(19)
(11)EP 2 279 241 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
08.04.2020 Bulletin 2020/15

(21)Application number: 09743560.6

(22)Date of filing:  06.05.2009
(51)International Patent Classification (IPC): 
C12N 1/19(2006.01)
C12P 7/06(2006.01)
C12N 15/56(2006.01)
C12R 1/01(2006.01)
(86)International application number:
PCT/US2009/042975
(87)International publication number:
WO 2009/137574 (12.11.2009 Gazette  2009/46)

(54)

DEVELOPMENT OF STRAINS OF THE THERMOTOLERANT YEAST HANSENULA POLYMORPHA CAPABLE OF ALCOHOLIC FERMENTATION OF STARCH AND XYLAN BY EXPRESSION OF STARCH AND XYLAN DEGRADING ENZYMES

ENTWICKLUNG VON STÄMMEN DER WÄRMETOLERANTEN HEFE HANSENULA POLYMORPHA ZUR ALKOHOLISCHEN FERMENTIERUNG VON STÄRKE UND XYLAN DURCH EXPRESSION VON STÄRKE- UND XYLANABBAUENDEN ENZYMEN

DÉVELOPPEMENT DE SOUCHES DE LA LEVURE HANSENULA POLYMORPHA THERMOTOLERANTE CAPABLE DE LA FERMENTATION ALCOOLIQUE DE L'AMIDON ET XYLAN PAR L'EXPRESSION DE AMIDON ET ENZYMES XYLAN DÉGRADANTS


(84)Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

(30)Priority: 06.05.2008 US 50685 P

(43)Date of publication of application:
02.02.2011 Bulletin 2011/05

(73)Proprietor: Archer Daniels Midland Company
Decatur, IL 62526 (US)

(72)Inventors:
  • ABBAS, Charles
    Champaign, IL 61820 (US)
  • SIBIRNY, Andriy
    Lviv 79000 (UA)
  • VORONOVSKY, Andriy, Y.
    Lviv 79052 (UA)

(74)Representative: dompatent von Kreisler Selting Werner - Partnerschaft von Patent- und Rechtsanwälten mbB 
Deichmannhaus am Dom Bahnhofsvorplatz 1
50667 Köln
50667 Köln (DE)


(56)References cited: : 
EP-A2- 0 260 404
WO-A2-01/38510
US-A- 5 935 789
WO-A1-03/095653
DE-A1- 4 329 969
US-B1- 6 204 012
  
  • GELLISEN G ET AL: "HETEROLOGOUS GENE EXPRESSION IN HANSENULA POLYMORPHA: EFFICIENT SECRETION OF GLUCOAMYLASE", BIO/TECHNOLOGY, NATURE PUBLISHING CO. NEW YORK, US, vol. 9, 1 March 1991 (1991-03-01), pages 291-295, XP002062735, ISSN: 0733-222X, DOI: 10.1038/NBT0391-291
  • GELLISSEN ET AL: "PROGRESS IN DEVELOPING METHYLOTROPHIC YEASTS AS EXPRESSION SYSTEMS", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 10, no. 12, 1 December 1992 (1992-12-01), pages 413-417, XP001019012, ISSN: 0167-7799, DOI: 10.1016/0167-7799(92)90288-7
  • OLENA B RYABOVA ET AL: "Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha", FEMS YEAST RESEARCH, WILEY-BLACKWELL PUBLISHING LTD, GB, NL, vol. 4, no. 2, 1 November 2003 (2003-11-01), pages 157-164, XP002625819, ISSN: 1567-1356, DOI: 10.1016/S1567-1356(03)00146-6 [retrieved on 2006-01-09]
  • DMYTRUK O. ET AL.: "overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast h. polymorpha", FEMS YEAST RES, vol. 8, no. 1, 28 February 2008 (2008-02-28), pages 165-173, XP002674026,
  • VORONOVSKY, A.Y. ET AL.: 'Development of strains of the thermotolerant yeast Hansenula polymorpha capable of alcoholic fermentation of starch and xylan' METABOLIC ENGINEERING vol. 11, no. 4-5, 18 April 2009, pages 234 - 242, XP026439226
  • HEO, SY. ET AL.: 'Xylan Hydrolysis by Treatment with Endoxylanase and beta-Xylosiade Expressed in Yeast' J. MICROBIOL. BIOTECHNOL. vol. 14, no. 1, 2004, pages 171 - 177, XP008135574
  • RYABOVA, O.B. ET AL.: 'Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha' FEMS YEAST RESEARCH vol. 4, no. 2, November 2003, pages 157 - 164, XP002625819
  • OHTA, K. ET AL.: 'Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.' APPL ENVIRON MICROBIOL. vol. 57, no. 4, April 1991, pages 893 - 900, XP009005587
  • STRASSER, AW ET AL.: 'Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera.' EUR J BIOCHEM. vol. 184, no. 3, October 1989, pages 699 - 706, XP008004120
  • LA GRANGE DC ET AL.: 'Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta- xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes.' APPL ENVIRON MICROBIOL. vol. 67, no. 12, December 2001, pages 5512 - 5519, XP008135576
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Technical Field



[0001] This invention relates to the field of cellulosic ethanol production by fermentation, particularly to fermentation of starch carbon sources, still more particularly to recombinant H. polymorpha strains useful for the production of ethanol by fermentation on starch, and still more particularly to strains of H. polymorpha that excrete recombinant α-amylase and glucoamylase, to achieve ethanol production by fermentation on starch containing media.

Introduction



[0002] Fuel ethanol production from renewable raw materials, such as plant biomass, is of great economic and ecological significance. Plant lignocellulosics have the potential as alternative feedstocks to sucrose and starch-based polysaccharides that are currently widely in use for bioethanol production. Lignocellulosics and other plant derived polysaccahrides represent a renewable sustainable energy resource that can be reproduced by the bioconversion of carbon dioxide. One of the many touted environmental benefits of biofuels produced from plants over fossil fuels is the significant reduction in greenhouse gases [6, 24].

[0003] Most ethanol produced in the world today is derived from starch or sucrose. Starches and sugars are abundant in many crops, but expansion of ethanol production as a liquid transportation fuel will require feedstocks that do not compete directly with food or animal feed uses. Such feedstocks include lignocellulosic byproduct residues from agriculture and silviculture [14].

[0004] Lignocellulose is a generic term for plant matter derived from wood and agricultural residues. It is composed mainly of lignin and cellulose as well as significant amounts of hemicellulose with lesser quantities of structural proteins and organic solvent extractable matter [14]. Hemicellulose is a substituted polysaccharide that consist of xylan as the backbone and is present in plant cell walls [11].

[0005] Processing of lignocellulosics and starch to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/purification. Bioconversion of the starch involves enzymatic hydrolysis and fermentation of the resulting glucose to ethanol with the production of animal feed co-products. Hydrolysis of lignocellulose is more difficult by reason of its more complex structure and the great variation in its composition in different: plants (e.g., cereals, softwoods, hardwoods) and within a plant (e.g. stems, hulls, straws, cobs, stover, leaves, kernels) [14]. Xylose is the
major pentose sugar obtained upon hydrolysis of the hemicellulose fraction with the C5 L-arabinose and other C6 sugars such as glucose, mannose, galactose as the primary hexoses [11].

[0006] Due to the many steps involved and the high energy inputs required to process lignocellulosics, the development of more direct and less expensive technologies are needed for commercial viability of lignocelluloscis as feedstocks. The direct microbial conversion (DMF, Direct Microbial Fermentation) of carbohydrate polymers is one option that can improve the economics of bioethanol production from lignocellulosics. One of the key prerequisites for development of this technology is obtaining microorganisms capable of direct starch and xylan fermentation to ethanol at elevated temperatures [15]. The optimal temperature of the current hydrolytic enzymes involved in DMF is approximately 50 °C whereas most of the microorganisms currently used for bioethanol production from lignocellulosic and starchy sugars are mesophiles with optimum of growth and fermentation temperatures between 28 and 40 °C [6].

[0007] Recent studies in our lab indicate that the thermotolerant methylotrophic yeast, Hansenula polymorpha, is able to ferment D-xylose and D-glucose to ethanol at elevated temperatures (37-48 °C). In view of its high optimum temperature for growth and fermentation, H. polymorpha is a good candidate for further development of DMF technology [3, 26]. Since H. polymorpha cannot utilize starch and xylan as carbon and energy sources, the cloning and overexpression of heterologous xylanolytic and amylolytic genes in this yeast is necessary.

[0008] β-1,4-xylans are heterogeneous polysaccharides found in almost all parts of the cell wall of plants. The β-1,4-linked xylose monomers form a backbone chain to which several substituents are attached [30]. Hydrolysis of the xylan backbone is catalyzed by endo-β-1,4-xylanases (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) and β-D-xylosidases (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37). Endo-p-xylanases act on xylans and xylooligosaccharides, producing mainly mixtures of xylooligosaccharides. β-D-Xylosidases hydrolyze xylo-oligosaccharides to D-xylose [19]. The fungi Trichoderma and Aspergillus secrete large amounts of efficient xylan-degrading enzymes. Trichoderma reesei is a filamentous mesophilic fungus known for its cellulolytic and xylanolytic activities [3]. The two major inducible endo-xylanases secreted by this fungus are Xynl and Xyn2. Xyn2 represents more than 50% of the total xylanolytic activity of T. reesei cultivated on xylan. Members of the genus Aspergillus are also efficient producers of cellulolytic and xylanolytic enzymes. The xlnD gene of A. niger encoding the 804-amino acid β-xytosidase was successfully expressed in yeast [19].

[0009] Starch is composed of two high molecular weight fractions: amylose and amylopectin. Amylose, the minor component (20-30%) is a linear polysaccharide formed by α-1,4-linked glucose residues and some α-1,6- branching points, while amylopectin represents the major fraction of starch (70-80%) and is highly branched [4]. Starch is degraded by two secreted amylases: α-amylase and a glucoamylase [25]. α-Amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) catalyzes the endoamylolytic cleavage of α-1,4-glucosidic linkages of starch and similar substrates releasing maltose, oligosaccharides and limit dextrins. Glucoamylase (1,4-α-D-glucanglucohydrolase, EC 3.2.1.3) hydrolyzes glucooligosaccharides and maltose to D-glucose. The yeast Schwanniomyces occidentalis produces amylolytic enzymes and ferments starch to ethanol with high efficiency [34]. The α-Amylase secreted by this yeast is encoded by the SWA2 gene. The GAM1 gene encodes the secretory glucoamylase.

[0010] Some agricultural lignocellulosic residues obtained from the processing of cereal grains such as corn (example: corn fiber hulls) contain a significant quantity of starch. For this reason developing microbial strains capable of active direct conversion of both starch and lignocellulose to ethanol is of great economical significance.

[0011] EP 0260404 discloses a yeast strain comprising a gene encoding an alpha-amylase and a gene encoding a glucoamylase for fermentation using starch as a carbon source and teaches chromosome integration of the genes of interest and secretion of said enzymes.

[0012] Heo, SY., et al., (2004) J. Microbiol. Biotechnol. Vo1.14(1): 171-177 discloses a yeast strain comprising a gene encoding an endoxylanase and a gene encoding a beta-xylosidase for fermentation using xylan as a carbon source.

[0013] Ryabova, O.B., et al., (Nov 2003) FEMS Yeast Research, Vol.4(2):157-164 teaches that wild-type strains of the thermotolerant methylotrophic yeast H. polymorpha are able to ferment glucose, cellobiose and xylose to ethanol.

SUMMARY



[0014] Described herein are amylolytic and xylanolytic strains of H. polymorpha capable of direct alcoholic fermentation of starch and xylan. Here we describe construction of the strains by successful insertion into the chromosome and expression of the genes Sew. occidentalis SWA2 and GAM1, T. reesei XYN2 and A. niger xlnD in this yeast. Also, a strain that over expresses pyruvate decarboxylase (PDC) was engineered with one or more of these genes. In each case the strains were able to grow solely on media containing either solubilized starch or solubilized xylan and were able to ferment the same into ethanol at various levels. The strains that also over expressed PDC gave higher ethanol titers than strains that only overexpressed the SWA2 and GAM1 genes.

[0015] The present invention provides a H. polymorpha strain comprising a gene encoding an a-amylase enzyme according to SEQ ID NO: 2 and a gene encoding a glucoamylase enzyme according to SEQ ID NO: 4, the genes being operably linked to a HpGAP promoter obtained from H. polymorpha that expresses said genes in the H. polymorpha strain, each of the genes encoding the α-amylase and glucoamylase are integrated into the H. polymorpha chromosome, and further including a gene encoding a pyruvate decarboxylase enzyme from H. polymorpha operably linked to a HpGAP promoter that expresses said pyruvate decarboxylase in the H. polymorpha strain.

[0016] The present invention further provides a process for making ethanol comprising growing the H. polymorpha strain in a media comprising soluble starch under conditions that cause the H. polymorpha to make ethanol.

[0017] In a particular embodiment, the H. polymorpha strain further comprises a gene encoding an endoxylanase enzyme according to SEQ ID NO: 8, and a gene encoding a β-xylosidase enzyme according to SEQ ID NO: 6, each gene being operably linked to a HpGAP promoter obtained from H. polymorpha that expresses said genes in the H. polymorpha strain.

Description of the Drawings



[0018] 

Figure 1. Linear schemes of the plasmids pGAM1 (∼6.63 kb), pGAM1SWA2 (∼ 9.1 kb), pOR1 (∼10.8 kb) and pOR11 (∼10 kb). The S. cerevisiae genome fragment containing the LEU2 gene is shown as grey box, the HpGAP promoter is shown as green box, the HpAOX terminator: orange box, the ORF of the GAM1 gene is shown as blue box, the ORF of the SWA2 gene: red box, the aminoglycoside 3-phosphotransferase gene (APH): black box. Restriction sites: H, Hind III; RV, EcoRV; K, Kpn I; RI, EcoR I; Sl, Sal I; BI, BamH I; Bg, Bgl II; Sc, Sac I; P, Pst I; Sm, Sma I.

Figure 2. Linear schemes of the plasmids pKO8-GAPpr (7.6 kb), pKO8-GAPpr_SWA2 (9.1 kb) and pKO8-GAPpr_XYN2 (8.27 kb). The S. cerevisiae genome fragment containing the LEU2 gene is shown as grey box, the HpGAP promoter is shown as yellow box, the HpAOX terminator: blue box, the ORF of the SWA2 gene: red box, the ORF of TrXYN2 gene: orange box. Restriction sites: H, Hind III; Nd, Nde I; K, Kpn I; RI, EcoR I; Sl, Sal I; BI, BamH I; Bg, Bgl II; Sc, Sac I; P, Pst I; Nt, Not I.

Figure 3. Linear schemes of plasmids pXYN2 (∼ 4.24 kb), pXYN2xlnD (∼7.61 kb) and pOR2 (∼9.57 kb). Grey box, the S. cerevisiae genome fragment containing the LEU2 gene; green box, the HpGAP promoter; orange box, the HpAOX terminator; blue box, the fragment containing ORF of A. niger xlnD gene; red box, the fragment containing ORF of T. reseei gene XYN2. Restriction sites: H, Hind III; RV, EcoRV; K, Kpn I; RI, EcoR I; Sl, Sal I; B, BamH I; Bg, Bgl II; Sc, Sac I; P, Pst I; Sm, Sma I; Xb, XbaI; Sp, Sph I.

Figure 4. Ethanol production by the H. polymorpha recombinants 2Eth- leu1-1/pOR1 # 14' (A) and # 7 (B) in the minimal YNB medium with the 2 % soluble starch as sole carbon source with different medium pH: 1 - without pH adjacent; 2 - pH 6; 3 - pH 5.5; 48°C, 135 rpm.

Figure 5. Ethanol production by the H. polymorpha recombinant strain 495 2Eth-leu1-1/pOR2 in the minimal YNB medium with 3% or 9% birchwood xylan with different pH medium (A) and aeration conditions (B). 48°C.

Figure 6. Demonstration of genes GAM1, SWA2, XYN2, and xlnD under the HpGAP promoter in genomic DNA of H. polymorpha transformants by means of PCR. Lane ## 2, 3: the pair of primers K43, Ko51 was used for analysis of the transformants obtained by transformation with the plasmid pOR2 to show the artificial construct: T. reesei XYN2 ORF fused with the HpGAP promoter and HpAOX terminator. Lane ## 4, 5: the pair of primers K43, Ko47 was used for analysis of the transformants obtained by plasmid pOR2 to show the construct: A. niger xlnD fused with the HpGAP promoter. Lane ## 6, 7: the pair of primers K43, Ko49 was used for analysis the transformant obtained by plasmids pOR1 and pOR11 to show the construct: GAM1 of Sew. occidentalis fused with the HpGAP1 promoter. Lane ## 8, 9: the pair of primers K43, Ko50 was used for analysis of the transformants obtained by plasmids pOR1 and pOR11 to show the construct: ORF of Sew. occidentalis SWA2 fused with the HpGAP1 promoter and HpAOX term inator. Lane ## 1, 10: DNA markers.

Figure 7. Formation of the clear halos by the H. polymorpha recombinants that express the Scw.occidentalis SWA2 and GAM1 genes driven by the HpGAP1 promoter. The control Scw.occidentalis strain does not produce a halo on the medium supplemented with glucose (B) because of repression of native promoters of SWA2 and GAM1 genes. The second control 2Eth- leu1-1 strain cannot grow on the medium with starch as sole carbon source (A).

Figure 8. Ethanol production by the H. polymorpha recombinant strains 2Eth- leu1-1/pOR1 ## 7 and 14' in the minimal YNB medium with 2% of the soluble starch, 48°C, 135 rpm.

Figure 9. SDS-PAGE analysis of cultural media of the recombinant strains # 7, 14 (495 2Eth- leu1-1/pOR1), 1', 2' 4', 2g (495 2Eth- leu1-1/pOR11) and recipient strain 495 2Eth- leu1-1. Electrophoresis was carried out using 8% separation gel, protein bands were visualized by Coomasie and Silver staining. A, visualization of glucoamylase; B, visualization of α-amylase; the diffuse bands of α-amylase presumably are because of differing degrees of glycosylation. L, protein molecular weight marker.

Figure 10. Formation of clear halos by the H. polymorpha recombinants # #7, 14 (495 2Eth- leu1-1/pOR1) and multicopy integrants ## 1', 2', 3', 4' and 2g (495 2Eth- leu1-1/pOR11).

Figure 11. Results of dot-blot Southern hybridization that illustrate a copy number of genes encoding amylolytic enzymes in H. polymorpha transformants. The promoter of HpGAP was used as a probe. Strains: 1 - wt (1 copy standard); 2, 3 - ## 7, 14 (about 3 - 4 copies); 4, 5, 7 - ##1', 2', 4', respectively (about 6 - 8 copies); 6 - # 3' (∼3 copies).

Figure 12. Specific activity of α-amylase (A) and glucoamylase (B) in cultural media of the recombinant yeast strains # # 7, 14, 6 (495 2Eth- leu1-1/pOR1) containing 3 - 4 copies of amylase genes and # # 1', 2' 4', 2g (495 2Eth- leu1-1/pOR11) containing 6 - 8 copies of the genes.

Figure 13. Ethanol production by the H. polymorpha recombinants 2Eth- leu1-1/pOR1 (# 1: an average of ethanol production of the strains ## 7 and 14) and 2Eth- leu1-1/pOR11 (# 2: an average of ethanol production of the strains ## 1', 2', 4', 2g). The strains were cultivated in the minimal YNB medium with 3% of soluble starch at pH 5.5, 48°C, 135 rpm.

Figure 14. Ethanol production by the H. polymorpha strains overexpressing genes of amylolytic enzymes and PDC1 gene in the minimal medium with 3% soluble starch; 48°C, 135 rpm. 4', the transformant 495 2Eth- leu1-1/pOR11 (recipient strain, control); 6p, 10, 12, the transformants 4'/ploxZeoloxPDC1Hp.

Figure 15. Formation of clear halos by the H. polymorpha recombinant #6p (4'/ploxZeoloxPDC1Hp) and its derivatives: integrants ## 1, 2, 2-1, 3, 4, 5, 7, 9, 10 (6p/pOR1).

Figure 16. Ethanol production by the H. polymorpha strains overexpressing amylolytic enzymes and Pdclp in the minimal medium with 3% soluble starch at 47°C with restricted aeration. 4', the transformant 495 2Eth- leul1-1/pOR11; 6p, the transformant 4'/pZeoloxPDC1Hp; 3, 4, 5, the transformants 6p/pOR1.

Figure 17. Growth of the H. polymorpha recombinants expressing endoxylanase and β-xylosidase genes on the medium with xylan as sole carbon source. A, Growth of the recombinant strains expressing endoxylanase and β-xylosidase genes (strains ## 6x and 8x) on the solid medium with xylan as sole carbon source. B, Biomass accumulation by the strains expressing endoxylanase and β-xylosidase genes during growth in the liquid minimal medium with 3% xylan from birchwood (B1) or 2% xylose (B2), 48°C, 240 rpm. 6x, 8x, the transformants 495 2Eth- leu1-1/pOR2.

Figure 18. Formation of yellow (A) or clear (B) halos by the H. polymorpha recombinants expressing the A. niger xlnD and T. reseei XYN2 genes.

Figure 19. Specific activity of the endoxylanase (A) and β-xylosidase (B) in culture medium of the H. polymorpha recombinant strains 495 2Eth-/pOR2 ## 6x and 8x.

Figure 20. A. Sequence of a recombinant construct containing the H. polymorpha GAP promoter (single underscore) operably linked to a SWA2 gene (SEQ. ID NO 1) and to the H. polymorpha AOX terminator (double underscore). B. Amino acid sequence of the α-amylase (SEQ. ID NO2) from Schwanniomyces occidentalis encoded by the SWA2 gene.

Figure 21. A. Sequence of a recombinant construct containing the H. polymorpha GAP promoter (single underscore) operably linked to a GAM1 gene (SEQ. ID NO 3) and to the H. polymorpha AOX terminator (double underscore). B. Amino acid sequence of the glucoamylase (SEQ. ID NO 4) from Schwanniomyces occidentalis encoded by the GAM1 gene.

Figure 22. A. Sequence of a recombinant construct containing the H. polymorpha GAP promoter (single underscore) operably linked to an xlnD gene (SEQ. ID NO 5) of A. niger including its endogenous terminator sequence (SEQ. ID NO 9, double underscore). B. Amino acid sequence of the β-xylosidase (SEQ. ID NO 6) encoded by the xlnD gene.

Figure 23. A. Sequence of a recombinant construct containing the H. polymorpha GAP promoter (single underscore) operably linked to an Xyn2 gene (SEQ. ID NO 7) from Trichoderma reesei including its endogenous terminator sequence (SEQ. ID NO 10, double underscore). B. Amino acid sequence of the endo-β-xylanase (SEQ. ID NO 8) encoded by the Xyn2 gene.


Detailed Description of Methods, Strains, and Results


Strains and media.



[0019] The H. polymorpha strain 495 2Eth- leu1-1 deficient in β-isopropylmalate dehydrogenase and unable to grow on ethanol [14], was used as a recipient for isolation of the amylolytic and xylanolytic recombinants. This strain is a derivative of NCYC 495 leu1-1 [8]. Yeast strains and transformants were grown on YPD (0.5% yeast extract, 1% peptone, 2% glucose) or minimal medium (0.67% YNB without amino acids, 2% glucose, 3% soluble starch (Sigma S2630-500G) or 3% xylan from birchwood (Fluka X0502-100G) at 37 °C or 48 °C. For the 495 2Eth- leu1-1 strain, leucine (40 mg/l) was added to the medium. For the selection of yeast transformants on YPD, 0.2 mg/l of G418 (geneticin) or 150 µg/ml of zeocine were added.

[0020] The E. coli DH5α strain [ΦD80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(r-K, m+K), supE44, relA1, deoR, Δ(lac- ZYA-argF)U169] was used as a host for propagation of plasmids. The strain was grown at 37 °C in LB medium as described previously [27]. Transformed E. coli cells were maintained on a medium containing 100 mg/l of ampicillin.

DNA techniques.



[0021] Standard cloning techniques were applied [27]. Plasmid DNA isolation from E. coli was performed with the Wizards Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA). Genomic DNA of H. polymorpha, Scw. occidentalis, T. reesei and A. niger was isolated using the Wizards Genomic DNA Purification Kit (Promega, Madison, WI, USA). Restriction endonucleases, T4 DNA ligase and T4 DNA polymerase (Fermentas, Vilnius, Lithuania) were used according to the manufacturer specifications. DNA fragments were separated on 0.8% agarose (Fisher Scientific, Fair Lawn, NJ, USA) gel in 1xTAE [27]. Isolation of fragments from the gel was carried out with a DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). Taq DNA polymerase and High Fidelity Mix Polymerase (both Fermentas, Vilnius, Lithuania) were used for analytical and preparative PCR, respectively. PCRs were performed in GeneAmps PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). Transformation of the yeast H. polymorpha by electroporation was carried as described before [5].

Construction of plasmids carrying the α-amylase and glucoamylase genes of Scw. occidentalis.



[0022] The open reading frame (ORF) together with native terminator of the GAM1 gene encoding glucoamylase (∼3.27 kb) was isolated from the genomic DNA of the Schw. occidentalis strain NRRL Y-2470 by PCR using primers Ko48 (CCC AAG CTT ATG ATT TTT CTG AAG CTG) and Ko49 (GGA AGA TCT TTC TTT ACA AGA CCA ATG). Restriction sites Hind III and Bgl II were incorporated into the primers Ko48 and Ko49 (the cleavage sites are underlined). The PCR product was digested with Hind III and Bgl II restriction endonucleases and put under the strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase gene (HpGAPpr), digested with Hind III and Bam HI. The construct HpGAPpr+GAM1 was inserted into the Bam HI site of the plasmid pUC57 by means of double ligation. The resulting plasmid was named pGAM1 and used as a vector for the following constructions (Figure 1).

[0023] ORF of the SWA2 gene encoding α-amylase (∼2 kb) was amplified by PCR using primers SWA1 (TAG TCG CA TAT GAG ATT TTC AAC TGA AGG), SWA2 (CTA TTG ATT GCA GAT GCC AGA TCC C) and genomic DNA of Sew. occidentalis NRRL Y-247 as a template. The Nde I restriction site was incorporated into the SWA1 primer. 5'-end of the PCR product was digested with Nde I, whereas 3'-end was blunted. The product was inserted into the plasmid pKO8-GAPpr (Figure 2). ORF of the SWA2 gene was put under the HpGAPpr and fused with the HpAOX terminator (HpAOXtr). The constructed plasmid pKO8-GAPpr_SWA2 was used as a template for amplification by PCR of the DNA fragment containing HpGAPpr+SWA2_ORF+HpAOXtr using primers K43 (CCG GAT CCC AAT TAT CAT TAA TAA TC), Ko51 (CGC GGA TCC AAT CTT GCC TTT AAA ATG). The resulting PCR product was digested with Bam HI restriction endonuclease and inserted into the Bam HI site of the plasmid pGAM1. The constructed plasmid was named pGAM1SWA2 (Figure 1). The Saccharomyces cerevisiae LEU2 gene (selection marker) was inserted into the Pst I restriction site of the plasmid pGAM1SWA2 and resulting construct was named pOR1 (Figure 1).

[0024] The aminoglycoside 3-phosphotransferase gene (APH) conferring resistance to G418 in yeasts was isolated from the plasmid pGLG61 after its digesting by Pst I restriction endonuclease. The Pst I-fragment of pGLG61 [30] containing the gene was ligated with Pst I-portion of the plasmid pOR1 carrying recombinant genes GAM1, SWA2 and bacterial part, but without the S. cerevisiae LEU2 gene. Resulting plasmid was named pOR11 (Figure 1).

[0025] The sequence of the SWA2 and GAM1 recombinant constructs for expression in H. polymorpha are shown in Figures 20 and 21 respectively.

Construction of plasmids carrying the endoxylanase and β-xylosidase genes of T. reesei and A. niger, respectively.



[0026] The gene xlnD coding for β-xylosidase was derived from the fungus A. niger. ORF of the xlnD gene together with the native terminator (∼2.79 kb) was isolated from the genomic DNA of the A. niger strain NRRL 3 using primers Ko46 (TGC TCT AGA ATG GCG CAC TCA ATG TCT CG) and Ko47 (CCC GAG CTC AGC TAT GCT AGC AAG CAG C). The PCR product was treated with Sac I and Xba I restriction endonucleases (the sites of these endonucleases flank the product) and put under the HpGAPpr. The construct HpGAPpr+xlnD was inserted into the SacI site of the plasmid pUC57. The resulting plasmid was named pxlnD and used as a vector for the following constructions (Figure 3).

[0027] ORF of the XYN2 gene encoding endoxylanase (∼0.67 kb) without intron region was amplified by PCR using primers TR1 (TTC TCA CAT ATG GTT GCC TTT TCC AGC CCT CAT CTG CGC), TR2 (CTA GTT GCT GAC ACT CTG TGA GGC AGA ACC ACT ACC ACC), TRir (GAG CCG CCA AAG TTG ATG GGA GCA GAA GAT CCA GTC GTC), TRif (GAC GAC TGG ATC TTC TGC TCC CAT CAA CTT TGG CGG CTC) and genomic DNA of T. reseei NRRL 11460 as a template. 5'-end of the PCR product was digested with Nde I, whereas 3'-end was blunted. The product was inserted into the plasmid pKO8-GAPpr (Figure 2). ORF of the XYN2 gene was put under the HpGAPpr and terminated by the HpAOXtr. The resulting plasmid pKO8-GAPpr_XYN2 (Figure 2) was used as a template for amplification by PCR of the DNA fragment containing HpGAPpr+ORF_XYN2+HpAOXtr using primers K43 (CCG GAT CCC AAT TAT CAT TAA TAA TC), Ko50 (GGA AGA TCT AAT CTT GCC TTT AAA ATG). The resulting PCR product was digested with restriction endonucleases Bam HI and Bgl II and inserted into the Bam HI site of the plasmid pxlnD. The constructed plasmid was named pxlnDXYN2 (Figure 3).

[0028] The S. cerevisiae LEU2 gene was inserted into the Pst I site of the plasmid pxlnD XYN2 and the final plasmid was designated as pOR2 (Figure 3).

[0029] The sequence of the XylD and XYL2 recombinant constructs for expression in H. polymorpha are shown in Figures 22 and 23 respectively.

Screening for amylase activity.



[0030] Recombinant strains obtained after transformations with plasmids carrying genes of α-amylase and glucoamylase were screened for amylolytic activity after plating on minimal medium containing 2% soluble starch (Sigma) as a carbon source. Plates were incubated at 37 °C for 2 days followed by holding at 4 °C overnight. Amylolytic clones were detected by clear halos around colonies [4, 16].

Screening for endoxylanase activity.



[0031] Corresponding transformants were screened for xylan-degrading ability after being plated on a minimal medium containing 0.2% of 4-O-methyl-D-glucurono-D-xylan - remazol brilliant blue R (RBB)-xylan (Sigma) and 2% glucose as carbon source. Plates were incubated at 37 °C for 3 - 4 days. Endoxylanase cleaves RBB-xylan into a colorless product forming clear halos around the colonies [11, 20].

Screening for β-xylosidase activity.



[0032] Corresponding transformants were screened for β-xylosidase activity after being plated on a minimal medium containing ImM p-nitrophenyl-p-D-xyloside (PNPX) and 2% glucose as a carbon source. Plates were incubated at 37 °C for 1 - 3 hours. Enzyme activity was detected by production of yellow halos around the colonies [19].

α-Amylase and glucoamylase activity assays.



[0033] Appropriate dilutions of the cell-free culture were used for enzymes assays.

[0034] Total amylase activity was measured using the 3,5-dinitrosalicylic acid (DNS) method. A 50 µl aliquot of culture supernatant was incubated with 200 µl of 0.4M sodium acetate buffer (pH 5.0) containing 2% soluble starch for 30 min at 50 °C. The mixture was boiled for 10 min to stop the reaction. One unit of α-amylase activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar per ml per min under the same culture conditions [17, 22].

[0035] In the assay of glucoamylase activity, after keeping 0.9 ml of the starch solution boiled in sodium acetate buffer (pH 5.5) at 30 °C for 5 min, 0.1 ml of the sample was added and the mixture was incubated for 15 min. The reaction was then stopped by boiling the reaction mixture for 10 min and the concentration of glucose produced was determined using the "Diagluc" assay kit (UBT, Lviv, Ukraine) [9]. Activity of α-amylase was calculated by subtracting glucoamylase activity from total amylase activity. One unit of glucoamylase activity was defined as the amount of enzyme required to release 1 µmol of glucose per min from substrate [28].

β-xytosidase and endoxylanase activity assays.



[0036] Enzyme producing cultures were grown in 3 ml YPD overnight. Cells were collected by centrifugation and supernatant was used for enzymes activity measuring.

[0037] Endo-β-1,4-xylanase activity was assayed by the method described by Bailey et al. [1] with 1% birchwood xylan (Fluka) as the substrate at 50 °C. Appropriate dilutions of the cell-free culture solution in 50 mM sodium citrate buffer (pH 5.0) were used as the enzyme source. The amount of released sugar was determined by the dinitrosalicylic acid method [20]. The β-xytosidase activity was quantified using the chromophoric substrate PNPX at concentration of 5 mM. The supernatant was used as source of β-xytosidase for the activity determination assays. All activities were expressed in kat/ml; one katal is the amount of enzyme needed to produce 1 mol of reducing sugar from birchwood xylan or chromophoric substrate per second [19].

Ethanol production assay.



[0038] For ethanol production, H. polymorpha transformants were grown during 4 days in a liquid minimal medium containing 3% soluble starch or 3% xylan from birchwood at 48 °C, in semi-aerobic conditions. Concentrations of ethanol in the medium were determined after every 24 hr using the "Alcotest" kit [10].

Effects of pH and aeration on effectiveness of fermentation of starch to ethanol.



[0039] Optimal conditions for the direct fermentation of starch into ethanol by isolated transformants were studied. Yeast strains were pre-cultured at 48 °C in 50 ml of YPD medium in 125 ml Erlenmeyer flasks for 48 hours with agitation set at 220 rpm in the shaker Inkubator 1000 Heidolph (Schwabach, Germany). The cells were inoculated at concentration of 2 mg/ml into 50 ml of the minimal medium containing 3 % potato soluble starch as sole carbon source.

[0040] The effect of pH of the medium on ethanol production during starch fermentation was investigated. The optimum pH for α-amylase is ∼ 6.0 and for glucoamylase is 5.2 - 5.5. The fermentation was carried out using media with pH 5.5 and 6.0. A 1 M potassium phosphate buffer was used for adjusting the medium pH. The best ethanol production was in the medium with pH 5.5 (Figure 4A & B).

[0041] Effect of aeration on the fermentation efficiency was studied. Rotation rates from 120 to 180 rpm were tested. The highest ethanol production was in the case of the 135 rpm rotation.

Effects of pH, aeration and substrate concentration on effectiveness of fermentation of xylan to ethanol.



[0042] Optimal conditions for the fermentation of birchwood xylan to ethanol by isolated transformants were studied. The yeast strains were pre-cultured at 48 °C in 50 ml of YPD medium in 125 ml Erlenmeyer flasks for 48 hours with agitation set at 220 rpm. Cells were removed by centrifugation at 4000 rpm for 5 min, washed and inoculated (at concentration of 2 mg/ml) into 50 ml of a minimal medium containing 3% or 9% birchwood xylan and 0.05% of glucose as carbon sources.

[0043] The effect of pH of the medium on ethanol production during the xylan fermentation was investigated. Fermentation was carried out using media with pH 4.5 and 5.8. A 1M solution of potassium phosphate buffer was used to adjust medium pH with final concentration 0.1M in the medium. The best ethanol production was achieved in the medium with pH 5.8 (Figure 5A).

[0044] Effect of aeration on the fermentation efficiency was studied. Rotation rates from 120 to 180 rpm were tested. The highest ethanol production was in the case of 120 rpm (Figure 5B). The higher xylan concentration (9%) resulted in better ethanol production (Figure 5).

Southern blot hybridization.



[0045] The labeling of probe DNA and hybridization was performed using the non-radioactive Amersham ECL Direct Nucleic Acid Labeling and Detection System (GE Healthcare, USA) according to the manufacturer's manual. For quantitative Southern dot-blot, preparations of serial dilutions of yeast genomic DNAs were denatured in 0.4 M NaOH, spotted onto dry nylon membrane (Hybond N+, Amersham Pharmacia Biotech) and labeled with appropriate DNA fragments, followed by visualization with the Amersham ECL detection kit as above. HpGAPpr was used as a probe.

Gel electrophoresis.



[0046] SDS-PAGE was performed by method of Laemmli [19]. Concentrated proteins from cell-free extract were visualized by Silver staining and staining with Coomassie brilliant blue. The running and stacking gel concentration was 12% and 5% of poly acryl amid respectively. 20 µl of Laemmli solution added to 20 µl of sample and 35 µl of mixture was injected to the running gel [31].

Results


Expression of SWA2 and GAM1 genes of Scw. occidentalis in H. polymorpha.



[0047] The H. polymorpha strain 495 2Eth- leu1-1 was used as a recipient for transformation by the Sph I linearized plasmid pOR1 (scheme of the plasmid is shown in Figure 1). After transformation cells were plated on a minimal medium supplemented with 2% glucose and 1% soluble starch. Among ∼ 140 transformants the 14 that formed the largest amylolytic clear halos were picked out. Presence of SWA2 and GAM1 genes under the HpGAPpr in these transformants was shown by PCR using corresponding primers (Figure 6). The transformants were able to grow on soluble starch and ferment the substrate to ethanol at 37 and 48 °C. Efficient secretion of amylases with the integrants was shown by formation of clear halos around the colonies (Figure 7A & B). The best selected integrant (2Eth- leu1-1/pOR1 # 7) produced over 3 g/l of ethanol after 48 hours of fermentation in a minimal medium with 3% soluble starch at pH 5.5, 48 °C (Figure 8). To obtain H. polymorpha strains with improved amylolytic activity we tried to increase the SWA2 and GAM1 copy numbers. With this aim the plasmid pOR11 (Figure 3) carrying the dominant marker (APH gene that confers resistance to G418) and genes SWA2 and GAM1 was used. The H. polymorpha strain 495 2Eth- leu1-1 was used as a recipient for transformation by the Sph I linearized plasmid pOR11. After transformation, cells were plated on a YPD medium with 0.2 g/l G418. 5 stable transformants were found and picked out among ∼ 80 resulting G418-resistant colonies. Presence of the SWA2 and GAM1 genes under the HpGAPpr in these transformants was shown by PCR using corresponding primers (Figure 6). Production of recombinant enzymes (α- and glucoamylase) by these strains was demonstrated by the SDS-PAGE (Figure 9A & B).

[0048] Isolates following transformation with the plasmid pOR11 formed larger amylolytic clear halos in comparison with the best transformants isolated earlier from the pOR1 plasmid (Figure 10). Southern hybridization demonstrated presence of approx. 6 - 8 copies of the SWA2 and GAM1 genes in the isolated transformants (Figure 11).

[0049] The transformants 495 2Eth- leu1-1/pOR11 showed higher activity of both α- and glucoamylase when compared with strains which contain only 3 - 4 copies of the amylase genes (Figure 12A & B).

[0050] Effectiveness of alcoholic starch fermentation of isolated transformants was studied. These transformants showed elevated ethanol production (6.5 g/l after 72 hr of cultivation) as compared with transformants isolated earlier by the pOR1 transformation. The fermentation was carried out in the minimal YNB medium with 3% of soluble starch at pH 5.5, 48 °C, 135 rpm (Figure 13).

Isolating the strains of H. polymorpha with improved properties of starch fermentation.



[0051] The plasmid ploxZeoloxPDC1Hp constructed earlier in our laboratory [13], was used for obtaining of H. polymorpha strains with improved amylolytic properties. Following overexpression of the pyruvate decarboxylase gene (PDC1), these strains were characterized with improved ethanol production when compared to the control strain 495 2Eth- [13] The plasmid ploxZeoloxPDC1Hp that contains the PDC1 gene driven with the HpGAPpr was linearized by BamHI and used for transformation of the strain #4', isolated earlier (495 2Eth-/pOR1). Zeocine resistant transformants were selected on a YPD medium supplemented with 150 µg/ml of zeocine. Some of the stable integrants were selected for further study. Effectiveness of the alcoholic fermentation of these recombinants was studied. All of the transformants showed higher levels of the ethanol production (7 - 8 g/l) compared with the 4' strain (up to 4 g/l ; Figure 14).

[0052] The strain #6 (the transformant 4'/ ploxZeoloxPDC1Hp, which showed the highest level of the ethanol production) was used as a recipient for transformation with the plasmid pOR1 linearized with Sph I. The Leu+ transformants were selected on a minimal medium without leucine and stabilized. Stable transformants were plated on a minimal medium supplemented with 2% soluble starch. Some of the integrants, which formed larger clear halos in compare with the strain #6, were selected for further study (Figure 15). Effectiveness of alcoholic fermentation of these recombinants was studied. All of the transformants showed higher levels of the ethanol production (9 - 10 g/l) when compared with the strain #6 (Figure 16).

Expression of T.reesei XYN2 and A. niger XLND genes in H. polymorpha.



[0053] The plasmid pOR2 containing the T. reesei XYN2 and A. niger xlnD genes driven with the HpGAPpr (Figure 3) was linearized by SphI and used for transformation of the H. polymorpha strain 495 2Eth- leu1-1. Leu+ transformants were stabilized. The presence of the XYN2 and xlnD genes under the HpGAPpr in transformants was tested by PCR using corresponding primers (Figure 6).

[0054] Transformants were able to grow on xylan as the sole carbon source (Figure 17, A, B1 and B2). Efficient secretion of the endoxylanase and β-xylosidase with the integrants was shown by formation of clear or yellow halos on media containing 0.2% RBB-xylan or ImM PNPX, respectively (Figure 18A and B). The activity of both enzymes was measured (Figure 19A and B). The transformants were able to ferment birchwood xylan to ethanol with low efficiency (approx. 0.35 g/l) at both 37 and 48°C.

Co-expression of T.reesei XYN2, A. nieer XLND, with Scw, occidentalis SWA2 and GAM1 genes of in H. polymorpha.



[0055] In a first case, the transformants 495 2Eth- leu1-1/pOR11 containing both the α-amylase and glucoamylase genes from Scw. occidentalis intetgrated into the chromosome of H. polymorpha is usedas the host strain. The plasmid pOR2 containing the T. reesei XYN2 and A. niger xlnD genes driven with the HpGAPpr is linearized by SphI and used for transformation of the H. polymorpha strain 495 2Eth- leu1-1/pOR11. The transformants are stabilized as before. The presence of the XYN2 and xlnD genes is tested by PCR using corresponding primers Transformants will be able to grow with soluble starch and/or soluble xylan as the sole carbon source. Efficient secretion of all four enzymes will be demonstrated essentially as described above. The transformants will be able ferment a mixed media containing both soluble starch and birchwood xylan into ethanol at both 37 and 48 °C.

[0056] In a second case, the transformant described above, having both the α-amylase and glucoamylase genes already transformed in to the strain, 4'/ ploxZeoloxPDC1Hp, which over expresses the PDC gene is used as the host strain. Again, the plasmid pOR2 containing the T. reesei XYN2 and A. niger xlnD genes driven with the HpGAPpr is linearized by SphI and used for transformation of the H. polymorpha host strain. The transformants are stabilized as before. The presence of the XYN2 and xlnD genes is tested by PCR using corresponding primers Transformants will be able to grow with soluble starch and/or soluble xylan as the sole carbon source. Efficient secretion of all four enzymes will be demonstrated essentially as described above. The transformants will be able ferment a mixed media containing both soluble starch and birchwood xylan into ethanol at both 37 and 48°C at a level higher than the transformants made in the first case
due to the complimentary over expression of the PDC enzyme in the host cell.

Discussion



[0057] Fuel ethanol production from renewable plant material has a great economic and ecological significance. One of the byproducts from the corn wet milling industry that is readily available in large quantities is the corn fiber hull fraction. This fraction is mixed and dried with other processing byproducts and stillage fraction from ethanol fermentation to produce corn gluten feed. Corn fiber hulls consist of 35% hemicellulose, 18% cellulose and 20% starch (protein, fiber oil and lignin are also present in this material) [7]. Xylan is the major component of hemicellulose. Industrial steps which include enzymatic hydrolysis of xylan and starch are very expensive. Therefore the direct microbial conversion of these polymers to ethanol is of great economical significance. For this reason developing microorganisms capable of simultaneous hydrolysis of starch and xylan and fermentation of the released sugars to ethanol at elevated temperatures has a great importance for fuel ethanol production from corn. Glucose and xylose are the main sugars released after starch and xylan hydrolysis, respectively. The yeast H. polymorpha ferments glucose and xylose to ethanol at high temperatures. However, H. polymorpha cannot utilize starchy materials and xylan and grow on them as on sole carbon sources.

[0058] We cloned two genes from the yeast Sew. occidentalis SWA2 and GAM1 which encode α-amylase and glucoamylase, respectively. Both of these enzymes are needed for starch hydrolysis. SWA2 and GAM1 genes were successfully expressed in H. polymorpha. Isolated recombinant strains are capable to grow on starch as sole carbon source. They are also able to ferment soluble starch to ethanol at 48 °C. We showed that increasing gene copy numbers improves the capability of recombinant strains to starch hydrolysis and ethanol production.

[0059] Genes of T. reesei XYN2 and A. niger xlnD encoding endoxylanase and β-xylosidase, respectively, were cloned and expressed in H. polymorpha. At least two of these enzymes
are necessary for xylan hydrolysis. Isolated integrants were capable of growth on xylan as sole carbon source and fermentation of it to ethanol at both 37 and 48 °C. The low
effectiveness of conversion of xylan to ethanol by isolated strains is most likely due to the initial low capability of H. polymorpha strains for xylose alcoholic fermentation. Further improvement in xylan fermentation by the strains constructed will require improved ethanolic fermentation of xylose as a prerequisite. These strains can then be used as recipients for construction of effective xylanolytic recombinants.

References



[0060] 
  1. 1. Bailey, M.J., P. Biely, and K. Poutanen. 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23: 257-270.
  2. 2. Banat, I.M., P. Nigam, D. Singh, P. Marchant, and A.P. McHale. 1998. Ethanol production at elevated temperatures and alcohol concentrations. Part I: yeasts in general. World J Microbiol Biotechnol. 14: 809-821.
  3. 3. Dmytruk, O. V., A. Y. Voronovsky, C. A. Abbas, K. V. Dmytruk, O. P. Ishchuk, and A. A. Sibirny. 2008. Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha. FEMS Yeast Res. 8: 165-173.
  4. 4. Eksteen, J.M., P. van Rensburg, R.R. Cordero Otero, and I.S. Pretorius. 2003. Starch fermentation by recombinant Saccharomyces cerevisiae strains expressing the α-amylase and glucoamylase genes from Lipomyces kononenkoae and Saccharomycopsis fibuligera. Biotechnol. Bioengineer. 84: 639-646.
  5. 5. Faber, KN, P. Haima, W. Harder, M. Veenhuis, and G. Ab. 1994. Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr. Genet. 25: 305-310.
  6. 6. Fujita, Y., and J. Ito. 2004. Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl. Environ. Microbiol. 70: 1207-1212.
  7. 7. Gaspar, M., G. Kalman, and K. Reczey. 2007. Corn fiber as a raw material for hemicellulose and ethanol production. Process Biochem. 42: 1135-1139.
  8. 8. Gellissen, G., G. Kunze, C. Gaillardin, J.M. Cregg, E. Berardi, M. Veenhuis, and I. van der Klei. 2005. New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica a comparison. FEMS Yeast Res. 5:1079-1096.
  9. 9. Gonchar, M.V. 1998. Sensitive method for quantitative determination of hydrogen peroxide and oxidase substrates in biological samples. Ukr. Biokhim. Zh. 70: 157-163.
  10. 10. Gonchar, M.V., M.M. Maidan and A.A. Sibirny. 2001. A new oxidase-peroxidase kit for ethanol assays in alcoholic beverages. Food Technol. Biotechnol. 39: 37-42.
  11. 11. den Haan R., and W. H. van Zyl. 2001. Differential expression of the Trichoderma reesei β-xylanase II (xyn2) gene in the xylose-fermenting yeast Pichia stipitis. Appl Microbiol Biotechnol. 57: 521-527.
  12. 12. Haki, G.D., and S.K. Rakshit. 2003. Developments in industrially important thermostable enzymes: a review. Bioresource Technology. 89: 17-34.
  13. 13. Ishchuk, O. P., A. Y. Voronovsky, O. V. Stasyk, G. Z. Gayda, M. V. Gonchar, C. A. Abbas, and A. A. Sibirny. 2008. Improvement of xylose high-temperature fermentation in Hansenula polymorpha due to overexpression of the PDC1 gene coding for pyruvate decarboxylase. FEMS Yeast Res. In press.
  14. 14. Jeffries, T. W., and Y. S. Jin. 2000. Ethanol and thermotolerance in the bioconversion of xylose by yeasts. Adv. Appl. Microbiol. 47: 221 - 268.
  15. 15. Kadam, K. L., and S. L. Schmidt. 1997. Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass. Appl. Microbiol. Biotechnol. 48:709-713.
  16. 16. Kang, N. Y., J. N. Park, J. E. Chin, H. B. Lee, S. Y. Im, and S. Bai. 2003. Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene. Biotechnol. Lett. 25: 1847-1851.
  17. 17. Kιlιç, D., and B. Özbek. 2004. α-Amylase inactivation by temperature during starch hydrolysis. Process Biochem. 39: 1137-1144.
  18. 18. Kulkarni, N., A. Shendye, and M. Rao. 1999. Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23: 411-456.
  19. 19. La Grange, D. C., I. S. Pretorius, M. Claeyssens, and W. H.van Zyl. 2001. Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger β-xytosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes. Appl. Environ. Microbiol. 67: 5512-5519.
  20. 20. La Grange, D. C., I. S. Pretorius, and W. H. van Zyl. 1996. Expression of a Trichoderma reesei β-xylanase gene (XYN2) in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 62: 1036-1044.
  21. 21. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680 - 685.
  22. 22. Liu, Z., G. Zhang, and S. Liu. 2004. Constructing an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing. J. Biosci. Bioeng. 98: 414-419.
  23. 23. Marin, D., A. Jimenez, and M. Fernandez Lobato. 2001. Construction of an efficient amylolytic industrial yeast strain containing DNA exclusively derived from yeast. FEMS Microbiol. Lett. 201: 249-253.
  24. 24. Mosier, N., and C. Wyman. 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96: 673-686.
  25. 25. Piontek, M., J. Hagedorn, C. P. Hollenberg, G. Gellissen, and A. W. M. Strasser. 1998. Two novel gene expression systems based on the yeasts Schwanniomyces occidentalis and Pichia stipitis. Appl. Microbiol. Biotechnol. 50: 331-338.
  26. 26. Ryabova, O. B., O. M.Chmil and A. A.Sibirny. 2003. Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha. FEMS Yeast Res. 4: 157-164.
  27. 27. Sambrook, J., E. F. Fritsh, and T. Maniatis. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1989.
  28. 28. Shigechi, H., Y. Fujita, J. Koh, M. Uedac, H. Fukuda, and A. Kondo. 2004. Energy-saving direct ethanol production from low-temperature-cooked corn starch using a cell-surface engineered yeast strain co-displaying glucoamylase and α-amylase. Biochem. Eng. J. 18: 149-153.
  29. 29. Shigechi, H., J. Koh, Y. Fujita, T. Matsumoto, Y. Bito, M. Ueda, E. Satoh, H. Fukuda, and A. Kondo. 2004. Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and α-amylase. Appl. Environ. Microbiol. 70: 5037-5040.
  30. 30. Sohn, J. H., E. S. Choi, H. A. Kang, J. S.Rhee, M. O. Agaphonov, M. D. Ter-Avanesyan, and S. K. Rhee. 1999. A dominant selection system designed for copynumber-controlled gene integration in Hansenula polymorpha DL-1. Appl Microbiol Biotechnol. 51: 800-807.
  31. 31. Torronen, A., L. R. Mach, R. Massner, R. Gonzales, N. Kalkkinen, A. Harkki, and C. P. Kubicek. 1992. The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes. Biothechnology. 10: 1461-1465.
  32. 32. Ulgen, K. O., and B. Saygιlι. 2002. Bioconversion of starch into ethanol by a recombinant Saccharomyces cerevisiae strain YPG-AB. Process Biochem. 37: 1157-1168.
  33. 33. de Vries, R. P. and J. Visser. 2001. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol. Mol. Biol. Rev. 65: 497-522.
  34. 34. Wang, T. T., L. L. Lin, and W. H. Hsu. 1989. Cloning and expression of a Schwanniomyces occidentalis α-amylase gene in Saccharomyces cerevisiae. Appl. Environment. Microbiol. 55: 3167-3172.
  35. 35. Zaldivar, J., J. Nielsen, and L. Olsson. 2001. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol. 56: 17-



Claims

1. A H. polymorpha strain comprising a gene encoding an α-amylase enzyme according to Fig.20B and a gene encoding a glucoamylase enzyme according to Fig.21B, the genes being operably linked to a HpGAP promoter obtained from H. polymorpha that expresses said genes in the H. polymorpha strain, each of the genes encoding the α-amylase and glucoamylase are integrated into the H. polymorpha chromosome, and further including a gene encoding a pyruvate decarboxylase enzyme from H. polymorpha operably linked to a HpGAP promoter that expresses said pyruvate decarboxylase in the H. polymorpha strain.
 
2. A process for making ethanol comprising growing the H. polymorpha strain of claim 1 in a media comprising soluble starch under conditions that cause the H. polymorpha to make ethanol.
 
3. The H. polymorpha strain of claim 1 further comprising a gene encoding an endoxylanase enzyme according to Fig. 23B, and a gene encoding a β-xytosidase enzyme according to Fig.22B, each gene being operably linked to a HpGAP promoter obtained from H. polymorpha that expresses said genes in the H. polymorpha strain.
 


Ansprüche

1. H.-polymorpha-Stamm, der ein Gen, das ein α-Amylase-Enzym codiert, gemäß Figur 20B und ein Gen, das ein Glucoamylase-Enzym codiert, gemäß Figur 21B umfasst, wobei die Gene funktionell mit einem HpGAP-Promotor verknüpft sind, der aus H. polymorpha erhalten wurde und die Gene in dem H.-polymorpha-Stamm exprimiert, wobei die Gene, die die α-Amylase bzw. die Glucoamylase codieren, in das H.-polymorpha-Chromosom integriert sind, und der weiterhin ein Gen umfasst, das ein Pyruvat-Decarboxylase-Enzym aus H. polymorpha codiert und funktionell mit einem HpGAP-Promotor verknüpft sind, der die Pyruvat-Decarboxylase in dem H.-polymorpha-Stamm exprimiert.
 
2. Verfahren zur Herstellung von Ethanol, das das Wachsenlassen des H.-polymorpha-Stamms gemäß Anspruch 1 in einem Medium, das lösliche Stärke umfasst, unter Bedingungen, die bewirken, das das H. polymorpha Ethanol produziert, umfasst.
 
3. H.-polymorpha-Stamm gemäß Anspruch 1, der weiterhin ein Gen, das ein Endoxylanase-Enzym codiert, gemäß Figur 23B und ein Gen, das ein β-Xylosidase-Enzym codiert, gemäß Figur 22B umfasst, wobei die Gene jeweils funktionell mit einem HpGAP-Promotor verknüpft sind, der aus H. polymorpha erhalten wurde und die Gene in dem H.-polymorpha-Stamm exprimiert.
 


Revendications

1. Souche de H. polymorpha comprenant un gène codant pour une enzyme α-amylase selon la Fig.20B et un gène codant pour une enzyme glucoamylase selon la Fig.21B, les gènes étant liés de manière fonctionnelle à un promoteur HpGAP obtenu à partir de H. polymorpha qui exprime lesdits gènes dans la souche de H. polymorpha, chacun des gènes codant pour l'α-amylase et la glucoamylase est intégré dans le chromosome de H. polymorpha, et incluant en outre un gène codant pour une enzyme pyruvate décarboxylase provenant de H. polymorpha lié de manière fonctionnelle à un promoteur HpGAP qui exprime ladite pyruvate décarboxylase dans la souche de H. polymorpha.
 
2. Procédé de production d'éthanol comprenant la croissance de la souche de H. polymorpha selon la revendication 1 dans un milieu comprenant de l'amidon soluble dans des conditions qui amènent H. polymorpha à produire de l'éthanol.
 
3. Souche de H. polymorpha selon la revendication 1 comprenant en outre un gène codant pour une enzyme endoxylanase selon la Fig.23B, et un gène codant pour une enzyme β-xylosidase selon la Fig.22B, chaque gène étant lié de manière fonctionnelle à un promoteur HpGAP obtenu à partir de H. polymorpha qui exprime lesdits gènes dans la souche de H. polymorpha.
 




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Cited references

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