Prokaryotic xylose isomerase for the construction of xylose-fermenting yeasts

Abstract

The present invention relates to the use of nucleic acid molecules coding for a bacterial xylose isomerase (XI), preferably coming from Clostridium phytofermentans, for reaction/metabolization, particularly fermentation, of recombinant microorganisms of biomaterial containing xylose, and particularly for the production of bioalcohols, particularly bioethanol, by means of xylose fermenting yeasts. The present invention further relates to cells, particularly eukaryotic cells, which are transformed utilizing a nucleic acid expression construct which codes for a xylose isomerase, wherein the expression of the nucleic acid expression construct imparts to the cells the capability to directly isomerize xylose into xylulose. Said cells are preferably utilized for reaction/metabolization, particularly fermentation, of biomaterial containing xylose, and particularly for the production of bioalcohols, particularly bioethanol. The present invention also relates to methods for the production of bioethanol, and to methods for the production of further metabolization products, comprising the metabolization of media containing xylose.

Claims

1. A method for the recombinant expression and production of a functional xylose isomerase or for the conversion of xylose to xylulose by an isolated host cell, wherein said isolated host cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bulderi, Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces uvarum, Saccharomyces diastaticus, Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces fragilis, and wherein said method comprises transforming said host cell with a nucleic acid molecule comprising a nucleic acid sequence that encodes a Clostridium phytofermentans xylose isomerase (XI) having an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO: 1.

2. The method according to claim 1, wherein said method is used for: the conversion or metabolization of biomaterial containing xylose, the production of bio-based chemicals, or the production of biobutanol, bioethanol or of both biobutanol and bioethanol.

3. The method according to claim 1, wherein the nucleic acid molecule is a nucleic acid expression construct, which comprises promoter and terminator sequences, the promoter being operatively linked with the nucleic acid sequence encoding the Clostridium phytofermentans xylose isomerase (XI).

4. The method according to claim 3, wherein the nucleic acid expression construct further comprises one or more of 5 recognition sequences, 3 recognition sequences, and selection markers.

5. An isolated host cell, wherein said host cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bulderi, Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces uvarum, Saccharomyces diastaticus, Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces fragilis, and said host cell is transformed with a nucleic acid expression construct comprising: (a) a nucleic acid sequence encoding a Clostridium phytofermentans xylose isomerase (XI), having an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO: 1, and said nucleic acid sequence being operatively linked to a promoter allowing for the expression of the Clostridium phytofermentans xylose isomerase (XI) in the cell; or (b) a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2 and which is operatively linked to a promoter allowing for the expression of the Clostridium phytofermentans xylose isomerase (XI) in the cell, wherein the expression of the nucleic acid expression construct produces a functional xylose isomerase in the host cell and imparts to the host cell the capability to isomerize xylose into xylulose.

6. The isolated host cell according to claim 5, wherein the cell is transformed with a nucleic acid expression vector that is a nucleic acid expression construct, which comprises promoter and terminator sequences, the promoter being operatively linked with the nucleic acid sequence coding for a Clostridium phytofermentans xylose isomerase (XI), said nucleic acid sequence being at least 95% identical to the nucleic acid sequence SEQ ID NO: 2; wherein the nucleic acid expression construct optionally further comprises 5 and/or 3 recognition sequences and/or selection markers.

7. The isolated host cell according to claim 5, wherein the cell further expresses one or more enzymes that impart to the cell the capability to produce further metabolization products, the further metabolization products being selected from lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerol, a -lactam antibiotic or a cephalosporin, alkanes, terpenes, isoprenoids and the precursor molecule amorphadiene of the antimalarial drug artemisinin.

8. A method for the conversion and metabolization of biomaterial containing xylose to ethanol or for the production of bioethanol wherein said method utilizes a cell of claim 5; and wherein the ethanol yield is at least 0.3 g of ethanol per g of xylose.

9. A method for the production of a metabolization product, wherein said method utilizes a cell according to claim 5 and wherein the metabolization product is selected from lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerol, a -lactam antibiotic or a cephalosporin, alkanes, terpenes, isoprenoids and the precursor molecule amorphadiene of the antimalarial drug artemisinin.

10. The method for the production of bioethanol according to claim 8 comprising the steps of: (a) converting a medium containing a xylose source with said cell, which converts xylose to ethanol, (b) optionally obtaining the ethanol.

11. The method according to claim 10, wherein the medium contains a further carbon source.

12. The method according to claim 10, wherein the production of bioethanol takes place at a rate of at least 0.03 g of ethanol per g of yeast dry weight an hour.

13. A method for the production of a metabolization product comprising the steps of: (a) converting a medium containing a xylose source with a cell according to claim 7, which converts xylose to produce the metabolization product, (b) optionally obtaining the metabolization product, the metabolization product being selected from lactic acid, acetic acid, succinic acid, malic acid, 1-butanol, isobutanol, 2-butanol, other alcohols, amino acids, 1,3-propanediol, ethylene, glycerol, a -lactam antibiotic or a cephalosporin, alkanes, terpenes, isoprenoids and the precursor molecule amorphadiene of the antimalarial drug artemisinin.

14. The method according to claim 13, wherein the medium contains a further carbon source.

15. The method according to claim 1, wherein the nucleic acid sequence that encodes a Clostridium phytofermentans xylose isomerase (XI) is at least 95% identical to the nucleic acid sequence SEQ ID NO:2.

16. A method for the recombinant expression and production of a functional xylose isomerase or for the conversion of xylose to xylulose by an isolated host cell, wherein said isolated host cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bulderi, Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces uvarum, Saccharomyces diastaticus, Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces fragilis, wherein said method comprises: (a) isolating the host cell and transforming the host cell with a nucleic acid expression construct comprising: (i) a nucleic acid sequence encoding a Clostridium phytofermentans xylose isomerase (XI), having an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO: 1, and (ii) a promoter operatively linked with the nucleic acid sequence, allowing for the expression of the Clostridium phytofermentans xylose isomerase (XI) in the cell, (b) expressing the nucleic acid encoding the Clostridium phytofermentans xylose isomerase, which imparts to the host cell the capability to isomerize xylose into xylulose.

17. An isolated host cell, wherein said host cell is a yeast cell selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bulderi, Saccharomyces barnetti, Saccharomyces exiguus, Saccharomyces uvarum, Saccharomyces diustaticus, Kluyveromyces lactis, Kluyveromyces marxianus, and Kluyveromyces fragilis and said host cell is transformed with a nucleic acid expression construct comprising: (a) a nucleic acid sequence encoding a Clostridium phytofermentans xylose isomerase (XI), having an amino acid sequence that is at least 95% identical to the amino acid sequence SEQ ID NO: 1, and (b) a promoter operatively linked with the nucleic acid sequence, allowing for the expression of the Clostridium phytofermentans xylose isomerase (XI) in the cell, wherein the expression of the nucleic acid expression construct produces a functional xylose isomerase in the host cell and imparts to the host cell the capability to isomerize xylose into xylulose.

18. The isolated host cell according to claim 5, wherein: the Clostridium phytofermentans xylose isomerase (XI) has the sequence of SEQ ID NO: 1, or the nucleic acid encoding the Clostridium phytofermentans xylose isomerase (XI) has the sequence of SEQ ID NO: 2.

19. The method according to claim 1, wherein the yeast cell is Saccharomyces cerevisiae.

20. The isolated host cell according to claim 5, wherein the yeast cell is Saccharomyces cerevisiae.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Composition of biomass. Biomass consists of cellulose, hemicellulose and lignin. The second most occurring hemicellulose is a highly branched polymer consisting of pentoses, uronic acids and hexoses. To a large proportion, the hemicellulose consists of the pentoses xylose and arabinose.

(2) FIG. 2. Diagram of the conversion of D-xylose in recombinant S. cerevisiae by means of direct isomerization

(3) FIG. 3. Genealogical tree of the different xylose isomerases

(4) The genealogical tree of the tested xylose isomerases is depicted. Comparisons with regard to the similarity of the xylose isomerases were performed with the program MEGA version 4.

(5) FIGS. 4A-4C. Used vectors. The starting plasmid for the construction of p426H7-XI-Clos (4B) or p426H7-opt.XI-Clos (4C) was the plasmid p426HXT7-6HIS (4A). Vector p426HXT7-6HIS is a 2 expression plasmid, which has a URA3 marker. The open reading frame (ORF) and its codon-optimized form of the xylose isomerase from C. phytofermentans according to the invention, respectively, was cloned behind the truncated strong HXT7 promoter and the CYC1 terminator of the plasmid p426HXT7-6HIS.

(6) FIG. 5A-5C. Growth behaviour on medium containing xylose using the different xylose isomerase genes

(7) Growth tests of recombinant S. cerevisiae strains, which include the bacterial D-xylose metabolism with the xylose isomerase from C. phytofermentans. Growth tests were performed on agar plates with SC medium and 2% xylose as the only carbon source. The native (5B) and the codon-optimized form (5C) of the xylose isomerase from C. phytofermentans were tested. The empty vector p426HXT7-6HIS (5A) served as the negative control.

(8) FIG. 6. Xylose conversion in recombinant yeast strains using a bacterial xylose isomerase

(9) The xylose conversion of recombinant yeast cells MKY09, which contained the native and the codon-optimized form of the xylose isomerase from C. phytofermentans was tested. The empty vector p426HXT7-6HIS served as a comparison. Growth curves were performed in liquid SC medium with 1.4% xylose under aerobic conditions. HPLC samples were taken in parallel to measure the optical density at 600 nm. See also table 2, example 3.

(10) FIG. 7. Enzyme kinetics

(11) Eadie-Hofstee Plot of the Xylose Conversion of the Native and the Codon-Optimized Xylose Isomerase from C. phytofermentans

(12) The strain CEN.PK2-1C transformed with the plasmid p426H7-XI-Clos and p426H7-opt.XI-Clos, respectively, was grown over night in synthetic complete medium with 2% glucose and no uracil. Raw extracts were prepared and quantitative enzyme tests were performed. A representative result is shown. The values indicated in table 3 are average values from at least 3 independent measurements.

EXAMPLES

(13) Methods

(14) 1. Strains and Media

(15) Bacteria

(16) E. coli SURE (Stratagene)

(17) E. coli DH5 (Stratagene)

(18) Bacillus licheniformis (37 C.)

(19) Agrobacterium tumefaciens (26 C.)

(20) Burkholderia xenovorans (28 C.)

(21) Clostridium phytofermentans (30 C., anaerobic)

(22) Lactobacillus pentosus (30 C.)

(23) Leifsonia xyli (28 C.)

(24) Pseudomonas syringae pv. phaseolicola (28 C.)

(25) Robiginitalea biformata (30 C.)

(26) Saccharophagus degradans (26 C.)

(27) Salmonella typhimurium LT2 (28 C.)

(28) Staphylococcus xylosus (37 C.)

(29) Streptomyces diastaticus (28 C.)

(30) Xanthomonas campestris (26 C.)

(31) Other Organisms

(32) Arabidopsis thaliana (genomic DNA)

(33) Media and Cultivation of E. coli

(34) Complete Medium LB: 1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.5 (see Maniatis, 1982).

(35) For the selection for a plasmid-coded antibiotic resistance, 40 g/ml of ampicillin was added to the medium after autoclaving. Solid culture media additionally contained 2% agar. The cultivation took place at 37 C.

(36) Media and Cultivation of Further Bacteria

(37) Composition of the media and cultivation conditions, see information from the DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen, Brunswick, Germany).

(38) Yeast

(39) Strain CEN.PK2-1C

(40) CEN.PK2-1C (MATa leu2-3, 112 ura3-52 trpl-289 his3-1MAL2-8c SUC2)

(41) Strain MYKO9

(42) MKY09 is based on the strain CEN.PK2-1C (MATa leu2-3, 112 ura3-52 trpl-289 his3-1MAL2-8C SUC2, PromTKL1::loxP-Prom-vkHXT7, PromRPE1::loxP-Prom-vkHXT7, PromRKI1::loxP-Prom-vkHXT7, Prom GAL2::loxP-Prom-vkHXT7, PromXKS1::loxP-Prom-vkHXT7), including further unknown mutations.

(43) Media and Cultivation of Yeasts

(44) Synthetic complete selective medium SC: 0.67% yeast nitrogen base w/o amino acids, pH 6.3, amino acid/nucleobase solution, carbon source in the concentration respectively given

(45) Synthetic minimal selective medium SM: 0.16% yeast nitrogen base w/o amino acid and ammonium sulphate, 0.5% ammonium sulphate, 20 mM of potassium dihydrogenphosphate, pH 6.3, carbon source in the concentration respectively given

(46) Concentration of the amino acids and nucleobases in the synthetic complete medium (according to Zimmermann, 1975): adenine (0.08 mM), arginine (0.22 mM), histidine (0.25 mM), isoleucine (0.44 mM), leucine (0.44 mM), lysine (0.35 mM), methionine (0.26 mM), phenylalanine (0.29 mM), tryptophan (0.19 mM), threonine (0.48 mM), tyrosine (0.34 mM), uracil (0.44 mM), valine (0.49 mM). L-arabinose and D-glucose were used as the carbon source.

(47) 2. Plasmids

(48) Plasmids used

(49) TABLE-US-00001 Plasmid Source/reference Description p426HXT7- Hamacher et al., 2 expression plasmid for the 6HIS 2002 overexpression of genes and for the (=p426H7) production of a His.sub.6 epitope; URA3 selection marker gene, truncated HXT7 promoter and CYC1 terminator

(50) Plasmids constructed in the course of this work

(51) TABLE-US-00002 Plasmid Description p426H7-XI- Cloning of the XI from A. tumefaciens in p426HXT7-6HIS Agro omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from A. thaliana in p426HXT7-6HIS Arab omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from B. licheniformis in p426HXT7- BaLi 6HIS omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from B. xenovorans in p426HXT7-6HIS Burk omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI fromC. phytofermentans in p426HXT7- Clos 6HIS omitting the His.sub.6 epitope p426H7- Cloning of the codon-optimized XI from C. opt.XI-Clos phytofermentans in p426HXT7-6HIS omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from L. pentosus in p426HXT7-6HIS Lacto omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from L. xyli in p426HXT7-6HIS Leif omitting the His.sub.6 epitope p426H7- Cloning of the codon-optimized XI from Piromyces sp. E2 opt.XI-Piro in p426HXT7-6HIS omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from P. syringae in p426HXT7-6HIS Pseudo omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from R. biformata in p426HXT7-6HIS Robi omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from S. degradans in p426HXT7-6HIS Saccha omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from S. typhimurium in p426HXT7-6HIS Salmo omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from S. xylosus in p426HXT7-6HIS Staph omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from S. diastaticus in p426HXT7-6HIS Strep omitting the His.sub.6 epitope p426H7-XI- Cloning of the XI from X. campestris in p426HXT7-6HIS Xantho omitting the His.sub.6 epitope
3. Transformation:
Transformation of E. coli

(52) The transformation of E. coli cells was performed with the electroporation method according to Dower et al. (1988) and Wirth (1993) by means of an Easyject prima instrument (EQUIBO).

(53) Transformation of S. cerevisiae

(54) The transformation of S. cerevisiae strains with plasmid DNA or DNA fragments was performed in accordance with the lithium acetate method according to Gietz and Woods (1994).

(55) 4. Preparation of DNA

(56) Isolation of plasmid DNA from E. coli

(57) The isolation of plasmid DNA from E. coli was performed in accordance with the method of alkaline lysis according to Birnboim and Doly (1979), modified according to Maniatis et al. (1982) or alternatively with the QIAprep Spin Miniprep Kit from the company Qiagen.

(58) High-purity plasmid DNA for sequencing was prepared with the Plasmid Mini Kit from the company Qiagen according to the manufacturer's instructions.

(59) Isolation of Plasmid DNA from S. cerevisiae

(60) The cells of a stationary yeast culture (5 ml) were harvested by centrifugation, washed and resuspended in 400 l of buffer B1 (Plasmid Mini Kit, company Qiagen). Following the addition of 400 l of buffer B2 and of a volume of glass beads (0.45 mm), the cell disruption was performed by shaking for 5 minutes on a Vibrax (Vibrax-VXR from Janke & Kunkel or IKA). of a volume of buffer B3 was added to the supernatant, it was mixed and incubated for 10 min on ice. After centrifuging for 10 minutes at 13,000 rpm, the plasmid DNA was precipitated at room temperature by adding 0.75 ml of isopropanol to the supernatant. The DNA pelleted by centrifugation for 30 min at 13,000 rpm was washed with 70% ethanol, dried and resuspended in 20 l of water. 1 l of the DNA was used for the transformation in E. coli.

(61) Colony PCR of B. licheniformis and S. degradans

(62) Minor amounts of cells were collected from bacterial cultures growing on a plate by means of a toothpick and transferred into a PCR reaction vessel. Following the addition of H.sub.2O, 0.2 mM dNTP mix, 1PCR buffer (contains 1.5 mM MgCl.sub.2) and in each case 10 pmol of the corresponding oligonucleotide primer, the cell disruption was performed in a thermocycler from the company Techne at 99 C. for 10 min. This batch was directly used in a PCR reaction as a template. By adding 1 U of polymerase, the polymerase chain reaction was started with a total volume of 50 l.

(63) Determination of the DNA Concentration

(64) The DNA concentration was measured spectrophotometrically in a wavelength range of 240-300 nm. If the purity of the DNA, determined with the quotient E.sub.260nm/E.sub.280nm, is 1.8, the extinction E.sub.260nm=1.0 corresponds to a DNA concentration of 50 g of dsDNA/ml (Maniatis et al., 1982).

(65) DNA Amplification by Means of PCR

(66) Use of the Phusion High Fidelity Systems

(67) The polymerase chain reaction was performed in a total volume of 50 l with the Phusion High Fidelity PCR System from the company Finnzymes according to the manufacturer's instructions. Each batch consisted of 1-10 ng of DNA or 1-2 yeast gcolonies as the synthesis template, 0.2 mM of dNTP mix, 1 buffer 2 (contains 1.5 mM of MgCl.sub.2), 1 U of polymerase and in each case 100 pmol of the corresponding oligonucleotide primer. The PCR reaction was performed in a thermocycler from the company Techne and the PCR conditions were chosen as follows, as required:

(68) TABLE-US-00003 1. 1x 30 sec, 98 C. Denaturation of the DNA 2. 30x 10 sec, 98 C. Denaturation of the DNA 30 sec, 52-62 C. Annealing/bonding of the oligonucleotides to the DNA 50 sec, 72 DNA synthesis/elongation 3. 1x 7 min, 72 C. DNA synthesis/elongation

(69) The polymerase was added after the first denaturation step (hot-start PCR). The number of synthesis steps, the annealing temperature and the elongation time were adapted to the specific melting temperatures of the oligonucleotides used or the size of the product to be expected, respectively. The PCR products were examined by means of an agarose gel electrophoresis and subsequently purified.

(70) DNA Purification of PCR Products

(71) The purification of the PCR products was performed with the QIAquick PCR Purification Kit from the company Qiagen according to the manufacturer's instructions.

(72) Gel Electrophoretic Separation of DNA Fragments

(73) The separation of DNA fragments having a size of 0.15-20 kb was performed in 0.5-1% agarose gels with 0.5 g/ml of ethidium bromide. 1TAE buffer (40 mM of Tris, 40 mM of acetic acid, 2 mM of EDTA) was used as the gel and running buffer (Maniatis et al., 1982). A lambda phage DNA cut with the restriction endonucleases EcoRI and HindIII served as a size standard. Before application, 1/10 of a volume of blue marker (1TAE buffer, 10% glycerine, 0.004% bromophenol blue) was added to the DNA samples and they were visualized after the separation by irradiation with UV light (254 nm).

(74) Isolation of DNA Fragments from Agarose Gels

(75) The desired DNA fragment was cut out from the TAE agarose gel under long-wave UV light (366 nm) and isolated with the QIAquick Gel Extraction Kit from the company Qiagen according to the manufacturer's instructions.

(76) 5. Enzymatic Modification of DNA

(77) DNA restriction

(78) Sequence-specific cleavage of the DNA with restriction endonucleases was performed for 1 hour with 2-5 U of enzyme per g of DNA under the incubation conditions recommended by the manufacturer.

(79) 6. Metabolite Analyses

(80) Samples were taken at different times and centrifuged at 4 C. for 15 min at 13,000 rpm and 450 l were collected from the supernatant. The protein precipitation was performed with 50% sulphosalicylic acid. 1/10 of a volume of sulphosalicylic acid was added onto the samples, mixed and centrifuged for 20 min at 13,000 rpm at 4 C. The supernatant was collected and the samples could be used for the measurement after another dilution with water. Samples with D-glucose, D-xylose, xylitol, acetate, glycerine and ethanol served as standards, which were employed in concentrations of 0.05% w/w, 0.1% w/v, 0.5% w/v, 1.0% w/v and 2.0% w/v.

(81) The sugar concentration and the ethanol concentration were measured by means of BioLC (Dionex). The autosampler AS50, the column heater TCC-100, the RI detector RI-101 (Shodex) and the gradient pump GS50 were used in the measurement. The measurement of the samples was performed with the column VA 300/7.7 Nucleogel Sugar 810 H (Macherey-Nagel). The column was eluted at a temperature of 65 C. with 5 mM H.sub.2SO.sub.4 as the eluent and at a flow rate of 0.6 ml.Math.min.sup.1. The evaluation of the data was performed with the program Chromeleon Version 6.50 (version 6.50, Dionex).

(82) 7. Measurement of Enzyme Activities in S. cerevisiae

(83) Preparation of Raw Extracts

(84) 50 ml of cultures of yeast cells were grown to the exponential phase in synthetic minimal medium with 2% glucose. The cells were harvested, washed twice in Tris-HCl buffer (pH 7.5) and disrupted by means of glass beads (=0.45 nm) for 8 min on a Vibrax (Janke & Kunkel, Vibrax-VBR) at 4 C. Cell debris was removed by centrifugation for 10 min at 13,000 rpm. Subsequently, the supernatant was collected and filled up to 2 ml with cold Tris-HCl buffer (pH 7.5) and used as a raw extract for the protein determination and for the measurement of the enzyme activities or the xylitol inhibition.

(85) Protein Determination

(86) The protein concentration was determined with the kit Roti-Quant from the company Carl Roth GmbH+Co. according to the manufacturer's instructions on the basis of Bradford (1976). In this connection, bovine serum albumin (BSA) in concentrations of 0-100 g/ml served as the standard. After an incubation time of at least 5 min at room temperature, the samples were measured in microtiter plates with a microtiter plate photometer from the company Molecular Devices at OD.sub.590.

(87) Measurement of the Xylose Isomerase Activity

(88) To determine the xylose isomerase activity, recombinant yeast cells containing the vector p426H7-XI-Clos or p426H7-opt.XI-Clos, respectively, were grown, harvested and raw extracts were prepared. Recombinant yeast cells containing the empty vector p426HXT7-6HIS served as a comparison. In a total volume of 1 ml, the conversion of 6.25-500 mM of xylose with 100 l of raw extract, 0.23 mM of NADH, 10 mM of MgCl.sub.2, 2 U of sorbitol dehydrogenase in 100 mM of Tris-HCl buffer (pH 7.5) was continuously monitored. The acceptance of NADH as a measured variable was determined spectrophotometrically at a wave length of 340 nm. The reaction was started by adding xylose.

(89) Measurement of the Xylitol Inhibition

(90) To determine the xylitol inhibition of the xylose isomerase recombinant yeast cells containing the vector p426H7-XI-Clos were grown, harvested and raw extracts were prepared. Recombinant yeast cells with the vector p426H7-opt.XI-Piro or the vector p426HXT7-6HIS, respectively, served as a comparison. In a total volume of 1 ml, the conversion of 6.25-500 mM of xylose with 100 l of raw extract, 10-100 mM of xylitol, 0.23 mM of NADH, 10 mM of MgCl.sub.2, 2 U of sorbitol dehydrogenase in 100 mM of Tris-HCl buffer (pH 7.5) was continuously monitored. The acceptance of NADH as a measured variable was determined spectrophotometrically at a wave length of 340 nm. The reaction was started by adding xylose.

Example 1: Screen of a (Highly) Functional Prokaryotic Xylose Isomerase

(91) A) Construction of MKY09

(92) In the yeast strain CEN.PK2-1C, all the genes of the non-oxidative pentose phosphate pathway as well as the xylulokinase (XKS1) and GAL2 were overexpressed. To this end, the endogenous promoters were replaced with the truncated HXT7 promoter. This strain was named MKY09 and used for the screen for functional xylose isomerases.

(93) B) Selection of the Xylose Isomerases to be Tested

(94) To make a selection of the xylose isomerases to be tested, protein sequences of xylose isomerases from the database NCBI BLAST were compared. An excerpt of the xylose isomerase obtained is depicted in FIG. 3. 14 xylose isomerases from different organisms were selected to be tested on their functionality in yeast.

(95) C) Execution of the Screen

(96) To this end, genomic DNA was isolated from the organisms. The cells were grown, harvested and disrupted (see Isolation of plasmid DNA from S. cerevisiae and Colony PCR from B. licheniformis and S. degradans, respectively). The open reading frame (ORF) of XI from the mentioned organisms was amplified with primers additionally having homologous regions to the HXT7 promoter or CYC1 terminator.

(97) The obtained PCR products were together with the vector p426HXT7-6HIS linearized with EcoRI/BamHI transformed in yeast and cloned via in vivo recombination into the plasmid between the HXT7 promoter or CYC1 terminator, respectively (FIG. 4). The sequence of the plasmids obtained was verified by means of restriction analysis. Furthermore, the functionality of the new isomerases and its effect on the xylose conversion in yeast was to be studied. However, it was not possible to amplify the desired PCR product with the xylose isomerase from the organisms Streptomyces diastaticus and Leifsonia xyli. Both xylose isomerases thus could not be tested on functionality in yeast.

(98) D) Growth Behaviour (Plate)

(99) Out of the 12 different tested xylose isomerases, a xylose isomerase was found, which was functional in yeast strain MKY09. Recombinant yeasts containing the xylose isomerase from C. phytofermentans showed good growth on plates containing xylose (FIG. 5).

Example 2: Codon Optimization of the Gene for Xylose Degradation in Yeast Codon Optimization of Genes According to the Codon Usage of the Glycolysis Genes from S. cerevisiae

(100) The preferred codon usage of the glycolysis genes from S. cerevisiae was determined and is listed in table 1. The ORF of the gene XI from C. phytofermentans was codon-optimized. That is, the sequences of the open reading frame were adapted to the preferred codon usage indicated below. The protein sequence of the enzymes remained unchanged. The genes were synthesized by an external company and supplied in dried form in company-owned vectors. Further details about the synthesis of genes can be found under www.geneart.com.

(101) TABLE-US-00004 TABLE 1 Preferred codon usage of the glycolytic genes from S. cerevisiae Codon usage of Amino acid codon-optimized genes Ala GCT Arg AGA Asn AAC Asp GAC Cys TGT Gln CAA Glu GAA Gly GGT His CAC Ile ATT Leu TTG Lys AAG Met ATG Phe TTC Pro CCA Ser TCT Thr ACC Trp TGG Tyr TAC Val GTT Stop TAA
B) Introduction of the Codon-Optimized Xylose Isomerase Gene into the Strain MKY09

(102) To test the codon-optimized xylose isomerase gene in strain MKY09, the gene had to be subcloned into a yeast vector. To this end, the codon-optimized XI-ORF was amplified with primers and cloned into the linearized vector p426HXT7-6HIS (see Execution of the screen). The sequence of the obtained plasmid p426H7-opt.XI-Clos was verified by means of restriction analysis. To test the functionality of the codon-optimized isomerase, the plasmid p426H7-opt.XI-Clos was transformed in the strain MKY09. Recombinant yeast strains showed good growth on plates with medium containing xylose (FIG. 5). Further characterizations of the native and the codon-optimized XI from C. phytofermentans followed.

Example 3: Characterization of the Functional Prokaryotic Xylose Isomerase

(103) A) Growth Behaviour and Xylose Conversion

(104) The growth of the strain MKY09 with the native and the codon-optimized xylose isomerase from C. phytofermentans was investigated in growth tests on medium containing xylose under aerobic conditions. The empty vector p426HXT7-6HIS served as a comparison.

(105) The strains were grown in SC medium with 0.1% glucose and 1.4% xylose and inoculated with an OD.sub.600nm=0.2 in 50 ml of SC medium with 0.1% glucose and 1.4% xylose. The incubation was performed in shaking flasks under aerobic conditions at 30 C. Samples for the determination of the optical density and for the determination of the metabolite composition were taken several times.

(106) The growth curves showed that all the recombinant yeasts grew on glucose up to an OD.sub.600 of 2.5 (table 2). After another 50 h, the yeast strain containing the native xylose isomerase from C. phytofermentans began to grow further on xylose and reached a final OD.sub.600 of 3.5 at a maximum growth rate of 0.0058 h.sup.1 on medium containing xylose. The yeast strain with the codon-optimized xylose isomerase likewise reached a final OD.sub.600 of 3.5. The maximum growth rate was 0.0072 h.sup.1. Yeast transformants with the empty vector p426HXT7-6HIS showed no growth on xylose and began to die already after 150 h.

(107) The recombinant yeasts containing the native xylose isomerase from C. phytofermentans or the codon-optimized xylose isomerase, respectively, converted more than 2.6 g of xylose in 312 hours (FIG. 6).

(108) TABLE-US-00005 TABLE 2 Determination of the maximum growth rate on xylose () MKY09 transformed with plasmid Max. growth rate p426H7-XI-Clos 0.0058 p426H7-opt.XI-Clos 0.0072

(109) It could be shown with this experiment that the introduction of the native as well as the codon-optimized xylose isomerase from C. phytofermentans allows the recombinant S. cerevisiae strains growth on D-xylose and its conversion. By means of the codon optimization of the xylose isomerase, a higher max. growth rate could be achieved.

(110) B) Measurement of the Xylose Isomerase Activity

(111) Enzyme tests were performed directly after the raw extract preparation. The XI activity was performed at 30 C. in a reaction mix (100 mM of Tris-HCl, pH 7.5; 10 mM of MgCl.sub.2, 0.23 mM of NADH; 2 U of sorbitol dehydrogenase) with different raw extract concentrations. The reaction was started with 6.25-500 mM of xylose.

(112) The determination of the enzyme kinetics of the native form of the xylose isomerase resulted in a K.sub.m value of 61.853.41 mM and for the codon-optimized form a K.sub.m value of 66.011 mM (FIG. 7 and table 3). As expected, the K.sub.m values were thus the same as they do not differ significantly.

(113) V.sub.max (mol/min.sup.1 mg protein.sup.1) was 0.0076 for the native form of the xylose isomerase and 0.0344 for the codon-optimized form (FIG. 7). Therefore, V.sub.max could be increased by more than 450% by means of the codon optimization of the enzyme.

(114) TABLE-US-00006 TABLE 3 CEN.PK2-1C transformed V.sub.max (mol/min.sup.1 with plasmid protein.sup.1) mg K.sub.m (mM) p426H7-XI-Clos 0.0076 61.85 3.4 p426H7-opt.XI-Clos 0.0344 66.01 1.sup.

(115) The strain CEN.PK2-1C transformed with the plasmid p426H7-XI-Clos and p426H7-opt.XI-Clos, respectively, was grown over night in synthetic complete medium with 2% glucose and no uracil. Raw extracts were prepared and quantitative enzyme tests were performed.

(116) C) Measurement of the Xylitol Inhibition

(117) The determination of the xylitol inhibition of the xylose isomerases was performed directly after the raw extract preparation. The XI activity was performed at 30 C. in a reaction mix (100 mM of Tris-HCl, pH 7.5; 10 mM of MgCl.sub.2, 0.23 mM of NADH; 2 U of sorbitol dehydrogenase) with different raw extract concentrations. Additionally, different concentrations of xylitol (10-100 mM) were present in the reaction mix. The reaction was started with 6.25-500 mM of xylose.

(118) K was determined via the equation K.sub.m=K.sub.m=K.sub.m*(1+i/K.sub.i), i being the xylitol concentration used and K.sub.m being the apparent K.sub.m value at the corresponding xylitol concentration.

(119) The determination of the kinetics of the xylitol inhibition of the xylose isomerase form C. phytofermentans resulted in a K.sub.i value of 14.241.48 mM (table 4). As already described several times (Yamanaka et al., 1969 and references cited therein), it is a competitive inhibition.

(120) TABLE-US-00007 TABLE 4 CEN.PK2-1C transformed with plasmid K.sub.i (mM) p426H7-opt.XI-Piro 4.67 1.77 p426H7-opt.XI-Clos 14.51 1.08

(121) The strain CEN.PK2-1C transformed with the plasmid p426H7-opt.XI-Clos and p426H7-opt.XI-Piro, respectively, was grown over night in synthetic complete medium with 2% glucose and no uracil. Raw extracts were prepared and quantitative enzyme tests with constant xylitol concentrations of 10-100 mM were performed.

(122) The xylose isomerase from Piromyces sp.E2 and the empty vector p426HXT7-6HIS served as a comparison. The determined K.sub.i value of the xylose isomerase from Piromyces sp.E2 was 4.671.77 mM.

(123) It can be seen from the determined K.sub.i values that the xylose isomerase from C. phytofermentans is significantly less inhibited by xylitol than the xylose isomerase from Piromyces sp.E2.

(124) D) Examples of Vectors for the Xylose Isomerase

(125) The plasmid p426HXT7-6HIS was the starting plasmid for the construction of p426H7-opt.XI-Clos. The vector is a 2 expression plasmid, which has a URA3 marker.

(126) Further possible expression vectors are from the series of pRS303X, p3RS305 and p3RS306X. These are integrative vectors, which have a dominant antibiotic marker. Further details about these vectors can be found in Taxis and Knop (2006).

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