Coughlan MP. The xylan-degrading enzyme system of T. emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans. Biochem J 1993; 290:515-23.

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