WO2015091757A1 - Polypeptides with permease activity - Google Patents

Polypeptides with permease activity Download PDF

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Publication number
WO2015091757A1
WO2015091757A1 PCT/EP2014/078409 EP2014078409W WO2015091757A1 WO 2015091757 A1 WO2015091757 A1 WO 2015091757A1 EP 2014078409 W EP2014078409 W EP 2014078409W WO 2015091757 A1 WO2015091757 A1 WO 2015091757A1
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Prior art keywords
polypeptide
host cell
xylose
seq
gal2
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PCT/EP2014/078409
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French (fr)
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Ondrej REZNICEK
Sandra FACEY
Kassandra LINDENMAIER
Bernhard Hauer
Paulus Petrus DE WAAL
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Dsm Ip Assets B.V.
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Publication of WO2015091757A1 publication Critical patent/WO2015091757A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • the invention is directed to novel polypeptides and to recombinant organisms expressing the polypeptides.
  • the present invention relates to novel permease polypeptides with altered sugar specificity and/or sugar transport activity, more specifically to novel GAL2 polypeptides in Saccharomyces cerevisiae.
  • the plasma membrane of yeast cells and other eukaryotes is a complex bio- membrane, consisting of two layers of phospholipids, with a plethora of proteins embedded in it. Many molecules may cross the plasma membrane by diffusion and osmosis or with the aid of specific transport systems.
  • Transport systems allow the uptake of nutrients and ions, export of products of metabolism and undesired or harmful substances. Different mechanisms exist. Primary active transporters drive solute accumulation or extrusion by using for instance ATP hydrolysis. Secondary carriers, belonging to the Major Facilitator Superfamily (MFS) transporters, facilitate the transport of one or more molecular species across the membrane in response to chemi-osmotic gradients. In the yeast Saccharomyces cerevisiae, 186 MFS proteins have been identified (Nelissen, 1997) in strain S288c.
  • MFS Facilitator Superfamily
  • Hxt1 protein involved in hexose transport in Saccharomyces cerevisiae.
  • Permeases are membrane transport proteins, a class of multipass transmembrane proteins that facilitate the diffusion of a specific molecule, herein specifically one or more sugar, in or out of the cell by passive transport.
  • active transporters couple molecule transmembrane transport with an energy source such as ATP or a favorable ion gradient.
  • permease, facilitator, transporter or transport protein or related terms are all describing proteins with multiple membrane spanning domains that exhibit a function in transporting molecules across a membrane. This transport can be brought about by different mechanisms: uniport (transport of one molecule), symport (simultaneous co-transport of two different molecules in the same direction), antiport (simultaneous transport of two molecules in opposite directions) and facilitated diffusion.
  • uniport transport of one molecule
  • symport sinultaneous co-transport of two different molecules in the same direction
  • antiport susimultaneous transport of two molecules in opposite directions
  • facilitated diffusion The family of sugar transporters in yeast consists of 30-40 members (34 members in strain S288c (Nelissen, 1997)).
  • the sugar transporters can be divided in five clusters: hexose permeases ( ⁇ -genes, GAL2), disaccharide permeases, myo-inositol permeases, sugar receptors and a final cluster of transporters of which the substrate is unknown.
  • Lignocellulosic biomass an attractive alternative feedstock for the production of liquid transportation fuels, consists of several different sugars.
  • the hexose fraction of lignocellulose mainly glucose
  • yeast S. cerevisiae can in principle be readily fermented by non-recombinant versions of the yeast S. cerevisiae.
  • this organism is not able to metabolize the pentose sugars, such as xylose and arabinose, into ethanol.
  • Pentose transport in S. cerevisiae is mediated by the different members of the hexose transporter (Hxt) family.
  • Hxt4, Hxt5, Hxt7 and Gal2 have been described as the main xylose transporters in S. cerevisiae (Hamacher et al, 2002), and Gal2 is also known to mediate arabinose transport (Becker, et al, 2003).
  • the affinity for the respective pentose sugars is approximately 10 to 100 times lower than for the respective hexose sugars.
  • pentose-specific sugar transporters i.e. no glucose interference (pentose specificity) and high affinity to pentose, in an otherwise unchanged transporter landscape, in order to maintain the ability to convert hexoses at approximately the same level.
  • mutant hexose transporters that are able to transport pentose sugars more efficiently are known in the art. For instance, in WO/2012/049173, the isolation of mutant hexose transporter genes from cultures of pentose-fermenting S. cerevisiae cells is described.
  • the permease GAL2 transports galactose across the cell membrane. It is also known as a transporter of glucose across the membrane.
  • An object of the invention is to provide novel permease polypeptides with altered, in particular improved, sugar specificity. Another object of the invention is to provide recombinant strains expressing the permease polypeptide that have improved uptake of the molecule that the permease transports across the cell membrane. Another object is to provide a permease polypeptide that has a improved capacity for transport of C5 sugars, in particular xylose compared to a parent polypeptide. Another object is to provide a permease polypeptide that has reduced transport activity for C6 sugar, in particular glucose, compared to a parent polypeptide. Another object is to provide a method to identify mutations in other related permease polypeptides that have a beneficial effect on the improved capacity for transport of xylose or reduced transport activity for glucose.
  • MFS Major Facilitator Superfamily
  • the polypeptide has one or more substitution corresponding to L301 R/M/IA /F/Q/A, K310R/N/M/Q/T/D/E, L31 1 R/M/l/V/F/Q/A, N314D/H/S/K/E/T/Q/G/A, M435T/L/IA /K/R/Q/F and/or S468T/L/IA /K/R/Q/F of SEQ ID NO: 57.
  • the polypeptide has pentose transport activity.
  • the polypeptide has xylose transport activity.
  • the polypeptide has one or more substitution corresponding to L301 R, K310R, L31 1 R, N314D, M435T and/or S468T of SEQ ID NO: 57.
  • a polypeptide according to the invention having one or more of these mutations has an advantageous sugar consumption and/or fermentation product production. This this will be described in more detail below and will be illustrated by examples 1 -5 below.
  • FIG. 1 shows results of aerobic shake flask cultures hexose transporter mutants on Verduyn-urea + 15 g 1 glucose + 20 g ⁇ 1 xylose.
  • A Optical density measurements at 600 nm wavelength,
  • B glucose concentrations (g ⁇ 1 ),
  • C xylose concentrations during the culture period.
  • FIG. 2 shows results of aerobic shake flask cultures hexose transporter mutants on Verduyn-urea + 20 g ⁇ 1 xylose.
  • A Optical density measurements at 600 nm wavelength
  • B xylose concentrations (g ⁇ 1 ).
  • FIG. 3 shows results of micro-well plate cultures on Verduyn-urea-his supplemented with 2% glucose (A) or 2% xylose (B). Growth characteristics were determined for DS64973 ⁇ Aglkl, Ahxkl, Ahxk2, Agall) reference strain DS68616 ⁇ GLK1, HXK1, HXK2, GAL1), and all intermediate hexokinase mutants (single [Aglkl], double [Aglkl, Ahxkl], triple [Aglkl, Ahxkl, Ahxk2]). Every 15 minutes, OD600 was measured automatically by Bioscreen C apparatus. Data points are the average of measurements in triplicate.
  • FIG. 4 shows a S.
  • the cerevisiae Gal2 wild-type transporter protein topology model predicted by SOSUI The cytoplasm is located at the bottom of each model, the extra-cytoplasm is on top and the numbers depict the transmembrane segments.
  • the restriction enzyme sites BamYW and Mun ⁇ were used to excise the template DNA.
  • Eag ⁇ and Mun ⁇ were used to excise the template DNA for the third round of epPCR.
  • the highlighted black areas indicate the identified amino acid substitutions after two rounds of random mutagenesis.
  • the black crosses indicate the identified alterations in the amino acid sequence after the third round of random mutagenesis.
  • the black arrow shows the mutation in TM8 in candidate 3.1 that led to significantly impaired uptake of glucose but not of xylose.
  • C Plasmid map of expression vector pRS313- GAL2 used as host vector to insert mutagenic PCR fragments; for first and second round mutagenesis fragments were inserted between BamYW and Mun ⁇ , and for third round an Eagl site was added between TM2 and TM3 on GAL2 to expand the diversity of variant GAL2 sequences.
  • FIG. 5 shows the comparison of DS68625 expressing wild-type Gal2 (square) and the identified variant 1 .1 (circle) from the first round of mutagenesis in Verduyn-urea with 2% xylose as carbon source.
  • DS6825 expressing an empty vector (diamond) was included as control. Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from three independent experiments.
  • FIG. 6 shows the comparison of growth (A and B) and consumption (C and D) of Gal2 (wild-type) and identified mutants after two rounds of random mutagenesis at low xylose concentrations.
  • DS68625 expressing wild-type Gal2 (square) and the variants 1 .1 (circle), 2.1 (up triangle), 2.2 (down triangle), 2.3 (cross) and 2.4 (pentagon) in Verduyn- urea with 0.45% xylose (A and C) and with 0.1 % xylose (B and D) as carbon source.
  • DS68625 (diamond) expressing an empty vector was included as a control. Growth was determined measuring the optical density at 600 nm. At different time points the sugar consumption was determined via HPLC. The error bars represent standard deviations calculated from three independent experiments.
  • FIG. 7 shows the comparison of growth facilitated by GAL2 variants after three rounds of directed evolution.
  • DS68625 expressing wild-type Gal2p (square) and the variants with amino acid substitutions 2.1 (up-triangle), 3.1 (cross), 3.2 (down triangle), 3.5 (left triangle), 3.6 (right triangle) and 3.7 (pentagon) in Verduyn-urea with 2% glucose (A) and with 2% xylose (B) as sole carbon source.
  • DS68625 black, diamond
  • Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from two independent experiments.
  • FIG. 8 shows the growth and consumption measurements with xylose in the presence of glucose.
  • A shows growth in Verduyn-urea with 2% xylose and 2% glucose
  • B shows growth in Verduyn-urea with 0.45% xylose and 0.45% glucose: wild-type Gal2 (square), mutants 2.1 (triangle) and 3.1 (star), DS68625 (diamond).
  • (C) shows consumption measurements for 2% xylose with 2% glucose and (D) for 0.45% xylose with 0.45% glucose, respectively: wild-type Gal2 with glucose (empty square), wild-type Gal2 with xylose (filled square), mutants 2.1 with glucose (empty triangle), 2.1 with xylose (filled triangle), 3.1 with glucose (empty star), 3.1 with xylose (filled star), DS68625 with glucose (empty diamond), DS68625 with xylose (black, diamond).
  • Gal2 and mutants were expressed in hxt1-7, gal2 deletion strain DS68625. DS68625 expressing the empty vector was included as control. Growth was determined measuring the optical density at 600 nm. Sugar consumption was determined via HPLC. The error bars represent standard deviations calculated from three independent experiments, glc: glucose; xyl: xylose.
  • FIG. 9 displays growth facilitated by wild-type Gal2 in comparison to mutants 2.1 , 3.1 and Gal2_T386A.
  • DS68625 expressing wild-type Gal2 (square) and mutants 2.1 (triangle), 3.1 (cross) and Gal2_T386A (circle) in Verduyn-urea with 2% glucose (A), 2% xylose (B), 0.1 % glucose (C) and 0.1 % xylose (D) as carbon sources.
  • DS68625 expressing the empty vector (diamond) was included as control. Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from two independent experiments.
  • FIG. 10 Growth behavior facilitated by Gal2 mutants of S. cerevisiae.
  • DS68625 expressing Gal2 wild-type (square), with a single mutation M435T (circle), and double mutations M435T+L301 R (up triangle), M435T+K310R (down triangle), M435T+N314D (left triangle), M435T+S468T (right triangle), K310R+N314D (pentagon) in Verduyn-urea with 2% xylose (A), 0.45% xylose (B) and 0.1 % xylose (C) as carbon source.
  • DS68625 expressing the empty vector (diamond) was included as control. Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from two independent experiments.
  • SEQ ID NO 2 primer 5035-kanr
  • SEQ ID NO 5 primer 51 15-lf 1
  • SEQ ID NO 7 pRN201 ; TOPO-BLUNT-loxP-kanMX-loxP
  • SEQ ID NO 8 pRN251 ; TOPO-BLUNT-loxP-hphMX-loxP
  • SEQ ID NO 1 1 primer 1 16-natr
  • SEQ ID NO 1 2 pRN447; TOPO-BLUNT-loxP-zeoMX-loxP
  • SEQ ID NO 1 5 pRN247 (TOPO- BLUNT-HIS3::loxPkanMXIoxP)
  • SEQ ID NO 16 primer 201 -Hx2uf
  • SEQ ID NO 1 I 7 primer 202-Hx2ur
  • SEQ ID NO 1 18 pri mer 203-Hx2df
  • SEQ ID NO 26 pri mer 229-Hx7df
  • SEQ ID NO 29 primer 244-Gal2urn
  • SEQ ID NO 30 primer 233-Ga2df
  • SEQ ID NO 34 pRN569: TOPO-BLUNT-HXT514::loxP-natMX-loxP
  • SEQ ID NO 35 pRN635; TOPO-BLUNT-HXT2::loxP-kanMX-loxP
  • SEQ ID NO 36 pi imer 281 -Hx3inr2
  • SEQ ID NO 40 pi imer 324-Ga2inf1
  • SEQ ID NO 54 pRN774; TOPO-BLUNT-loxP-hphMX-loxP (loxP sites in opposite orientation)
  • SEQID 55 pRN775; TOPO-BLUNT-loxP-natMX-loxP (loxP sites in opposite orientation)
  • SEQ ID NO 56 WT-GAL2 DNA sequence
  • SEQ ID NO 57 WT Gal2p amino acid sequence
  • SEQ ID NO 58 pRS313-GAL2 (GAL2 expression vector uSEQ as host vector for error prone mutagenesis library
  • SEQ ID NO 59 pRN187 (pSH65-derived CRE recombinase expressing vector)
  • SEQ ID NO 60 Primer Gal2_Bamf
  • the invention relates to identification of amino acid positions in permease polypeptides, preferably hexose permease polypeptides, more preferably hexose permease polypeptides from yeast and fungi, even more preferably in Saccharomyces cerevisiae Hxt or Gal2 permease polypeptides, which are mutated to alter the sugar transport activity or sugar specificity of the permease.
  • MFS Facilitator Superfamily
  • the transport of solutes by primary active transporters is energy-driven in the first place, such as by energy supplied from ATP hydrolysis, photon absorption, electron flow, substrate decarboxylation, or methyl transfer. If charged molecules are pumped in one direction as a consequence of the consumption of a primary cellular energy source, an electrochemical potential is the result. The resulting chemiosmotic gradient can then be used to drive the transport of additional molecules via secondary carrier structures which just facilitate the transport of one or more molecules across the membrane.
  • ABS ATP-binding cassette
  • MFS major facilitator superfamily
  • ABC family permeases consist of multiple components and are primary active transporters, capable of transporting both small molecules and macromolecules only after generating energy through ATP hydrolysis
  • MFS transporters consist of a single polypeptide of a secondary carrier which facilitates transport of small solutes in response to a chemiosmotic ion gradient.
  • ABC superfamily and MFS proteins account for almost half of the solute transporters encoded within the microbe genomes (reviewed by Pao et al, 1998, Microbiol Mol Biol Rev.; 62 pp.1-34, and Saier et al, 1999, J Mol Microbiol Biotechnol, 1 pp.257-279).
  • Suitable permease polypeptide sequences can contain one or more of the following motifs:
  • Motif (a) is corresponds to residues 179-221 in Gal2; motif (b) is corresponds to residues 330-353 in Gal2; motif (c) is corresponds to residues 375-399 in Gal2.
  • Variant polypeptides of the invention may be found by the skilled person as described herein after. This includesmodeling a permease polypeptide sequence onto the published crystal structure of the xylose- or glucose-bound Escherichia coii xylose permease XylE (respectively, PDB code 4GBY & 4GBZ in the PDB database, http://www.pdb.org) to identify the amino acid positions in the channel of the permease that directly interact with the bound sugar (called the first-shell residues in the art), and the residues that interact with the first shell residues (called the second shell residues in the art).
  • Suitable modeling software to construct such models are YASARA, Prime (Schrodinger Inc.) or MODELLER using the default settings.
  • the sugar- specificity-altering first and second shell amino acid positions in a permease polypeptide sequence can be identified by a global pairwise alignment of the permease sequence with the Gal2 sequence SEQ ID NO: 57 using the NEEDLE protocol described below.
  • An example alignment for Gal2 and Hxt's from Saccharomyces cerevisiae is given in Fig. 10, which shows how alignment can be used to identify the corresponding amino acid positions in the different yeast Hxt's.
  • the amino acid positions herein thus refer to SEQ ID NO: 57 that describes Gal2 or to corresponding aminoacid positions in other polypeptides, in particular other permease polypeptides.
  • the corresponding position of the position N376 in Gal2 (SEQ ID NO; 57) in Hxt1 is N370, in Hxt2 N361 , in Hxt3 N367, in Hxt4SC N376, in Hxt4RN N376, in Hxt5 N391 , in Hxt6/7 N370, in Hxt8 N372, in Hxt9 N366, in Hxt10 N354, in Hxt1 1 N366, in Hxt12 N256, in Hxt13 N363, in Hxt14 N387, in Hxt15 N366, in Hxt16 N366 and in Hxt17 N363.
  • the corresponding position of N346 in Gal2 (SEQ ID NO:9) in Hxt1 is D340, in Hxt2 N331 , in Hxt3 D337, in Hxt4SC D346, in Hxt4RN D346, in Hxt5 D361 , in Hxt6/7 D340, in Hxt8 D342, in Hxt9 D336, in Hxt10 C324, in Hxt1 1 D336, in Hxt12 D226, in Hxt13 E333, in Hxt14 I357, in Hxt15 E336, in Hxt16 E336 and in Hxt17 E333.
  • This can be similary done for other MFS Superfamily transporters, so that corresponding positions in these polypeptides corresponding to the positions in SEQ ID NO: 57 can be obtained.
  • a person skilled in the art can subsequently mutate the identified amino acid positions in the permease polypeptide to all other 19 amino acids, and screen for improved C5 sugar uptake and/or reduced C6 sugar uptake of the mutant permease, as described in Example 4 and 5.
  • the mutations at the positions corresponding to N346 may be a substitution with C, P, G, A, V, L, I , M, F, W, Y, H, S, T, N, Q, D, E, K, R or a deletion.
  • X may be any aminoacid, X(2) means two X.
  • Gal2 is a facilitated diffusion transporter required for both the high-affinity galactokinase-dependent and low-affinity galactokinase-independent galactose transport processes. It belongs to the major facilitator superfamily, sugar transporter (TC 2.A.1 .1 ) family.
  • Permease polypeptide is also designated herein as “polypeptide permease” or “polypeptide”.
  • Periodease polypeptide polynucleotide is herein a polynucleotide that encodes the permease polypeptide.
  • the permease polypeptide has one or more substitution corresponding to a substitution on position 301 , 310, 31 1 , 314, 435 and/or 468 of SEQ ID NO:57 and has at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 57.
  • A6 indicates an amino acid (one letter code) at a certain position in SEQ ID NO: 57, here A (Alanine) at position 6 of the protein.
  • A6 (L/N/Q/GA /I Y/S/E/K) indicates herein mutation of amino acid at a certain position, here A (Alanine) at position 6 of the protein is exchanged for any of L (Leucine), N (Asparagine), Q (Glutamine), G (Glycine), V (Valine), I (Isoleucine), Y (Tyrosine), S (Serine), E (Glutamic acid) or K (Lysine).
  • the polypeptide has xylose transport activity.
  • polypeptide has one or more substitution corresponding to According to the present invention, there is provided a polypeptide having one or more substitution corresponding to a substitution on position 301 , 310, 31 1 , 314, 435 and/or 468 of SEQ ID NO:57 wherein the polypeptide is member of the Major Facilitator Superfamily (MFS). In an embodiment, the polypeptide has pentose transport activity.
  • MFS Major Facilitator Superfamily
  • the polypeptide has one or more substitution corresponding to L301 R/M/IA /F/Q/A, K310R/N/M/Q/T/D/E, L31 1 R/M/l/V/F/Q/A, N314D/H/S/K/E/T/Q/G/A, M435T/L/IA /K/R/Q/F and/or S468T/L/IA /K/R/Q/F of SEQ ID NO: 57.
  • the polypeptide has one or more substitution corresponding to L301 R, K310R, L31 1 R, N314D, M435T and/or S468T of SEQ ID NO: 57.
  • polypeptide has one or more substitution pair corresponding to L30 1 R and M435T, L310R and M435T, N314D and M435T, M435T and S468T and/or N314D and K310R of SEQ ID NO: 57.
  • polypeptide has reduced glucose transport activity compared to the polypeptide having SEQ I D NO: 57. In an embodiment the polypeptide has increased xylose transport activity compared to the polypeptide having SEQ ID NO: 57.
  • the permease polypeptide of the invention may have one or more alternative and/or additional activities other than that of sugar permease activity.
  • a permease polypeptide of the invention will typically have sugar permease activity.
  • a permease polypeptide of the invention may have one or more of the activities set out above in addition to or alternative to that activity.
  • the skilled person may determine suitable polynucleotides that encode the permease polypeptide.
  • the polynucleotide is a variant polynucleotide having at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 56, and encodes the polypeptide as described in claims 1 to 1 1.
  • the invention therefore provides polynucleotide sequences comprising the gene encoding the permease polypeptide, as well as its coding sequence.
  • the polynucleotides of the invention may be isolated or synthesized.
  • the permease polypeptides and permease polypeptide polynucleotides herein may be synthetic polypeptides, respectively polynucleotides.
  • the synthetic polynucleotides may be optimized in codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943, which are herein incorporated by reference.
  • PCT/EP2007/055943 addresses codon-pair optimization.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • a polynucleotide may either be present in isolated form, or be comprised in recombinant nucleic acid molecules or vectors, or be comprised in a host cell.
  • polypeptide is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art.
  • isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
  • recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31 -40 (1988).
  • polynucleotides of the present invention such as a polynucleotide encoding the permease polypeptide can be isolated or synthesized using standard molecular biology techniques and the sequence information provided herein.
  • the polynucleotide encoding the permease polypeptide of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Transformation
  • the polynucleotides according to the invention may be expressed in a suitable host cell.
  • the invention thus relates to a transformed host cell.
  • the host cell may be transformed with a nucleic acid construct that comprises a polynucleotide that encodes the polypeptide according to the invention defined before. Therefore standard transformation techniques may be used.
  • the transformed host cell comprises a heterologuous nucleotide that encodes a polypeptide as defined before an described in claim 1 or encodes a polypeptide having substitution
  • the transformed host is transformed with a polynucleotide that encodes a polypeptide that is a mutant of a polypeptide that is native in the untransformed host cell.
  • polypeptide that is native in the untransformed host eel is a member of the Major Facilitator Superfamily (MFS) transporters, in an embodiment a hexose transporter polypeptide.
  • MFS Major Facilitator Superfamily
  • polypeptide has one or more substitution corresponding to L301 R, K310R, L31 1 R, N314D, M435T and/or S468T of SEQ ID NO: 57.
  • he polypeptide that is native in the untransformed host cell is a transporter polypeptide chosen from the list consisting of Gal2, Hxt1 , Hxt2, Hxt3, Hxt4, Hxt5, Hxt6, Hxt7, Hxt8, Hxt9, Hxt10, Hxt1 1 , Hxt12, Hxt13, Hxt14, Hxt15, Hxt16 and Hxt17.
  • the invention further relates to a nucleic acid construct comprising the polynucleotide as described before, e.g. a vector.
  • vectors including cloning and expression vectors, comprising a polynucleotide of the invention encoding a permease polypeptide protein or a functional equivalent thereof and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a permease of the invention occurs.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell. Suitable host cells are described below.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the vectors, such as expression vectors, of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein.
  • the vectors, such as recombinant expression vectors, of the invention can be designed for expression of permease polypeptide proteins in prokaryotic or eukaryotic cells.
  • permease polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), filamentous fungi, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Representative examples of appropriate hosts are described hereafter.
  • the vector or expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants.
  • suitable episomal vectors are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2 ⁇ and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively, or vectors containing an AMA sequence (e.g. AMA1 from Aspergillus).
  • the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.
  • expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage
  • an appropriate signal sequence can be added to the polypeptide in order to direct the de novo synthesized polypeptide to the secretion route of the host cell.
  • the person skilled in the art knows to select an appropriate signal sequence for a specific host.
  • the vector may further include sequences flanking the polynucleotide giving rise to RNA which comprise sequences homologous to eukaryotic genomic sequences or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of a host cell.
  • An integrative cloning vector may integrate at random or at a predetermined target locus in the chromosome(s) of the host cell into which it is to be integrated.
  • the vector system may be a single vector, such as a single plasmid, or two or more vectors, such as two or more plasmids, which together contain the total DNA to be introduced into the genome of the host cell.
  • the vector may contain a polynucleotide of the invention oriented in an antisense direction to provide for the production of antisense RNA.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 nd , ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
  • the invention provides an isolated polypeptide having the amino acid sequence according to SEQ ID NO: 57 with the mutations indicated in claim 1 .
  • Polypeptides of the present invention may include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the invention also features biologically active fragments of the polypeptides according to the invention.
  • host cells comprising a polynucleotide or vector of the invention.
  • the polynucleotide may be heterologous to the genome of the host cell.
  • heterologous usually with respect to the host cell, means that the polynucleotide does not naturally occur in the genome of the host cell or that the polypeptide is not naturally produced by that cell.
  • the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention.
  • a "transformed cell” or “recombinant cell” is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention.
  • prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are yeast cells including e.g. Saccharomyces, for example Saccharomyces cerevisiae.
  • a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
  • Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products.
  • Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
  • Such host cells are well known in the art.
  • a cell as described above may be used to in the preparation of a polypeptide according to the invention.
  • Such a method typically comprises cultivating a host cell (e. g. transformed or transfected with an expression vector as described above) under conditions to provide for expression (by the vector) of a coding sequence encoding the polypeptide, and optionally recovering the expressed polypeptide.
  • a host cell e. g. transformed or transfected with an expression vector as described above
  • Polynucleotides of the invention can be incorporated into a recombinant replicable vector, e. g. an expression vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector.
  • the vector may be recovered from the host cell.
  • the vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptide.
  • Amino acid or nucleotide sequences are said to be homologous when exhibiting a certain level of similarity.
  • Two sequences being homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "percent identity” or “percent similarity”, which is high or low respectively. Although disputed, to indicate "percent identity” or “percent similarity”, "level of homology” or “percent homology” are frequently used interchangeably.
  • a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1 -44 Addison Wesley).
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453).
  • the algorithm aligns amino acid sequences as well as nucleotide sequences.
  • the Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.
  • the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. LongdenJ. and BleasbyA Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/).
  • EBLOSUM62 is used for the substitution matrix.
  • EDNAFULL is used for nucleotide sequences.
  • Other matrices can be specified.
  • the optional parameters used for alignment of amino acid sequences are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
  • the homology or identity is the percentage of identical matches between the two full sequences over the total aligned region including any gaps or extensions.
  • the homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment including the gaps.
  • the identity defined as herein can be obtained from NEEDLE and is labelled in the output of the program as "IDENTITY".
  • the homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
  • the identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as "longest-identity".
  • the sugar composition is a mixture of the sugar composition
  • the sugar composition according to the invention comprises glucose, arabinose and xylose. Any sugar composition may be used in the invention that suffices those criteria. Optional sugars in the sugar composition are galactose and mannose.
  • the sugar composition is a hydrolysate of one or more lignocellulosic material. Lignocelllulose herein includes hemicellulose and hemicellulose parts of biomass. Also lignocellulose includes lignocellulosic fractions of biomass.
  • Suitable lignocellulosic materials may be found in the following list: orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, switch grass, miscanthus, sweet sorghum, canola stems, soybean stems, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from kernels, products
  • lignocelluloses include: corn cobs, corn fiber, rice hulls, melon shells, sugar beet pulp, wheat straw, sugar cane bagasse, wood, grass and olive pressings.
  • Table 1 Overview of sugar compositions from lignocellulosic materials.
  • Gal galactose
  • Xyl xylose
  • Ara arabinose
  • Man mannose
  • Glu glucose
  • the transformed host cell may comprise one or more copies of xylose isomerase (XI) gene (suitable Xl's are hereinafter described) and/or one or more copies of xylose reductase and/or xylitol dehydrogenase, and one or more, or two to ten copies of araA, araB and araD, genes, wherein these genes may be integrated into the cell genome.
  • XI xylose isomerase
  • the transformed host cell comprises genes, for example the above xylose isomerase gene and/or one or more copies of xylose reductase and/or xylitol dehydrogenase, and two to ten copies of araA, araB and araD, an the genes, are integrated into the transformed host cell genome.
  • the number of copies may be determined by the skilled person by any known method. In the examples, a suitable method is described.
  • the transformed host cell is able to ferment glucose, arabinose, xylose and galactose.
  • the cell is capable of converting 90% or more glucose, xylose arabinose, galactose and mannose available, into a fermentation product. In an embodiment, cell is capable of converting 91 % or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 100% of all glucose, xylose arabinose, galactose and mannose available, into a fermentation product.
  • the transformed host cell is able to ferment one or more additional sugar, preferably C5 and/or C6 sugar e.g. mannose or galactose.
  • the transformed host cell comprises one or more of: a xylA-gene, XYL1 gene and XYL2 gene and/or XKS7-gene, to allow the transformed host cell to ferment xylose; deletion of the aldose reductase (GRE3) gene; overexpression of PPP-genes TAL1, TKL1, RPE1 and RKI1 to allow the increase of the flux through the pentose phosphate pathway in the cell.
  • the transformed host cell is an industrial cell, more preferably an industrial yeast.
  • An industrial cell and industrial yeast cell may be defined as follows.
  • the living environments of (yeast) cells in industrial processes are significantly different from that in the laboratory.
  • Industrial yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include change in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of Saccharomyces cerevisiae.
  • the environmental tolerant strains should allow robust growth and production.
  • Industrial yeast strains are generally more robust towards these changes in environmental conditions which may occur in the applications they are used, such as in the baking industry, brewing industry, wine making and the ethanol industry.
  • the industrial transformed host cell is constructed on the basis of an industrial host cell, wherein the construction is conducted as described hereinafter.
  • industrial yeast S. cerevisiae
  • Ethanol Red® Fermiol®
  • DSM Fermiol®
  • Thermosacc® Longmand
  • the transformed host cell is inhibitor tolerant.
  • Inhibitor tolerance is resistance to inhibiting compounds.
  • the presence and level of inhibitory compounds in lignocellulose may vary widely with variation of feedstock, pretreatment method hydrolysis process.
  • Examples of categories of inhibitors are carboxylic acids, furans and/or phenolic compounds.
  • Examples of carboxylic acids are lactic acid, acetic acid or formic acid.
  • Examples of furans are furfural and hydroxy- methylfurfural.
  • Examples or phenolic compounds are vannilin, syringic acid, ferulic acid and coumaric acid.
  • the typical amounts of inhibitors are for carboxylic acids: several grams per liter, up to 20 grams per liter or more, depending on the feedstock, the pretreatment and the hydrolysis conditions.
  • For furans several hundreds of milligrams per liter up to several grams per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions.
  • phenolics several tens of milligrams per liter, up to a gram per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions.
  • the transformed host cells according to the invention may be inhibitor tolerant, i.e. they can withstand common inhibitors at the level that they typically have with common pretreatment and hydrolysis conditions, so that the transformed host cells can find broad application, i.e. it has high applicability for different feedstock, different pretreatment methods and different hydrolysis conditions.
  • the industrial transformed host cell is constructed on the basis of an inhibitor tolerant host cell, wherein the construction is conducted as described hereinafter.
  • Inhibitor tolerant host cells may be selected by screening strains for growth on inhibitors containing materials, such as illustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, 847-858, wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was selected.
  • the transformed host cell is marker-free.
  • the term "marker” refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. Marker-free means that markers are essentially absent in the transformed host cell. Being marker-free is particularly advantageous when antibiotic markers have been used in construction of the transformed host cell and are removed thereafter. Removal of markers may be done using any suitable prior art technique, e.g intramolecular recombination. A suitable method of marker removal is illustrated in the examples.
  • a transformed host cell may be able to convert plant biomass, celluloses, hemicelluloses, pectins, starch, starch derivatives,, for example into fermentable sugars. Accordingly, a transformed host cell may express one or more enzymes such as a cellulase (an endocellulase or an exocellulase), a hemicellulase (an endo- or exo- xylanase or arabinase) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, a pectinase able to convert pectins into glucuronic acid and galacturonic acid or an amylase to convert starch into glucose monomers.
  • a cellulase an endocellulase or an exocellulase
  • a hemicellulase an endo- or exo- xylanase or arabinase
  • the transformed host cell further may comprise those enzymatic activities required for conversion of pyruvate to a desired fermentation product, such as ethanol, butanol, lactic acid, di-terpene, glycosylated di-terpene, 3 -hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid, itaconic acid, an amino acid, 1 ,3- propane-diol, ethylene, glycerol, a ⁇ -lactam antibiotic or a cephalosporin.
  • a desired fermentation product such as ethanol, butanol, lactic acid, di-terpene, glycosylated di-terpene, 3 -hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid, itaconic acid, an amino acid, 1 ,3- propane-diol, ethylene, glycerol, a ⁇
  • the transformed host cell is a cell that is naturally capable of alcoholic fermentation, preferably, anaerobic alcoholic fermentation.
  • a transformed host cell preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of growth at a pH lower than about 5, about 4, about 3, or about 2.5) and towards organic and/or a high tolerance to elevated temperatures.
  • Any of the above characteristics or activities of a transformed host cell may be naturally present in the cell or may be introduced or modified by genetic modification.
  • the genes may be introduced in the host cell by introduction into a host cell:
  • a cluster consisting of PPP-genes TAL1, TKL1, RPE1 and RKI1, optionally under control of strong constitutive promoter; and deletion of an aldose reductase gene; c) a cluster consisting of a xylA-gene and a XKS7-gene under control of strong constitutive promoter;
  • a construct comprising a xylA gene under control of a strong constitutive promoter, which has the ability to integrate into the genome on multiple loci;
  • the above cell may be constructed using recombinant expression techniques.
  • the transformed host cell is a recombinant cell. That is to say, a transformed host cell comprises, or is transformed with or is genetically modified with a nucleotide sequence that does not naturally occur in the cell in question.
  • the nucleic acid construct may be a plasmid, for instance a low copy plasmid or a high copy plasmid.
  • the cell according to the present invention may comprise a single or multiple copies of the nucleotide sequence encoding a enzyme, for instance by multiple copies of a nucleotide construct or by use of construct which has multiple copies of the enzyme sequence.
  • the nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence sequence.
  • a suitable episomal nucleic acid construct may e.g. be based on the yeast 2 ⁇ or pKD1 plasmids (Gleer et al., 1991 , Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489).
  • each nucleic acid construct may be integrated in one or more copies into the genome of the cell.
  • nucleic acid construct may be integrated into the cell's genome by homologous recombination as is well known in the art (see e.g. WO90/14423, EP-A-0481008, EP-A-0635 574 and US 6,265,186).
  • Most episomal or 2 ⁇ plasmids are relatively unstable in yeast, being lost in approximately 10 "2 or more cells after each generation. Even under conditions of selective growth, only 60% to 95% of the cells retain the episomal plasmid.
  • the copy number of most episomal plasmids ranges from 20-100 per cell of cir + hosts. However, the plasmids are not equally distributed among the cells, and there is a high variance in the copy number per cell in populations.
  • Strains transformed with integrative plasmids are extremely stable, even in the absence of selective pressure. However, plasmid loss can occur at approximately 10 "3 to 10 "4 frequencies by homologous recombination between tandemly repeated DNA, leading to looping out of the vector sequence.
  • the vector design in the case of stable integration is thus, that upon loss of the selection marker genes (which also occurs by intramolecular, homologous recombination) that looping out of the integrated construct is no longer possible.
  • the genes are thus stably integrated.
  • Stable integration is herein defined as integration into the genome, wherein looping out of the integrated construct is no longer possible.
  • selection markers are absent.
  • the enzyme encoding sequence will be operably linked to one or more nucleic acid sequences, capable of providing for or aiding the transcription and/or translation of the enzyme sequence.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a promoter or enhancer is operably linked to a coding sequence the said promoter or enhancer affects the transcription of the coding sequence.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences known to one of skilled in the art.
  • a "constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
  • the promoter that could be used to achieve the expression of a nucleotide sequence coding for an enzyme according to the present invention may be not native to the nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked.
  • the promoter may, however, be homologous, i.e. endogenous, to the host cell.
  • Promotors are widely available and known to the skilled person. Suitable examples of such promoters include e.g. promoters from glycolytic genes, such as the phosphofructokinase (PFK), triose phosphate isomerase (TPI), glyceraldehyde-3 - phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts or filamentous fungi; more details about such promoters from yeast may be found in (WO 93/03159).
  • PFK phosphofructokinase
  • TPI triose phosphate isomerase
  • GPD glyceraldehyde-3 - phosphate dehydrogenase
  • PYK pyruvate kinase
  • PGK phosphoglycerate kinase
  • promoters are ribosomal protein encoding gene promoters, the lactase gene promoter (LAC4), alcohol dehydrogenase promoters (ADH1 , ADH4, and the like), and the enolase promoter (ENO).
  • LAC4 lactase gene promoter
  • ADH1 , ADH4, and the like alcohol dehydrogenase promoters
  • ENO enolase promoter
  • Other promoters, both constitutive and inducible, and enhancers or upstream activating sequences will be known to those of skill in the art.
  • the promoters used in the host cells of the invention may be modified, if desired, to affect their control characteristics. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art.
  • Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, EN01, TPI1, and AOX1.
  • Other suitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3.
  • the 3 '-end of the nucleotide acid sequence encoding enzyme preferably is operably linked to a transcription terminator sequence.
  • the terminator sequence is operable in a host cell of choice, such as e.g. the yeast species of choice.
  • the choice of the terminator is not critical; it may e.g. be from any yeast gene, although terminators may sometimes work if from a non-yeast, eukaryotic, gene.
  • a nucleotide sequence encoding the enzyme comprises a terminator.
  • such terminators are combined with mutations that prevent nonsense mediated mRNA decay in the host transformed host cell (see for example: Shirley et al., 2002, Genetics 161 :1465-1482).
  • the transcription termination sequence further preferably comprises a polyadenylation signal.
  • a selectable marker may be present in a nucleic acid construct suitable for use in the invention.
  • the term "marker” refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker.
  • the marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed.
  • suitable antibiotic resistance markers include e.g. dihydrofolate reductase, hygromycin-B-phosphotransferase, 3'-0- phosphotransferase II (kanamycin, neomycin and G418 resistance).
  • Antibiotic resistance markers may be most convenient for the transformation of polyploid host cells, Also non- antibiotic resistance markers may be used, such as auxotrophic markers (URA3, TRP1, LEU2) or the S. pombe TPI gene (described by Russell P R, 1985, Gene 40: 125-130).
  • the host cells transformed with the nucleic acid constructs are marker gene free. Methods for constructing recombinant marker gene free microbial host cells are disclosed in EP-A-0 635 574 and are based on the use of bidirectional markers such as the A. nidulans amdS (acetamidase) gene or the yeast URA3 and LYS2 genes.
  • a screenable marker such as Green Fluorescent Protein, lacL, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into the nucleic acid constructs of the invention allowing to screen for transformed cells.
  • nucleic acid constructs suitable for use in the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromers, telomers and/or matrix attachment (MAR) sequences.
  • the nucleic acid constructs of the invention may further comprise a sequence for autonomous replication, such as an ARS sequence.
  • the recombination process may thus be executed with known recombination techniques.
  • Various means are known to those skilled in the art for expression and overexpression of enzymes in a transformed host cell.
  • an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the host cell, e.g. by integrating additional copies of the gene in the host cell's genome, by expressing the gene from an episomal multicopy expression vector or by introducing a episomal expression vector that comprises multiple copies of the gene.
  • overexpression of enzymes in the host cells of the invention may be achieved by using a promoter that is not native to the sequence coding for the enzyme to be overexpressed, i.e. a promoter that is heterologous to the coding sequence to which it is operably linked.
  • a promoter that is heterologous to the coding sequence to which it is operably linked
  • the promoter is also preferred that the promoter is homologous, i.e. endogenous to the host cell.
  • the heterologous promoter is capable of producing a higher steady state level of the transcript comprising the coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is the promoter that is native to the coding sequence.
  • Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters.
  • the transformed host cell is markerfree, which means that no auxotrophic or dominant markers, in particular antibiotic resistance markers, are present in the genome or extra-chromosomally.
  • the coding sequence used for overexpression of the enzymes mentioned above may preferably be homologous to the host cell. However, coding sequences that are heterologous to the host may be used.
  • Overexpression of an enzyme when referring to the production of the enzyme in a genetically modified cell, means that the enzyme is produced at a higher level of specific enzymatic activity as compared to the unmodified host cell under identical conditions. Usually this means that the enzymatically active protein (or proteins in case of multi-subunit enzymes) is produced in greater amounts, or rather at a higher steady state level as compared to the unmodified host cell under identical conditions. Similarly this usually means that the mRNA coding for the enzymatically active protein is produced in greater amounts, or again rather at a higher steady state level as compared to the unmodified host cell under identical conditions.
  • an enzyme to be overexpressed is overexpressed by at least a factor of about 1 .1 , about 1 .2, about 1 .5, about 2, about 5, about 10 or about 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.
  • Adaptation is the evolutionary process whereby a population becomes better suited (adapted) to its habitat or habitats. This process takes place over several to many generations, and is one of the basic phenomena of biology.
  • adaptation may also refer to a feature which is especially important for an organism's survival. Such adaptations are produced in a variable population by the better suited forms reproducing more successfully, by natural selection.
  • the transformed host cells may in their preparation be subjected to adaptive evolution.
  • a transformed host cell may be adapted to sugar utilisation by selection of mutants, either spontaneous or induced (e.g. by radiation or chemicals), for growth on the desired sugar, preferably as sole carbon source, and more preferably under anaerobic conditions. Selection of mutants may be performed by techniques including serial transfer of cultures as e.g. described by Kuyper et al. (2004, FEMS Yeast Res. 4: 655-664) or by cultivation under selective pressure in a chemostat culture. E.g.
  • the cell in a preferred host cell at least one of the genetic modifications described above, including modifications obtained by selection of mutants, confer to the host cell the ability to grow on the xylose as carbon source, preferably as sole carbon source, and preferably under anaerobic conditions.
  • the cell When XI is used as gene to convert xylose, preferably the cell produce essentially no xylitol, e.g. the xylitol produced is below the detection limit or e.g. less than about 5, about 2, about 1 , about 0.5, or about 0.3 % of the carbon consumed on a molar basis.
  • a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in different media is applied, e.g. three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose. See Wisselink et al. (2009) Applied and Environmental Microbiology, Feb. 2009, p. 907-914.
  • shuttle vectors contain genetic material derived from E.coli vectors consisting of an origin of replication and a selectable marker (often the ⁇ 3 ⁇ .3 ⁇ 38 ⁇ gene, ampR), which enable them to be propagated in E.coli prior to transformation into yeast cells. Additionally, the shuttle vectors contain a selectable marker for selection in yeast.
  • Markers can be genes encoding enzymes for the synthesis of a particular amino acid or nucleotide, so that cells carrying the corresponding genomic deletion (or mutation) are complemented for auxotrophy or autotrophy.
  • these vectors contain heterologous dominant resistance markers, which provides recombinant yeast cells (i.e. the cells that have taken up the DNA and express the marker gene) resistance towards certain antibiotics, like g418 (Geneticin), hygromycinB or phleomycin.
  • these vectors may contain a sequence of (combined) restriction sites (multiple cloning site or MCS) which will allow to clone foreign DNA into these sites, although alternative methods exist as well.
  • shuttle vectors can be distinguished by the absence or presence of additional genetic elements:
  • Integrative plasmids which by homologous recombination are integrated into the host genome at the locus of the marker or another gene, when this is opened by restriction and the linearized DNA is used for transformation of the yeast cells. This generally results in the presence of one copy of the foreign DNA inserted at this particular site in the genome.
  • CEN plasmids which carry in addition to an ARS sequence a centromeric sequence (derived from one of the nuclear chromosomes) which normally guarantees stable mitotic segregation and usually reduces the copy number of self- replicated plasmid to just one.
  • plasmids are being introduced into the yeast cells by transformation. Transformation of yeast cells may be achieved by several different techniques, such as permeabilization of cells with lithium acetate (Ito et al, 1983) and electroporation methods. In commercial application of recombinant microorganisms, plasmid instability is the most important problem. Instability is the tendency of the transformed cells to lose their engineered properties because of changes to, or loss of, plasmids. This issue is discussed in detail by Zhang et al (Plasmid stability in recombinant Saccharomyces cerevisiae. Biotechnology Advances, Vol. 14, No. 4, pp. 401 -435, 1996).
  • the heterologous DNA is usually introduced into the organism in the form of extra-chromosomal plasmids (YEp, YCp and YRp).
  • extra-chromosomal plasmids YEp, YCp and YRp.
  • antibiotics are often not compatible with production processes.
  • the use of antibiotics may not be desired from a registration point of view (the possibility that trace amounts of the antibiotic end up in the end product) or for economic reasons (costs of the use of antibiotics at industrial scale).
  • this may be accomplished using a vector with parts homologous to repeated sequences (transposons), of the host cell.
  • suitable repeated sequences are the long terminal repeats (LTR) of the Ty element, known as delta sequence.
  • LTR long terminal repeats
  • Ty elements fall into two rather similar subfamilies called Ty1 and Ty2. These elements are about 6 kilobases (kb) in length and are bounded by long terminal repeats (LTR), sequences of about 335 base pairs (Boeke JD et al, The Saccharomyces cerevisiae Genome Contains Functional and Nonfunctional Copies of Transposon Ty1. Molecular and Cellular Biology, Apr. 1988, p. 1432-1442 Vol. 8, No. 4). In the fully sequenced S.
  • the retrotransposition mechanism of the Ty retrotransposon has been exploited to integrate multiple copies throughout the genome (Boeke et al., 1988; Jacobs et al., 1988).
  • the long terminal repeats (LTR) of the Ty element known as delta sequences, are also good targets for integration by homologous recombination as they exist in about 150-200 copies that are either Ty associated or solo sites (Boeke, 1989; Kingsman and Kingsman, 1988). (Parekh R.N. (1996). An Integrating Vector for Tunable, High Copy, Stable Integration into the Dispersed Ty DELTA Sites of Saccharomyces cerevisiae. Biotechnol. Prog. 1996, 12, 16-21 ).
  • the number of copies may change.
  • the host cell The host cell
  • the host cell may be any host cell suitable for production of a useful product.
  • a host cell may be any suitable cell, such as a prokaryotic cell, such as a bacterium, or a eukaryotic cell.
  • the cell will be a eukaryotic cell, for example a yeast or a filamentous fungus.
  • Yeasts are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina (Alexopoulos, C. J. ,1962, In : Introductory Mycology, John Wiley & Sons, Inc. , New York) that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism.
  • a preferred yeast as a transformed host cell may belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia.
  • the yeast is one capable of anaerobic fermentation, more preferably one capable of anaerobic alcoholic fermentation.
  • Filamentous fungi are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina. These fungi are characterized by a vegetative mycelium composed of chitin, cellulose, and other complex polysaccharides.
  • the filamentous fungi of the suitable for use as a cell of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Filamentous fungal cells may be advantageously used since most fungi do not require sterile conditions for propagation and are insensitive to bacteriophage infections. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism of most filamentous fungi is obligately aerobic.
  • Preferred filamentous fungi as a host cell may belong to the genus Aspergillus, Trichoderma, Humicola, Acremoniurra, Fusarium or Penicillium. More preferably, the filamentous fungal cell may be a Aspergillus niger, Aspergillus oryzae, a Penicillium chrysogenum, or Rhizopus oryzae cell.
  • the host cell may be yeast.
  • the host is an industrial host, more preferably an industrial yeast.
  • An industrial host and industrial yeast cell may be defined as follows. The living environments of yeast cells in industrial processes are significantly different from that in the laboratory. Industrial yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include change in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of Saccharomyces cerevisiae. Under adverse industrial conditions, the environmental tolerant strains should allow robust growth and production. Industrial yeast strains are generally more robust towards these changes in environmental conditions which may occur in the applications they are used, such as in the baking industry, brewing industry, wine making and the ethanol industry. Examples of industrial yeast (S.
  • Inhibitor tolerant host cells may be selected by screening strains for growth on inhibitors containing materials, such as illustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, 847-858, wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was selected.
  • a transformed host cell is capable of using arabinose.
  • a transformed host cell is therefore, be capable of converting L-arabinose into L-ribulose and/or xylulose 5- phosphate and/or into a desired fermentation product, for example one of those mentioned herein.
  • Organisms for example S. cerevisiae strains, able to produce ethanol from L- arabinose may be produced by modifying a cell introducing the araA (L-arabinose isomerase), araB (L-ribulokinase) and araD (L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes may be introduced into a transformed host cell is order that it is capable of using arabinose. Such an approach is given is described in WO2003/095627. araA, araB and araD genes from Lactobacillus plantarum may be used and are disclosed in WO2008/041840.
  • araA L-arabinose isomerase
  • araB L-ribulokinase
  • araD L-ribulose-5-P4-epimerase
  • araA gene from Bacillus subtilis and the araB and araD genes from Escherichia coli may be used and are disclosed in EP1499708.
  • araA, araB and araD genes may derived from of at least one of the genus Clavibacter, Arthrobacter and/or Gramella, in particular one of Clavibacter michiganensis, Arthrobacter aurescens, and/or Gramella forsetii, as disclosed in WO 200901 1591 .
  • a transformed host cell may comprise one or more genetic modifications that increases the flux of the pentose phosphate pathway.
  • the genetic modification(s) may lead to an increased flux through the non-oxidative part of the pentose phosphate pathway.
  • a genetic modification that causes an increased flux of the non- oxidative part of the pentose phosphate pathway is herein understood to mean a modification that increases the flux by at least a factor of about 1.1 , about 1.2, about 1.5, about 2, about 5, about 10 or about 20 as compared to the flux in a strain which is genetically identical except for the genetic modification causing the increased flux.
  • the flux of the non-oxidative part of the pentose phosphate pathway may be measured by growing the modified host on xylose as sole carbon source, determining the specific xylose consumption rate and subtracting the specific xylitol production rate from the specific xylose consumption rate, if any xylitol is produced.
  • the flux of the non- oxidative part of the pentose phosphate pathway is proportional with the growth rate on xylose as sole carbon source, preferably with the anaerobic growth rate on xylose as sole carbon source. There is a linear relation between the growth rate on xylose as sole carbon source ⁇ ma x) and the flux of the non-oxidative part of the pentose phosphate pathway.
  • One or more genetic modifications that increase the flux of the pentose phosphate pathway may be introduced in the host cell in various ways. These including e.g. achieving higher steady state activity levels of xylulose kinase and/or one or more of the enzymes of the non-oxidative part pentose phosphate pathway and/or a reduced steady state level of unspecific aldose reductase activity. These changes in steady state activity levels may be effected by selection of mutants (spontaneous or induced by chemicals or radiation) and/or by recombinant DNA technology e.g. by overexpression or inactivation, respectively, of genes encoding the enzymes or factors regulating these genes.
  • the genetic modification comprises overexpression of at least one enzyme of the (non-oxidative part) pentose phosphate pathway.
  • the enzyme is selected from the group consisting of the enzymes encoding for ribulose-5- phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase.
  • Various combinations of enzymes of the (non-oxidative part) pentose phosphate pathway may be overexpressed. E.g.
  • the enzymes that are overexpressed may be at least the enzymes ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase; or at least the enzymes ribulose-5-phosphate isomerase and transketolase; or at least the enzymes ribulose-5-phosphate isomerase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase and transketolase; or at least the enzymes ribulose-5- phosphate epimerase and transaldolase; or at least the enzymes transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase, transketolase and transaldolase; or at least the enzymes ribulose-5- phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose-5- phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate
  • each of the enzymes ribulose-5- phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase are overexpressed in the host cell. More preferred is a host cell in which the genetic modification comprises at least overexpression of both the enzymes transketolase and transaldolase as such a host cell is already capable of anaerobic growth on xylose. In fact, under some conditions host cells overexpressing only the transketolase and the transaldolase already have the same anaerobic growth rate on xylose as do host cells that overexpress all four of the enzymes, i.e.
  • ribulose-5-phosphate isomerase ribulose-5-phosphate epimerase
  • transketolase transaldolase
  • host cells overexpressing both of the enzymes ribulose-5-phosphate isomerase and ribulose-5- phosphate epimerase are preferred over host cells overexpressing only the isomerase or only the epimerase as overexpression of only one of these enzymes may produce metabolic imbalances.
  • ribulose 5-phosphate epimerase (EC 5.1 .3.1 ) is herein defined as an enzyme that catalyses the epimerisation of D-xylulose 5-phosphate into D-ribulose 5- phosphate and vice versa.
  • the enzyme is also known as phosphoribulose epimerase; erythrose-4-phosphate isomerase; phosphoketopentose 3-epimerase; xylulose phosphate 3-epimerase; phosphoketopentose epimerase; ribulose 5-phosphate 3- epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphate epimerase; D- ribulose-5-P 3-epimerase; D-xylulose-5-phosphate 3-epimerase; pentose-5-phosphate 3-epimerase; or D-ribulose-5-phosphate 3-epimerase.
  • a ribulose 5-phosphate epimerase may be further defined by its amino acid sequence.
  • a ribulose 5- phosphate epimerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5-phosphate epimerase.
  • the nucleotide sequence encoding for ribulose 5-phosphate epimerase is herein designated RPE1.
  • ribulose 5-phosphate isomerase (EC 5.3.1.6) is herein defined as an enzyme that catalyses direct isomerisation of D-ribose 5-phosphate into D-ribulose 5- phosphate and vice versa.
  • the enzyme is also known as phosphopentosisomerase; phosphoriboisomerase; ribose phosphate isomerase; 5-phosphoribose isomerase; D- ribose 5-phosphate isomerase; D-ribose-5-phosphate ketol-isomerase; or D-ribose-5- phosphate aldose-ketose-isomerase.
  • a ribulose 5-phosphate isomerase may be further defined by its amino acid sequence.
  • a ribulose 5-phosphate isomerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5- phosphate isomerase.
  • the nucleotide sequence encoding for ribulose 5-phosphate isomerase is herein designated RKI1.
  • transketolase (EC 2.2.1.1 ) is herein defined as an enzyme that catalyses the reaction: D-ribose 5-phosphate + D-xylulose 5-phosphate ⁇ -> sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate and vice versa.
  • the enzyme is also known as glycolaldehydetransferase or sedoheptulose-7-phosphate:D- glyceraldehyde-3-phosphate glycolaldehydetransferase.
  • a transketolase may be further defined by its amino acid.
  • transketolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transketolase.
  • the nucleotide sequence encoding for transketolase is herein designated TKL1.
  • transaldolase (EC 2.2.1.2) is herein defined as an enzyme that catalyses the reaction: sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate ⁇ -> D-erythrose 4-phosphate + D-fructose 6-phosphate and vice versa.
  • the enzyme is also known as dihydroxyacetonetransferase; dihydroxyacetone synthase; formaldehyde transketolase; or sedoheptulose-7- phosphate :D-glyceraldehyde-3 -phosphate glyceronetransferase.
  • a transaldolase may be further defined by its amino acid sequence.
  • transaldolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transaldolase.
  • the nucleotide sequence encoding for transketolase from is herein designated TALL
  • one or more copies of one or more xylose isomerase gene and/or one or more xylose reductase and xylitol dehydrogenase may be introduced in the host cell, e.g. into the genome of the host cell.
  • the presence of these genetic elements confers on the cell the ability to convert xylose by isomerisation or reduction.
  • the one or more copies of one or more xylose isomerase gene are introduced into the genome of the host cell.
  • a "xylose isomerase” (EC 5.3.1.5) is herein defined as an enzyme that catalyses the direct isomerisation of D-xylose into D-xylulose and/or vice versa. The enzyme is also known as a D-xylose ketoisomerase.
  • a xylose isomerase herein may also be capable of catalysing the conversion between D-glucose and D-fructose (and accordingly may therefore be referred to as a glucose isomerase).
  • a xylose isomerase herein may require a bivalent cation, such as magnesium, manganese or cobalt as a cofactor.
  • such a transformed host cell is capable of isomerising xylose to xylulose.
  • the ability of isomerising xylose to xylulose is conferred on the host cell by transformation of the host cell with a nucleic acid construct comprising a nucleotide sequence encoding a defined xylose isomerase.
  • a transformed host cell isomerises xylose into xylulose by the direct isomerisation of xylose to xylulose.
  • a unit (U) of xylose isomerase activity may herein be defined as the amount of enzyme producing 1 nmol of xylulose per minute, under conditions as described by Kuyper ei a/. (2003, FEMS Yeast Res. 4: 69-78).
  • the Xylose isomerise gene may have various origin, such as for example Piromyces sp. as disclosed in WO2006/009434.
  • Other suitable origins are Bacteroides, in particular Bacteroides uniformis as described in PCT/EP2009/52623, Bacillus, in particular Bacillus stearothermophilus as described in PCT/EP2009/052625.
  • one or more copies of one or more xylose reductase and xylitol dehydrogenase genes are introduced into the genome of the host cell.
  • the conversion of xylose is conducted in a two step conversion of xylose into xylulose via a xylitol intermediate as catalysed by xylose reductase and xylitol dehydrogenase, respectively.
  • xylose reductase (XR), xylitol dehydrogenase (XDH), and xylokinase (XK) may be overexpressed, and optionally one or more of genes encoding NADPH producing enzymes are up-regulated and one or more of the genes encoding NADH consuming enzymes are up-regulated, as disclosed in WO 2004085627.
  • a transformed host cell may comprise one or more genetic modifications that increase the specific xylulose kinase activity.
  • the genetic modification or modifications causes overexpression of a xylulose kinase, e.g. by overexpression of a nucleotide sequence encoding a xylulose kinase.
  • the gene encoding the xylulose kinase may be endogenous to the host cell or may be a xylulose kinase that is heterologous to the host cell.
  • a nucleotide sequence used for overexpression of xylulose kinase in the host cell is a nucleotide sequence encoding a polypeptide with xylulose kinase activity.
  • the enzyme is also known as a phosphorylating xylulokinase, D-xylulokinase or ATP :D- xylulose 5- phosphotransferase.
  • a xylulose kinase of the invention may be further defined by its amino acid sequence.
  • a xylulose kinase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a xylulose kinase.
  • a genetic modification or modifications that increase(s) the specific xylulose kinase activity may be combined with any of the modifications increasing the flux of the pentose phosphate pathway as described above. This is not, however, essential.
  • a host cell may comprise only a genetic modification or modifications that increase the specific xylulose kinase activity.
  • the various means available in the art for achieving and analysing overexpression of a xylulose kinase in the host cells of the invention are the same as described above for enzymes of the pentose phosphate pathway.
  • a xylulose kinase to be overexpressed is overexpressed by at least a factor of about 1 .1 , about 1 .2, about 1 .5, about 2, about 5, about 10 or about 20 as compared to a strain which is genetically identical except for the genetic modification(s) causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.
  • GRE3 Aldose reductase
  • a transformed host cell may therefore comprise one or more genetic modifications that reduce unspecific aldose reductase activity in the host cell.
  • unspecific aldose reductase activity is reduced in the host cell by one or more genetic modifications that reduce the expression of or inactivates a gene encoding an unspecific aldose reductase.
  • the genetic modification(s) reduce or inactivate the expression of each endogenous copy of a gene encoding an unspecific aldose reductase in the host cell (herein called GRE3 deletion).
  • Transformed host cells may comprise multiple copies of genes encoding unspecific aldose reductases as a result of di-, poly- or aneu-ploidy, and/or the host cell may contain several different (iso)enzymes with aldose reductase activity that differ in amino acid sequence and that are each encoded by a different gene. Also in such instances preferably the expression of each gene that encodes an unspecific aldose reductase is reduced or inactivated.
  • the gene is inactivated by deletion of at least part of the gene or by disruption of the gene, whereby in this context the term gene also includes any non-coding sequence up- or down-stream of the coding sequence, the (partial) deletion or inactivation of which results in a reduction of expression of unspecific aldose reductase activity in the host cell.
  • a nucleotide sequence encoding an aldose reductase whose activity is to be reduced in the host cell is a nucleotide sequence encoding a polypeptide with aldose reductase activity.
  • a host cell comprising only a genetic modification or modifications that reduce(s) unspecific aldose reductase activity in the host cell is specifically included in the invention.
  • aldose reductase (EC 1 .1 .1 .21 ) is herein defined as any enzyme that is capable of reducing xylose or xylulose to xylitol.
  • an aldose reductase may be any unspecific aldose reductase that is native (endogenous) to a host cell of the invention and that is capable of reducing xylose or xylulose to xylitol.
  • Unspecific aldose reductases catalyse the reaction:
  • the enzyme has a wide specificity and is also known as aldose reductase; polyol dehydrogenase (NADP + ); alditokNADP oxidoreductase; alditol:NADP + 1 - oxidoreductase; NADPH-aldopentose reductase; or NADPH-aldose reductase.
  • aldose reductase polyol dehydrogenase (NADP + ); alditokNADP oxidoreductase; alditol:NADP + 1 - oxidoreductase; NADPH-aldopentose reductase; or NADPH-aldose reductase.
  • an aldose reductase of the invention may be further defined by its amino acid sequence.
  • an aldose reductase may be defined by the nucleotide sequences encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding an aldose reductase. Bioproducts production
  • yeasts of the genus Saccharomyces as ethanol producer. This is due to the many attractive features of Saccharomyces species for industrial processes, i. e. , a high acid-, ethanol-and osmo- tolerance, capability of anaerobic growth, and of course its high alcoholic fermentative capacity.
  • Preferred yeast species as host cells include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K fragilis.
  • a transformed host cell may be a cell suitable for the production of ethanol.
  • a transformed host cell may, however, be suitable for the production of fermentation products other than ethanol
  • Such non-ethanolic fermentation products include in principle any bulk or fine chemical that is producible by a eukaryotic microorganism such as a yeast or a filamentous fungus.
  • a transformed host cell that may be used for production of non-ethanolic fermentation products is a host cell that contains a genetic modification that results in decreased alcohol dehydrogenase activity.
  • the transformed host cell may be used in a process wherein sugars originating from lignocellulose are converted into ethanol.
  • Lignocellulose which may be considered as a potential renewable feedstock, generally comprises the polysaccharides cellulose (glucans) and hemicelluloses (xylans, heteroxylans and xyloglucans). In addition, some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks.
  • glucans polysaccharides cellulose
  • hemicelluloses xylans, heteroxylans and xyloglucans
  • some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks.
  • the enzymatic hydrolysis of these polysaccharides to soluble sugars, including both monomers and multimers, for example glucose, cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and other hexoses and pentoses occurs under the action of different enzymes
  • pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins).
  • the lignocellulosic material Before enzymatic treatment, the lignocellulosic material may be pretreated.
  • the pretreatment may comprise exposing the lignocellulosic material to an acid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding, grinding, milling or rapid depressurization, or a combination of any two or more thereof.
  • This chemical pretreatment is often combined with heat-pretreatment, e.g. between 150-220 °C for 1 to 30 minutes.
  • the pretreated material is commonly subjected to enzymatic hydrolysis to release sugars that may be fermented according to the invention.
  • This may be executed with conventional methods, e.g. contacting with cellulases, for instance cellobiohydrolase(s), endoglucanase(s), beta-glucosidase(s) and optionally other enzymes.
  • the conversion with the cellulases may be executed at ambient temperatures or at higher tempatures, at a reaction time to release sufficient amounts of sugar(s).
  • the result of the enzymatic hydrolysis is hydrolysis product comprising C5/C6 sugars, herein designated as the sugar composition.
  • the fermentation process may be an aerobic or an anaerobic fermentation process.
  • An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors.
  • NADH produced in glycolysis and biomass formation cannot be oxidised by oxidative phosphorylation.
  • many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD + .
  • pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, butanol, lactic acid, di-terpene, glycosylated di-terpene, 3 -hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1 ,3-propane-diol, ethylene, glycerol, a ⁇ -lactam antibiotic and a cephalosporin.
  • the fermentation process is preferably run at a temperature that is optimal for the cell.
  • the fermentation process is performed at a temperature which is less than about 42°C, preferably less than about 38°C.
  • the fermentation process is preferably performed at a temperature which is lower than about 35, about 33, about 30 or about 28°C and at a temperature which is higher than about 20, about 22, or about 25°C.
  • the ethanol yield on xylose and/or glucose in the process preferably is at least about 50, about 60, about 70, about 80, about 90, about 95 or about 98%.
  • the ethanol yield is herein defined as a percentage of the theoretical maximum yield.
  • the invention also relates to a process for producing a fermentation product.
  • the fermentation process according to the present invention may be run under aerobic and anaerobic conditions. In an embodiment, the process is carried out under micro-aerophilic or oxygen limited conditions.
  • An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, and wherein organic molecules serve as both electron donor and electron acceptors.
  • An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid.
  • the degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used.
  • the rate of oxygen consumption is at least about 5.5, more preferably at least about 6, such as at least 7 mmol/L/h.
  • a process of the invention may comprise recovery of the fermentation product.
  • the cell ferments both the xylose and glucose, preferably simultaneously in which case preferably a cell is used which is insensitive to glucose repression to prevent diauxic growth.
  • the fermentation medium will further comprise the appropriate ingredient required for growth of the cell. Compositions of fermentation media for growth of microorganisms such as yeasts are well known in the art
  • the fermentation processes may be carried out in batch, fed-batch or continuous mode.
  • a separate hydrolysis and fermentation (SHF) process or a simultaneous saccharification and fermentation (SSF) process may also be applied.
  • SHF hydrolysis and fermentation
  • SSF simultaneous saccharification and fermentation
  • a combination of these fermentation process modes may also be possible for optimal productivity.
  • the reaction time for liquefaction/hydrolysis or presaccharification step is dependent on the time to realize a desired yield, i.e. cellulose to glucose conversion yield.
  • a desired yield i.e. cellulose to glucose conversion yield.
  • Such yield is preferably as high as possible, preferably 60% or more, 65% or more, 70% or more, 75% or more 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, even 99.5% or more or 99.9% or more.
  • the glucose concentration is 25g/L or more, 30 g/L or more, 35g/L or more, 40 g/L or more, 45 g/L or more, 50 g/L or more, 55 g/L or more, 60 g/L or more, 65 g/L or more, 70 g/L or more , 75 g/L or more, 80 g/L or more, 85 g/L or more, 90 g/L or more, 95 g/L or more, 100 g/L or more, 1 10 g/L or more, 120g/L or more or may e.g.
  • the product concentration (g/L) is dependent on the amount of glucose produced, but this is not visible since sugars are converted to product in the SSF, and product concentrations can be related to underlying glucose concentration by multiplication with the theoretical maximum yield (Yps max in gr product per gram glucose)
  • the theoretical maximum yield (Yps max in gr product per gram glucose) of a fermentation product can be derived from textbook biochemistry.
  • Butanol MW 74 gr/mole
  • the product concentration is 25g * Yps g/L /L or more, 30 * Yps g/L or more, 35g * Yps /L or more, 40 * Yps g/L or more, 45 * Yps g/L or more, 50 * Yps g/L or more, 55 * Yps g/L or more, 60 * Yps g/L or more, 65 * Yps g/L or more, 70 * Yps g/L or more , 75 * Yps g/L or more, 80 * Yps g/L or more, 85 * Yps g/L or more, 90 * Yps g/L or more, 95 * Yps g/L or more, 100 * Yps g/L or more, 1 10 * Yps g/L or more, 120g/L
  • Yps or more or may e.g. be 25 * Yps g/L-250 * Yps g/L, 30 * Yps gl/L-200 * Yps g/L, 40
  • the invention provides a method for the preparation of a fermentation product, which method comprises:
  • the fermentation product of the invention may be any useful product.
  • it is a product selected from the group consisting of ethanol, n-butanol, isobutanol, lactic acid, di-terpene, glycosylated di-terpene, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid, itaconic acid, maleic acid, citric acid, adipic acid, an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid, 1 ,3-propane-diol, ethylene, glycerol, a ⁇ -lactam antibiotic and a cephalosporin, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, including biofuels and biogas or organic polymers, and an industrial enzyme, such as a protease, a cellulase, an amylase
  • n-butanol may be produced by cells as described in WO2008121701 or WO2008086124; lactic acid as described in US201 1053231 or US2010137551 ; 3-hydroxy-propionic acid as described in WO2010010291 ; acrylic acid as described in WO2009153047.
  • Plasmids were amplified and maintained in chemically competent TOP10 cells (TOPO® TA Cloning® Kit, Life Techonologies) following manufacturer's instructions. Plasmids were isolated from E. coli mini cultures using the GeneJETTM Plasmid Miniprep Kit (Fermentas).
  • Genomic DNA was isolated from yeast using the YeaStarTM Genomic DNA Kit (ZymoResearch) following manufacturer's instructions.
  • Escherichia coli XL1 -blue strain was used for site-directed mutagenesis, sub- cloning and plasmid amplification, and grown in Luria-Bertani (LB) medium containing 150 mg/liter ampicillin (agar plates and liquid media). Cultivation of E. coli was performed at 37°C.
  • Yeast transformants were pre-cultured in 100 ml shake flasks containing 10 ml of Verduyn-urea with 2% maltose (DS68625) or 3% xylose (DS69473).
  • xylose 250 ml shake flasks with 50 ml of the same Verduyn- urea supplemented with various concentrations of this sugar (2%, 0.45% and 0.1 %) were inoculated with the corresponding pre-culture at an initial optical density (OD) at 600 nm of 0.1 .
  • oligonucleotides SEQ ID NO: 60 and SEQ ID NO: 61 , were used as the forward and reverse primers, respectively.
  • the PCR reaction with a total volume of 50 ⁇ contained 2 mM MgCI2, 0.05-0.07 mM MnCI2, 0.4 mM dATP, 2 mM dCTP/dGTP/dTTP, 40 pmol of each oligonucleotide primer, 40 ng of template DNA, and 5 units of Taq DNA polymerase.
  • the PCR program consisted of one cycle of 5 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 52°C, 3 min at 72°C and finally one cycle of 4 min at 72°C.
  • the mutagenic PCR fragments were digested with BamYW and Mun ⁇ prior to ligation into the same enzyme-digested pRS313-GAL2 vector. After ligation the mutant library was transformed directly into the S. cerevisiae strain DS68625 and screened for improved uptake of xylose.
  • plasmid pRS313-GAL2 was used as the template.
  • the subsequently identified GAL2 variant Gal2-1 .1 served as the template for the second round of epPCR. Therefore, all further variants in the next two rounds of epPCR include the alterations in the DNA sequence from the mutant Gal2-1 .1 .
  • the identified variant Gal2-2.1 from the second round of epPCR was chosen as the template for the third round of mutagenesis and therefore all further variants of Gal2 identified from this round include the alterations in the DNA sequence from both the variants Gal2-1 .1 and Gal2-2.1.
  • the silent mutation L343L in Gal2-2.1 which encodes for an additional Muni restriction site was removed and changed back into the wild-type sequence by site- directed mutagenesis prior to creation of the third mutant library.
  • a new Eag ⁇ restriction site located within the putative cytoplasmic loop region between TM2 and TM3 was introduced into Gal2-2.1 via site-directed mutagenesis with no change in amino acid sequence.
  • oligonucleotides SEQ ID NO: 62 and SEQ ID NO: 60 were used as the forward and reverse primers, respectively.
  • the resulting mutagenic DNA was digested with the appropriate restriction enzymes and substituted for the corresponding region of pRS313- GAL2-2.1/£agl.
  • the mutant libraries were transformed into the S. cerevisiae transporter deletion strain DS68625 for the first and second round of mutagenesis and into the hexokinase deletion strain DS69473 for the third round of mutagenesis, respectively.
  • the transformed yeast cells of the first two mutant libraries were plated out in 100 ⁇ aliquots on agar plates with Verduyn-urea containing 2% xylose and incubated for 3 days at 30°C. All plates were floated with 5 ml of Verduyn-urea without urea, vitamins and carbon source for 4 min.
  • the cell suspension was then supplemented with the appropriate amount of urea, vitamins and 1 % xylose and incubated overnight at 30°C at 180 rpm.
  • the cells were then washed twice with Verduyn without urea, vitamins and carbon source.
  • the cell analysis of the grown culture was performed on a flow cytometer (BD FACSDiVa, Becton Dickinson, Heidelberg, Germany) and budding yeast cells were sorted out on agar plates with exactly 25 ml of Verduyn-urea supplemented with 0.1 % xylose as carbon source. Exactly 100 single cells were sorted per agar plate to guarantee the same growth conditions for every single yeast cell.
  • the third screening round of random mutagenesis was carried out in the S. cerevisiae hexokinase deletion strain DS69473.
  • the mutant library was transformed and plated out on agar plates with Verduyn-urea containing 3% xylose. All plates were floated with 5 ml of Verduyn-urea as mentioned above.
  • the cell analysis of the grown culture was performed on a flow cytometer. Only budding yeast cells were sorted out on agar plates with exactly 30 ml of Verduyn-urea supplemented with 2% xylose and 10% glucose as carbon sources. In total 131 agar plates were incubated at 30°C and observed for several days.
  • OD600 and HPLC analysis in shake flask culture were sampled regularly during culture. For OD600 measurements, cultures were diluted appropriately for accurate measurement and optical density was measured at 600 nm wavelength in a Perkin Elmer Spectrophotometer K2 instrument. Remaining sample was filtrated to separate medium from yeast.
  • the filtrate was inserted into the appropriate vials for HPLC analysis.
  • concentrations of glucose, xylose, glycerol, acetic acid and ethanol in the medium were determined using a Shimadzu HPLC system.
  • the system is equipped with column oven CTO-I OA-vp and Autoinjector SIL-10AD-vp with a guard column (Bio-Rad H cartridge, Bio-Rad) and an Aminex HPX-87H column (300 x 7.8 mm; Bio-Rad). Elution took place at 80 °C with 5 mM H2S04 at 0.6 mL/min.
  • the eluate was monitored using a Refractive Index detector RID-10A (Shimadzu).
  • Microwell plate culture for growth curve profiling For micro-well cultivation of strains, the Bioscreen C (Growth Curves Ltd.) was used. Overnight pre-cultures were pelleted, washed with demi water and diluted in demi water to twice the desired OD600 for inoculation. Medium was prepared in twice the concentration as desired. In one well of a honeycomb wellplate, 150 ⁇ medium was mixed with 150 ⁇ cell suspension. Measurements were conducted in triplicate. Settings for the Bioscreen C were maintained at 30°C incubation T, measurements every 15 min, shaking at type Continuous, amplitude Maximum, and speed Normal. Shaking was set to stop 5 sec before measurement.
  • Variant 1.1 was used as the template for 2 nd round of random mutagenesis (further variants include these mutations)
  • Variant 2.1 was used as template for 3 rd round of random mutagenesis (further variants include these mutations except L343L)
  • Example 1 - Hexose transporter gene deletions Deletion cassettes construction. Primers used in plasmid constructions are shown in Table 3; generated plasmids are shown in Table 4. Schemes with restriction sites used for cloning and sites used to release deletion constructs from the plasmid backbone are shown in Table 5.
  • the kanMX marker was amplified from the plasmid pFA6-kanMX4 (http://www- sequence.stanford.edu/qroup/yeast deletion project/kan mx4.txt) using primers SEQ ID NO's 1 and 2. Subsequently, the kanMX marker was floxed through adding loxP flanks by PCR amplification with primers SEQ ID NO's 3 and 4. Re-amplification was done with primers SEQ ID NO's 5 and 6. The resulting loxP-kanMX-loxP fragment was cloned in pCR-BLUNT resulting in pRN201 (SEQ ID NO: 7).
  • hphMX was isolated from pGRE3:hphMX (Kuyper et al, 2005, Metabolic engineering of a xylose-isomerase- expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res, 5, pp. 399-409).
  • pGRE3:hphMX was cut with Eco32l and re-ligated.
  • hphMX was cloned as Xho ⁇ -Mlu ⁇ fragment into pRN201 digested with Sa/I and Mlu ⁇ to replace kanMX.
  • the Streptomyces noursei natl gene was PCR-amplified from pYL16 (Werner Bioagents) using primers with SEQ ID NO:'s 10 and -1 1.
  • the Psc ⁇ -Sca ⁇ natl fragment together with the Acc65 ⁇ -Nco ⁇ pRN201 - fragment were cloned into pRN201 , already linearized with Acc65 ⁇ and Seal, in order to replace kanR for natl.
  • pRN201 was digested with Pml ⁇ . This served two ends. Firstly, the Streptoalloteichus hindustanus ble (zeocin or phleomycin resistance gene) ORF was isolated, and secondly, after re-ligation of the Pm/l-digested pRN201 an ⁇ /col site was deleted. Subsequently, ble as Nco ⁇ -Pml ⁇ fragment and part of pRN201 as BamY ⁇ -Nco ⁇ vector fragment were cloned into the re- ligated pRN201 (missing ble), digested with SamHI and Seal resulting in pRN447.
  • Streptoalloteichus hindustanus ble zeocin or phleomycin resistance gene
  • primers SEQ ID13 and-14 were used to amplify the HIS3 locus from yeast genomic DNA . Sites used to cut out the HIS3 flanks and to ligate these to the floxed kanMX marker are shown in Table 5.
  • the ligation product was digested with Sacl and Apa ⁇ and cloned into pCR-BLUNT digested with Sacl and Apa ⁇ .
  • the resulting plasmid is pRN247 (SEQ ID NO: 15).
  • HXT1-7 and GAL2 main hexose transporters
  • S. cerevisiae four deletion constructs were generated (see Table 4). Each deletion construct contained a different floxed dominant resistance marker.
  • HXT gene 400-700 bp flanks were amplified using the primers listed in Table 3 (SEQ ID NO:'s 16- 31 ) using DS68616 genomic DNA as template.
  • the upstream flank, the dominant resistance marker and the downstream marker were ligated using the fragments and cloning sites listed under Table 5.
  • the ligations were amplified using the forward primers of the upstream flank and the reverse primer of the downstream flank (primer combinations SEQ ID NO:'s 16+19, SEQ ID NO:'s 20+27, SEQ ID NO:'s 24+23, and SEQ ID NO:'s 28+31 ).
  • the fused PCR fragments were cloned into pCR-BLUNT to obtain pRN485, pRN566, pRN569, pRN635 (SEQ ID NO:'s 32-35, respectively).
  • the plasmids were isolated from 50 mL £. coli cultures using NucleoBond® Xtra Midi kit (Bioke, Leiden, the Netherlands). Before transformation to yeast, deletion constructs were released from plasmid backbone by digestion with the release restriction sites listed in Table 5.
  • the xylose-fermenting strain DS68616 was made histidine auxotroph by the insertion of loxP-kanMX-loxP (released from pRN247; SEQ NO ID15) at the HIS3 locus. Subsequently, the marker was removed through transient expression of plasmid pRN187 (derived from pSH65 expressing galactose-inducible ere recombinase; SEQ ID NO 60). Introduction of pRN187 was selected on phleo and CRE recombinase expression was induced on YP-medium supplemented with galactose.
  • the hexose transporters were deleted in the following order: 1 ) HXT3-HXT6-HXT7 cluster, 2) HXT5-HXT1-HXT4 cluster, 3) GAL2, 4) HXT2.
  • the deletion constructs were linearized or released from the plasmid backbone by cutting with the enzyme combinations listed in Table 5 and these were integrated in the genome. All transformations were plated on yeast extract (10 g/L), peptone (20g/L) agar (15g/L) medium supplemented with 20g/L maltose.
  • DS68625 did not grow on Verduyn-urea (+ 20 g 1-1 % xylose during the culturing period (Fig. 2) indicating the strain is useable as model strain for testing putative xylose transporters. DS68625 was further maintained on YPM.
  • oligonucleotides were designed (SEQ ID NO:'s in Table 3) comprised of 60 nucleotide flanking sequences homologous to the hexokinase gene locus and of 20 nucleotides homologous to a floxed dominant resistance marker cassette. The oligonucleotides were used to amplify the deletion constructs. Subsequent PCR products were column filter- purified (Fermentas GeneJet Kit) and used for transformations experiments. Two types of deletion cassettes were used:
  • a bipartite system was used.
  • One fragment consisted of a lox66 site, kanMX, GAL1 promoter upstream of CRE, and the 5'-part of CRE (CRE1 ) amplified from pSUC227 with one gene-specific primer (SEQ ID NO: 44 for GLK1 and SEQ ID NO: 45 for HXK2) and one pSUC227-specific primer (SEQ ID NO: 46);
  • the second fragment consisted of the 3'-part of CRE (CRE2) with overlap on CRE1 , and a /ox71 site, amplified from pSUC225 with again one gene-specific primers (SEQ ID NO: 47 for GLK1 and SEQ ID48 for HXK2) and one pSUC225- specific primer SEQ ID NO: 49.
  • a floxed dominant resistance marker (DRM) was amplified with flanking sequences homologous to the respective hexokinase to replace the coding region at the locus; as templates for the PCR amplifications of the DRM cassettes pRN774 ⁇ loxP-hphUX-loxP; SEQ ID NO: 54) and pRN775 ⁇ loxP-natMX-loxP; SEQ ID NO: 55) were used, respectively.
  • Strain construction For the generation of a strain incapable of hexose metabolism but capable of hexose transport, four hexokinase gene deletions were made in the xylose-fermenting strain DS68616 (Table 1 ).
  • HIS3 was deleted in DS68616.
  • the histidine-auxotroph strain (DS68616-/?/s3:.'/oxP) was the same strain, as was constructed in the DS68625 lineage.
  • the disruption cassettes were bi-partite. Through homologous recombination the two fragments integrate as lox66-kanMX-CRE-lox71 at the hexokinase locus. The integration was selected on YPD supplemented with G418.
  • the disruption cassettes for HXK1 and GAL1 consisted of one fragment: either loxP- natMX-loxP or loxP-hphMX-loxP, respectively.
  • the DS68616-/?/s3.'.7oxP was transformed with the purified PCR products and the integration was selected on the appropriate antibiotic.
  • the hexokinase genes were deleted in the following order: 1 ) GLK1, 2) HXK1, 3) HXK2, 4) GALL After the deletion of GLK1, the kanMX marker was recycled by galactose-induced Cre-mediated recombination. After deletion of HXK2, the intermediate strain was maintained on xylose-containing rich medium (YPX). After GAL1 deletion, the integrated markers were removed by galactose-induced CRE recombination. To ensure growth of the strain, 2% xylose was added to YP 2% galactose + hygromycin (YPGX).
  • GAL2 GAL2
  • SEQ ID NO: 56 a distinct part of GAL2 (SEQ ID NO: 56) was selected for mutagenesis, coding for the Gal2p protein sequence (SEQ ID NO: 57) between amino acid positions Asp292 and Ser477.
  • This segment which contains 185 amino acids, includes a large part of the proposed central cytoplasmic loop between the transmembrane (TM) segments 6 and 7, TM7 to TM10, and the corresponding cytoplasmic and extracellular loops (see Fig. 4A).
  • This part of the Gal2 transport protein was chosen based on relevant amino acids concerning hexose sugar transport identified in related hexose transporter proteins in S. cerevisiae (Kasahara & Maeda, 1998.
  • the library of mutagenic PCR fragments digested with the proper restriction zymes was inserted into pRS313-GAL2 (Fig. 4C) also digested with the same restriction enzymes.
  • the library of GAL2 variant plasmids was transformed into the S. cerevisiae deletion strain DS68625 lacking the hexose transporters HXT1-HXT7 and GAL2 (see Example 1 ).
  • this strain has been engineered for xylose utilization by introduction of the xylose isomerase gene ⁇ xylA) from the anaerobic fungus Piromyces sp. E2. All transformants harboring the mutant library were collected from the selective agar plates by floating the plates, transferring all yeast cells and growing the cell suspension overnight with 1 % xylose. Prior to screening for high-affinity xylose candidates, the overnight culture was analyzed with cell flow cytometry for budding yeast cells only. Subsequently, budding yeast cells were sorted out on agar plates containing exactly 25 ml of Verduyn-urea with 0.1 % xylose as sole carbon source.
  • Gal2-1 .1 One candidate, designated as Gal2-1 .1 , facilitated a faster growth as compared to wild-type Gal2 permease (Fig. 5).
  • DNA sequencing of this variant revealed a single amino acid substitution at position 31 1 where a leucine, located within the cytoplasmic loop region between TM6/7, was exchanged to an arginine. Additionally three silent mutations were also identified (L362L, D363D, K469K) (Table 2).
  • Gal2-2.2 showed another amino acid substitution at position 468 where a serine was substituted to a threonine within the loop region between TM10 and TM1 1.
  • the conditions were changed to improve the uptake of xylose in the presence of glucose.
  • the strain DS69473 is an ideal host for screening for transporters able to take up xylose in the presence of glucose competition for transport.
  • the new mutant library was transformed into the hexokinase deletion strain DS69473.
  • the mutant library was plated on Verduyn-urea agar plates containing 2% xylose and 10% glucose as carbon sources. In total 131 agar plates with single budding yeast cells were incubated at 30°C. After 12 days of incubation at 30°C only eight colonies could be identified. Out of these eight candidates DNA sequencing revealed five new Gal2 mutants with amino acid substitutions, two mutants with silent mutations and two candidates were found to be the same (Table 2).
  • the 2 nd round mutant 2.1 which was included as a control showed a significant decreased growth compared to that of the wild-type Gal2 (Fig. 7A).
  • the 3 rd round mutant 3.7 which was developed from mutant 2.1 displayed an almost identical growth as the wild-type Gal2.
  • mutant 3.2 showed an increased growth at this glucose concentration as compared to 2.1 but not as fast as 3.7 or the wild-type.
  • the mutants 3.1 , 3.5 and 3.6 revealed a strong impaired growth on 2% glucose up to 48 h.
  • the mutant 2.1 displayed the fastest growth among all tested candidates and the wild-type (Fig. 7B).
  • mutants 3.2 and 3.7 showed a slightly reduced growth than 2.1 , but a clearly faster growth than the wild-type.
  • mutant 3.1 which showed no growth at 2% glucose for the first 48 h grew almost as fast as 3.2 and 3.7 at 2% xylose.
  • the variants 3.5 and 3.6 showed no growth at 2% xylose over the tested period of time.
  • the two mutants Gal2-2.1 and 3.1 were evaluated under this aspect. After retransformation into DS68625 these two evolved mutants were analyzed for growth at 2% xylose in the presence of 2% glucose (ratio 1 :1 ) to test for improved uptake at higher sugar concentrations. In parallel, the mutants were also tested for improved affinity towards xylose at 0.45% xylose in the presence of 0.45% glucose (ratio 1 :1 ). Besides measuring the optical density at 600 nm the consumption of both sugars was analyzed by HPLC by measuring the remaining sugar concentration in the medium over time (Fig. 8A-D).
  • the diauxic sugar consumption profile in which glucose is consumed first, was detectable for both the Gal2 wild-type and mutant 2.1 under the growth conditions of 2% xylose with 2% glucose.
  • the variant 2.1 showed a faster growth than the Gal2 wild-type. This was confirmed by the HPLC analyses which revealed a faster consumption of both sugars for this mutant (compare Fig. 8A and 8C).
  • Mutant 2.1 showed also a faster cell growth than the wild-type in medium containing 0.45% xylose and 0.45% glucose (Fig. 8B).
  • Fig. 8D mutant 2.1 showed an almost simultaneous consumption of xylose and glucose.
  • the mutation at position 435 Compared to the wild-type Gal2 the mutation at position 435, where a methionine was substituted by a threonine, showed the best growth results among all the tested single mutants under the different conditions.
  • the mutation M435T L301 R+M435T, K310R+M435T, N314D+M435T and M435T+S468T.
  • the combination N314D+M435T showed the fastest growth at the three tested concentrations.
  • the mutation K310R in combination with N314D showed also an improvement of xylose uptake.

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Abstract

The invention relates to a polypeptide having one or more substitution corresponding to a substitution on position 301, 310, 311, 314, 435 and/or 468 of SEQ ID NO:57 wherein the polypeptide is member of the Major Facilitator Superfamily (MFS).

Description

POLYPEPTIDES WITH PERMEASE ACTIVITY
Field of the invention
The invention is directed to novel polypeptides and to recombinant organisms expressing the polypeptides. In an embodiment, the present invention relates to novel permease polypeptides with altered sugar specificity and/or sugar transport activity, more specifically to novel GAL2 polypeptides in Saccharomyces cerevisiae.
Background of the invention
The plasma membrane of yeast cells and other eukaryotes is a complex bio- membrane, consisting of two layers of phospholipids, with a plethora of proteins embedded in it. Many molecules may cross the plasma membrane by diffusion and osmosis or with the aid of specific transport systems.
Transport systems allow the uptake of nutrients and ions, export of products of metabolism and undesired or harmful substances. Different mechanisms exist. Primary active transporters drive solute accumulation or extrusion by using for instance ATP hydrolysis. Secondary carriers, belonging to the Major Facilitator Superfamily (MFS) transporters, facilitate the transport of one or more molecular species across the membrane in response to chemi-osmotic gradients. In the yeast Saccharomyces cerevisiae, 186 MFS proteins have been identified (Nelissen, 1997) in strain S288c.
An example of such a carrier is the Hxt1 protein, involved in hexose transport in Saccharomyces cerevisiae.
Permeases are membrane transport proteins, a class of multipass transmembrane proteins that facilitate the diffusion of a specific molecule, herein specifically one or more sugar, in or out of the cell by passive transport. In contrast, active transporters couple molecule transmembrane transport with an energy source such as ATP or a favorable ion gradient.
The terms permease, facilitator, transporter or transport protein or related terms are all describing proteins with multiple membrane spanning domains that exhibit a function in transporting molecules across a membrane. This transport can be brought about by different mechanisms: uniport (transport of one molecule), symport (simultaneous co-transport of two different molecules in the same direction), antiport (simultaneous transport of two molecules in opposite directions) and facilitated diffusion. The family of sugar transporters in yeast consists of 30-40 members (34 members in strain S288c (Nelissen, 1997)). The sugar transporters can be divided in five clusters: hexose permeases (ΗΧΓ-genes, GAL2), disaccharide permeases, myo-inositol permeases, sugar receptors and a final cluster of transporters of which the substrate is unknown.
Lignocellulosic biomass, an attractive alternative feedstock for the production of liquid transportation fuels, consists of several different sugars. The hexose fraction of lignocellulose, mainly glucose, can in principle be readily fermented by non-recombinant versions of the yeast S. cerevisiae. However, this organism is not able to metabolize the pentose sugars, such as xylose and arabinose, into ethanol.
Methods of creating microorganisms that are able to metabolize pentose sugars are known in the art. For instance, in WO/2009/109630 the construction of expression cassettes and the transformation of S. cerevisiae cells into pentose fermenting cells by expressing xylose isomerase are illustrated.
Native pentose-utilizing organisms exist but are lacking well-developed genetic tools and/or low product tolerances, which limit their suitability as hosts for lignocellulosic conversion processes.
As a consequence, efforts have focused on the introduction of pentose conversion pathways in the yeast S. cerevisiae, which is still the organism of choice in the ethanol industry, in order to enable pentose fermentation.
Despite the vast amount of progress achieved in the past years, the transport of pentose sugars is still considered to be (one of) the rate-limiting step in pentose metabolism.
Pentose transport in S. cerevisiae is mediated by the different members of the hexose transporter (Hxt) family. Hxt4, Hxt5, Hxt7 and Gal2 have been described as the main xylose transporters in S. cerevisiae (Hamacher et al, 2002), and Gal2 is also known to mediate arabinose transport (Becker, et al, 2003). However, the affinity for the respective pentose sugars is approximately 10 to 100 times lower than for the respective hexose sugars. The lack of a dedicated xylose or arabinose transporter in recombinant yeast cells thus limits the capacity for co-utilization of hexoses and pentoses in sugar mixtures, and prohibits a high pentose catabolic flux. As a consequence, conversion of biomass sugars may be considered bi-phasic: in the first phase, a relatively fast conversion of hexoses (glucose) takes place, while in the second phase, which starts when the hexoses have been exhausted from the medium, pentose fermentation commences, but at a far lower rate as compared to the rate of hexose conversion.
It is therefore a long-felt desire to express pentose-specific sugar transporters, i.e. no glucose interference (pentose specificity) and high affinity to pentose, in an otherwise unchanged transporter landscape, in order to maintain the ability to convert hexoses at approximately the same level.
One way of solving this problem is to screen for heterologous sugar (pentose) transporters which are pentose specific and have a (moderately) high affinity for pentose. However, such efforts have been with limited success so far. Only a few have been shown to be able to facilitate pentose transport upon expression in S. cerevisiae, but all favour glucose above xylose (Young et al, 201 1 , and references therein), as is the case with the S. cerevisiae Hxt-proteins, as indicated above.
Another approach is to re-engineer hexose transporters to pentose transporters. For instance, the works by Kasahara et al (2000; 2009; 2010) indicate which residues in several sugar transporters play a key role in the determination of the substrate affinity to the natural substrate.
Mutant hexose transporters that are able to transport pentose sugars more efficiently are known in the art. For instance, in WO/2012/049173, the isolation of mutant hexose transporter genes from cultures of pentose-fermenting S. cerevisiae cells is described.
In Saccharomyces cerevisiae, the permease GAL2 transports galactose across the cell membrane. It is also known as a transporter of glucose across the membrane.
Summary of the invention
An object of the invention is to provide novel permease polypeptides with altered, in particular improved, sugar specificity. Another object of the invention is to provide recombinant strains expressing the permease polypeptide that have improved uptake of the molecule that the permease transports across the cell membrane. Another object is to provide a permease polypeptide that has a improved capacity for transport of C5 sugars, in particular xylose compared to a parent polypeptide. Another object is to provide a permease polypeptide that has reduced transport activity for C6 sugar, in particular glucose, compared to a parent polypeptide. Another object is to provide a method to identify mutations in other related permease polypeptides that have a beneficial effect on the improved capacity for transport of xylose or reduced transport activity for glucose.
One or more of these objects are attained according to the invention. According to the present invention, there is provided a polypeptide having one or more substitution corresponding to a substitution on position 301 , 310, 31 1 , 314, 435 and/or 468 of SEQ ID NO:57 wherein the polypeptide is member of the Major Facilitator Superfamily (MFS). In an embodiment, the polypeptide has one or more substitution corresponding to L301 R/M/IA /F/Q/A, K310R/N/M/Q/T/D/E, L31 1 R/M/l/V/F/Q/A, N314D/H/S/K/E/T/Q/G/A, M435T/L/IA /K/R/Q/F and/or S468T/L/IA /K/R/Q/F of SEQ ID NO: 57. In an embodiment, the polypeptide has pentose transport activity. In an embodiment, the polypeptide has xylose transport activity. In an embodiment, the polypeptide has one or more substitution corresponding to L301 R, K310R, L31 1 R, N314D, M435T and/or S468T of SEQ ID NO: 57.
A polypeptide according to the invention having one or more of these mutations has an advantageous sugar consumption and/or fermentation product production. This this will be described in more detail below and will be illustrated by examples 1 -5 below.
Brief description of the drawings
FIG. 1 shows results of aerobic shake flask cultures hexose transporter mutants on Verduyn-urea + 15 g 1 glucose + 20 g Γ1 xylose. (A) Optical density measurements at 600 nm wavelength, (B) glucose concentrations (g Γ1), (C) xylose concentrations during the culture period.
FIG. 2 shows results of aerobic shake flask cultures hexose transporter mutants on Verduyn-urea + 20 g Γ1 xylose. (A) Optical density measurements at 600 nm wavelength, (B) xylose concentrations (g Γ1).
FIG. 3 shows results of micro-well plate cultures on Verduyn-urea-his supplemented with 2% glucose (A) or 2% xylose (B). Growth characteristics were determined for DS64973 {Aglkl, Ahxkl, Ahxk2, Agall) reference strain DS68616 {GLK1, HXK1, HXK2, GAL1), and all intermediate hexokinase mutants (single [Aglkl], double [Aglkl, Ahxkl], triple [Aglkl, Ahxkl, Ahxk2]). Every 15 minutes, OD600 was measured automatically by Bioscreen C apparatus. Data points are the average of measurements in triplicate. FIG. 4 shows a S. cerevisiae Gal2 wild-type transporter protein topology model predicted by SOSUI. The cytoplasm is located at the bottom of each model, the extra-cytoplasm is on top and the numbers depict the transmembrane segments. For the first two rounds of epPCR, the restriction enzyme sites BamYW and Mun\ were used to excise the template DNA. Eag\ and Mun\ were used to excise the template DNA for the third round of epPCR. (A) The highlighted black areas indicate the identified amino acid substitutions after two rounds of random mutagenesis. (B) The black crosses indicate the identified alterations in the amino acid sequence after the third round of random mutagenesis. The black arrow shows the mutation in TM8 in candidate 3.1 that led to significantly impaired uptake of glucose but not of xylose. (C) Plasmid map of expression vector pRS313- GAL2 used as host vector to insert mutagenic PCR fragments; for first and second round mutagenesis fragments were inserted between BamYW and Mun\, and for third round an Eagl site was added between TM2 and TM3 on GAL2 to expand the diversity of variant GAL2 sequences.
FIG. 5 shows the comparison of DS68625 expressing wild-type Gal2 (square) and the identified variant 1 .1 (circle) from the first round of mutagenesis in Verduyn-urea with 2% xylose as carbon source. DS6825 expressing an empty vector (diamond) was included as control. Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from three independent experiments.
FIG. 6 shows the comparison of growth (A and B) and consumption (C and D) of Gal2 (wild-type) and identified mutants after two rounds of random mutagenesis at low xylose concentrations. DS68625 expressing wild-type Gal2 (square) and the variants 1 .1 (circle), 2.1 (up triangle), 2.2 (down triangle), 2.3 (cross) and 2.4 (pentagon) in Verduyn- urea with 0.45% xylose (A and C) and with 0.1 % xylose (B and D) as carbon source. DS68625 (diamond) expressing an empty vector was included as a control. Growth was determined measuring the optical density at 600 nm. At different time points the sugar consumption was determined via HPLC. The error bars represent standard deviations calculated from three independent experiments.
FIG. 7 shows the comparison of growth facilitated by GAL2 variants after three rounds of directed evolution. DS68625 expressing wild-type Gal2p (square) and the variants with amino acid substitutions 2.1 (up-triangle), 3.1 (cross), 3.2 (down triangle), 3.5 (left triangle), 3.6 (right triangle) and 3.7 (pentagon) in Verduyn-urea with 2% glucose (A) and with 2% xylose (B) as sole carbon source. DS68625 (black, diamond) expressing the empty vector was included as control. Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from two independent experiments.
FIG. 8 shows the growth and consumption measurements with xylose in the presence of glucose. (A) shows growth in Verduyn-urea with 2% xylose and 2% glucose, (B) shows growth in Verduyn-urea with 0.45% xylose and 0.45% glucose: wild-type Gal2 (square), mutants 2.1 (triangle) and 3.1 (star), DS68625 (diamond). (C) shows consumption measurements for 2% xylose with 2% glucose and (D) for 0.45% xylose with 0.45% glucose, respectively: wild-type Gal2 with glucose (empty square), wild-type Gal2 with xylose (filled square), mutants 2.1 with glucose (empty triangle), 2.1 with xylose (filled triangle), 3.1 with glucose (empty star), 3.1 with xylose (filled star), DS68625 with glucose (empty diamond), DS68625 with xylose (black, diamond). Gal2 and mutants were expressed in hxt1-7, gal2 deletion strain DS68625. DS68625 expressing the empty vector was included as control. Growth was determined measuring the optical density at 600 nm. Sugar consumption was determined via HPLC. The error bars represent standard deviations calculated from three independent experiments, glc: glucose; xyl: xylose.
FIG. 9 displays growth facilitated by wild-type Gal2 in comparison to mutants 2.1 , 3.1 and Gal2_T386A. DS68625 expressing wild-type Gal2 (square) and mutants 2.1 (triangle), 3.1 (cross) and Gal2_T386A (circle) in Verduyn-urea with 2% glucose (A), 2% xylose (B), 0.1 % glucose (C) and 0.1 % xylose (D) as carbon sources. DS68625 expressing the empty vector (diamond) was included as control. Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from two independent experiments.
FIG. 10. Growth behavior facilitated by Gal2 mutants of S. cerevisiae. DS68625 expressing Gal2 wild-type (square), with a single mutation M435T (circle), and double mutations M435T+L301 R (up triangle), M435T+K310R (down triangle), M435T+N314D (left triangle), M435T+S468T (right triangle), K310R+N314D (pentagon) in Verduyn-urea with 2% xylose (A), 0.45% xylose (B) and 0.1 % xylose (C) as carbon source. DS68625 expressing the empty vector (diamond) was included as control. Growth was determined measuring the optical density at 600 nm. The error bars represent standard deviations calculated from two independent experiments.
Brief description of the sequence listing
SEQ ID NO 1 : primer 5034-kanf
SEQ ID NO 2: primer 5035-kanr
SEQ ID NO 3: primer 51 16-lf2
SEQ ID NO 4: primer 51 18-lr2
SEQ ID NO 5: primer 51 15-lf 1
SEQ ID NO 6: primer 51 17-I
SEQ ID NO 7: pRN201 ; TOPO-BLUNT-loxP-kanMX-loxP
SEQ ID NO 8: pRN251 ; TOPO-BLUNT-loxP-hphMX-loxP
SEQ ID NO 9. pRN365; TOPO-BLUNT-loxP-natMX-loxP
SEQ ID NO 1 0: primer 1 15-natf
SEQ ID NO 1 1 : primer 1 16-natr
SEQ ID NO 1 2: pRN447; TOPO-BLUNT-loxP-zeoMX-loxP
SEQ ID NO 13: primer 28-H3f
SEQ ID NO 14: primer 29-H3r
SEQ ID NO 1 5: pRN247 (TOPO- BLUNT-HIS3::loxPkanMXIoxP)
SEQ ID NO 16: primer 201 -Hx2uf
SEQ ID NO 1 I 7: primer 202-Hx2ur
SEQ ID NO 1 18: pri mer 203-Hx2df
SEQ ID NO 119: pr mer 204-Hx2dr
SEQ ID NO 20: pr mer 205-Hx3uf
SEQ ID NO 21 : pr mer 206-Hx3ur
SEQ ID NO 22: pr mer 210-Hx4df
SEQ ID NO 23: pr mer 21 1 -Hx4dr
SEQ ID NO 24: pr mer 212-Hx5uf
SEQ ID NO 25: pr mer 213-Hx5ur
SEQ ID NO 26: pri mer 229-Hx7df
SEQ ID NO 27: pr mer 230-Hx7dr
SEQ ID NO 28: pr mer 243-Gal2ufn
SEQ ID NO 29: primer 244-Gal2urn SEQ ID NO 30: primer 233-Ga2df
SEQ ID NO 31 : primer 234-Ga2dr
SEQ ID NO 32: pRN485; TOPO-BLUNT-GAL2::loxPzeoMXIoxP
SEQ ID NO 33: pRN566; TOPO-BLUNT-HXT367::loxP-hphMX-loxP
SEQ ID NO 34: pRN569: TOPO-BLUNT-HXT514::loxP-natMX-loxP
SEQ ID NO 35: pRN635; TOPO-BLUNT-HXT2::loxP-kanMX-loxP
SEQ ID NO 36: pi imer 281 -Hx3inr2
SEQ ID NO 37: pi imer 323-Hx7inr1
SEQ ID NO 38: pi imer Hx4inr2
SEQ ID NO 39: pi imer Hx5inf
SEQ ID NO 40: pi imer 324-Ga2inf1
SEQ ID NO 41 : pi imer 325-Ga2inr1
SEQ ID NO 42: pi imer 289-Hx2inf
SEQ ID NO 43: pi imer 290-Hx2inr
SEQ ID NO 44: pi imer Glk1 -psuc227f
SEQ ID NO 45: pi imer Hxk2-psuc227f
SEQ ID NO 46: pi imer pSUC227r
SEQ ID NO 47: pi imer Glk1 -psuc225r
SEQ ID NO 48: pi imer Hxk2-psuc225r
SEQ ID NO 49: pi imer pSUC225f
SEQ ID NO 50: pi imer Hxk1_loxP_f
SEQ ID NO 51 : pi imer Hxk1_loxP_r
SEQ ID NO 52: pi imer Gal1_loxP_f
SEQ ID NO 53: pi imer Gal1_loxP_r
SEQ ID NO 54: pRN774; TOPO-BLUNT-loxP-hphMX-loxP (loxP sites in opposite orientation)
SEQID 55: pRN775; TOPO-BLUNT-loxP-natMX-loxP (loxP sites in opposite orientation) SEQ ID NO 56: WT-GAL2 DNA sequence
SEQ ID NO 57: WT Gal2p amino acid sequence
SEQ ID NO 58: pRS313-GAL2 (GAL2 expression vector uSEQ as host vector for error prone mutagenesis library
SEQ ID NO 59: pRN187 (pSH65-derived CRE recombinase expressing vector) SEQ ID NO 60: Primer Gal2_Bamf
SEQ ID NO 61 : Primer Gal2 Munr SEQ ID NO 62: Primer Gal2_Eagf
Detailed description of the invention
Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.
The invention relates to identification of amino acid positions in permease polypeptides, preferably hexose permease polypeptides, more preferably hexose permease polypeptides from yeast and fungi, even more preferably in Saccharomyces cerevisiae Hxt or Gal2 permease polypeptides, which are mutated to alter the sugar transport activity or sugar specificity of the permease.
The permeases belong to the Major Facilitator Superfamily (MFS). This is defined hereinbelow. Cellular transport systems allow the uptake of essential nutrients and ions, and excretion of products of metabolism and deleterious substances. In addition, transport systems play a role in the communication between cells and the environment. Also, they are an essential part of the cell system to yield or consume energy-supplying molecules, such as ATP.
The transport of solutes by primary active transporters is energy-driven in the first place, such as by energy supplied from ATP hydrolysis, photon absorption, electron flow, substrate decarboxylation, or methyl transfer. If charged molecules are pumped in one direction as a consequence of the consumption of a primary cellular energy source, an electrochemical potential is the result. The resulting chemiosmotic gradient can then be used to drive the transport of additional molecules via secondary carrier structures which just facilitate the transport of one or more molecules across the membrane.
The last two decades the existence of a multitude of previously unknown protein families of primary and secondary transporters has been clarified by the emergence of genomics strategies and making use of the many performed biochemical and molecular genetics studies. The two main transporter families of which proteins were found throughout all living organism are of the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS), also known as the uniporter-symporter-antiporter family. Whereas ABC family permeases consist of multiple components and are primary active transporters, capable of transporting both small molecules and macromolecules only after generating energy through ATP hydrolysis, the MFS transporters consist of a single polypeptide of a secondary carrier which facilitates transport of small solutes in response to a chemiosmotic ion gradient. ABC superfamily and MFS proteins account for almost half of the solute transporters encoded within the microbe genomes (reviewed by Pao et al, 1998, Microbiol Mol Biol Rev.; 62 pp.1-34, and Saier et al, 1999, J Mol Microbiol Biotechnol, 1 pp.257-279).
Suitable permease polypeptide sequences can contain one or more of the following motifs:
a) G-R-x(3)-G-x(3)-G-x(1 1 )-E-x(5)-[LIVM]-R-G-x(12)-[GA] ;
b) R-x(14)-G-x(2)-Y-x(2)-[YF]-[YF]-[GSAL] and/or
c) V-x(15)-[GNR]-[RH]-R-x(2)-[LM]-x(2)-[GA]
Motif (a) is corresponds to residues 179-221 in Gal2; motif (b) is corresponds to residues 330-353 in Gal2; motif (c) is corresponds to residues 375-399 in Gal2.
Variant polypeptides of the invention may be found by the skilled person as described herein after. This includesmodeling a permease polypeptide sequence onto the published crystal structure of the xylose- or glucose-bound Escherichia coii xylose permease XylE (respectively, PDB code 4GBY & 4GBZ in the PDB database, http://www.pdb.org) to identify the amino acid positions in the channel of the permease that directly interact with the bound sugar (called the first-shell residues in the art), and the residues that interact with the first shell residues (called the second shell residues in the art). Suitable modeling software to construct such models are YASARA, Prime (Schrodinger Inc.) or MODELLER using the default settings. Alternatively, the sugar- specificity-altering first and second shell amino acid positions in a permease polypeptide sequence can be identified by a global pairwise alignment of the permease sequence with the Gal2 sequence SEQ ID NO: 57 using the NEEDLE protocol described below. An example alignment for Gal2 and Hxt's from Saccharomyces cerevisiae is given in Fig. 10, which shows how alignment can be used to identify the corresponding amino acid positions in the different yeast Hxt's. The amino acid positions herein thus refer to SEQ ID NO: 57 that describes Gal2 or to corresponding aminoacid positions in other polypeptides, in particular other permease polypeptides. For example, the corresponding position of the position N376 in Gal2 (SEQ ID NO; 57) in Hxt1 is N370, in Hxt2 N361 , in Hxt3 N367, in Hxt4SC N376, in Hxt4RN N376, in Hxt5 N391 , in Hxt6/7 N370, in Hxt8 N372, in Hxt9 N366, in Hxt10 N354, in Hxt1 1 N366, in Hxt12 N256, in Hxt13 N363, in Hxt14 N387, in Hxt15 N366, in Hxt16 N366 and in Hxt17 N363. Similarly, the corresponding position of N346 in Gal2 (SEQ ID NO:9) in Hxt1 is D340, in Hxt2 N331 , in Hxt3 D337, in Hxt4SC D346, in Hxt4RN D346, in Hxt5 D361 , in Hxt6/7 D340, in Hxt8 D342, in Hxt9 D336, in Hxt10 C324, in Hxt1 1 D336, in Hxt12 D226, in Hxt13 E333, in Hxt14 I357, in Hxt15 E336, in Hxt16 E336 and in Hxt17 E333. This can be similary done for other MFS Superfamily transporters, so that corresponding positions in these polypeptides corresponding to the positions in SEQ ID NO: 57 can be obtained.
A person skilled in the art can subsequently mutate the identified amino acid positions in the permease polypeptide to all other 19 amino acids, and screen for improved C5 sugar uptake and/or reduced C6 sugar uptake of the mutant permease, as described in Example 4 and 5.
For instance, for a polypeptide having a mutation at a position corresponding to one or more position corresponding to N346 of SEQ ID NO: 57, the mutations at the positions corresponding to N346 may be a substitution with C, P, G, A, V, L, I , M, F, W, Y, H, S, T, N, Q, D, E, K, R or a deletion. X may be any aminoacid, X(2) means two X.
Herein, Gal2 is a facilitated diffusion transporter required for both the high-affinity galactokinase-dependent and low-affinity galactokinase-independent galactose transport processes. It belongs to the major facilitator superfamily, sugar transporter (TC 2.A.1 .1 ) family. "Permease polypeptide", is also designated herein as "polypeptide permease" or "polypeptide". "Permease polypeptide polynucleotide", is herein a polynucleotide that encodes the permease polypeptide.
In an embodiment of the invention, the permease polypeptide has one or more substitution corresponding to a substitution on position 301 , 310, 31 1 , 314, 435 and/or 468 of SEQ ID NO:57 and has at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with SEQ ID NO: 57.
Herein mutations are indicated by one letter aminoacids and positions of these amino acids. For example, A6 herein indicates an amino acid (one letter code) at a certain position in SEQ ID NO: 57, here A (Alanine) at position 6 of the protein. A6 (L/N/Q/GA /I Y/S/E/K) indicates herein mutation of amino acid at a certain position, here A (Alanine) at position 6 of the protein is exchanged for any of L (Leucine), N (Asparagine), Q (Glutamine), G (Glycine), V (Valine), I (Isoleucine), Y (Tyrosine), S (Serine), E (Glutamic acid) or K (Lysine).
In an embodiment, the polypeptide has xylose transport activity.
In an embodiment the polypeptide has one or more substitution corresponding to According to the present invention, there is provided a polypeptide having one or more substitution corresponding to a substitution on position 301 , 310, 31 1 , 314, 435 and/or 468 of SEQ ID NO:57 wherein the polypeptide is member of the Major Facilitator Superfamily (MFS). In an embodiment, the polypeptide has pentose transport activity. In an embodiment, the polypeptide has one or more substitution corresponding to L301 R/M/IA /F/Q/A, K310R/N/M/Q/T/D/E, L31 1 R/M/l/V/F/Q/A, N314D/H/S/K/E/T/Q/G/A, M435T/L/IA /K/R/Q/F and/or S468T/L/IA /K/R/Q/F of SEQ ID NO: 57. In an embodiment the polypeptide has one or more substitution corresponding to L301 R, K310R, L31 1 R, N314D, M435T and/or S468T of SEQ ID NO: 57.
In a further embodiment the polypeptide has one or more substitution pair corresponding to L30 1 R and M435T, L310R and M435T, N314D and M435T, M435T and S468T and/or N314D and K310R of SEQ ID NO: 57.
In an embodiment the polypeptide has reduced glucose transport activity compared to the polypeptide having SEQ I D NO: 57. In an embodiment the polypeptide has increased xylose transport activity compared to the polypeptide having SEQ ID NO: 57.
The permease polypeptide of the invention may have one or more alternative and/or additional activities other than that of sugar permease activity.
As set out above, a permease polypeptide of the invention will typically have sugar permease activity. However, a permease polypeptide of the invention may have one or more of the activities set out above in addition to or alternative to that activity.
Polynucleotide sequence
With the permease polypeptide and its amino acid sequence as disclosed herein, the skilled person may determine suitable polynucleotides that encode the permease polypeptide.
In an embodiment the polynucleotide is a variant polynucleotide having at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 56, and encodes the polypeptide as described in claims 1 to 1 1.
The invention therefore provides polynucleotide sequences comprising the gene encoding the permease polypeptide, as well as its coding sequence.
The polynucleotides of the invention may be isolated or synthesized. The permease polypeptides and permease polypeptide polynucleotides herein may be synthetic polypeptides, respectively polynucleotides. The synthetic polynucleotides may be optimized in codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943, which are herein incorporated by reference. PCT/EP2007/055943 addresses codon-pair optimization.
The term refers to a polynucleotide molecule, which is a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, either single stranded or double stranded. A polynucleotide may either be present in isolated form, or be comprised in recombinant nucleic acid molecules or vectors, or be comprised in a host cell.
The word "polypeptide" is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art.
By "isolated" polypeptide or protein is intended a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31 -40 (1988).
The polynucleotides of the present invention, such as a polynucleotide encoding the permease polypeptide can be isolated or synthesized using standard molecular biology techniques and the sequence information provided herein.
The polynucleotide encoding the permease polypeptide of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Transformation
The polynucleotides according to the invention may be expressed in a suitable host cell. The invention thus relates to a transformed host cell. In an embodiment, the host cell may be transformed with a nucleic acid construct that comprises a polynucleotide that encodes the polypeptide according to the invention defined before. Therefore standard transformation techniques may be used.
In an embodiment the transformed host cell comprises a heterologuous nucleotide that encodes a polypeptide as defined before an described in claim 1 or encodes a polypeptide having substitution
In an embodiment the transformed host is transformed with a polynucleotide that encodes a polypeptide that is a mutant of a polypeptide that is native in the untransformed host cell.
In an embodiment the polypeptide that is native in the untransformed host eel is a member of the the Major Facilitator Superfamily (MFS) transporters, in an embodiment a hexose transporter polypeptide. In an embodiment the polypeptide has one or more substitution corresponding to L301 R, K310R, L31 1 R, N314D, M435T and/or S468T of SEQ ID NO: 57.
In an embodiment he polypeptide that is native in the untransformed host cell is a transporter polypeptide chosen from the list consisting of Gal2, Hxt1 , Hxt2, Hxt3, Hxt4, Hxt5, Hxt6, Hxt7, Hxt8, Hxt9, Hxt10, Hxt1 1 , Hxt12, Hxt13, Hxt14, Hxt15, Hxt16 and Hxt17.
The invention further relates to a nucleic acid construct comprising the polynucleotide as described before, e.g. a vector.
Another aspect of the invention thus pertains to vectors, including cloning and expression vectors, comprising a polynucleotide of the invention encoding a permease polypeptide protein or a functional equivalent thereof and methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a permease of the invention occurs. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below.
It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The vectors, such as expression vectors, of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein. The vectors, such as recombinant expression vectors, of the invention can be designed for expression of permease polypeptide proteins in prokaryotic or eukaryotic cells.
For example, permease polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), filamentous fungi, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Representative examples of appropriate hosts are described hereafter.
Appropriate culture media and conditions for the above-described host cells are known in the art.
For most filamentous fungi and yeast, the vector or expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants. However, for certain yeasts also suitable episomal vectors are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2μ and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively, or vectors containing an AMA sequence (e.g. AMA1 from Aspergillus). In case the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.
Accordingly, expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
When the polypeptide according to the invention is to be secreted from the host cell into the cultivation medium, an appropriate signal sequence can be added to the polypeptide in order to direct the de novo synthesized polypeptide to the secretion route of the host cell. The person skilled in the art knows to select an appropriate signal sequence for a specific host.
The vector may further include sequences flanking the polynucleotide giving rise to RNA which comprise sequences homologous to eukaryotic genomic sequences or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of a host cell.
An integrative cloning vector may integrate at random or at a predetermined target locus in the chromosome(s) of the host cell into which it is to be integrated.
The vector system may be a single vector, such as a single plasmid, or two or more vectors, such as two or more plasmids, which together contain the total DNA to be introduced into the genome of the host cell.
The vector may contain a polynucleotide of the invention oriented in an antisense direction to provide for the production of antisense RNA.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.
As indicated before, the invention provides an isolated polypeptide having the amino acid sequence according to SEQ ID NO: 57 with the mutations indicated in claim 1 .
Polypeptides of the present invention may include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
The invention also features biologically active fragments of the polypeptides according to the invention.
Provided also are host cells, comprising a polynucleotide or vector of the invention. The polynucleotide may be heterologous to the genome of the host cell. The term "heterologous", usually with respect to the host cell, means that the polynucleotide does not naturally occur in the genome of the host cell or that the polypeptide is not naturally produced by that cell.
In another embodiment, the invention features cells, e.g., transformed host cells or recombinant host cells that contain a nucleic acid encompassed by the invention. A "transformed cell" or "recombinant cell" is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like, especially preferred are yeast cells including e.g. Saccharomyces, for example Saccharomyces cerevisiae.
A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such host cells are well known in the art.
If desired, a cell as described above may be used to in the preparation of a polypeptide according to the invention. Such a method typically comprises cultivating a host cell (e. g. transformed or transfected with an expression vector as described above) under conditions to provide for expression (by the vector) of a coding sequence encoding the polypeptide, and optionally recovering the expressed polypeptide. Polynucleotides of the invention can be incorporated into a recombinant replicable vector, e. g. an expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector. The vector may be recovered from the host cell.
The vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptide.
Herein standard isolation, hybridization, transformation and cloning techniques are used (e. g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Homology & Identity
Amino acid or nucleotide sequences are said to be homologous when exhibiting a certain level of similarity. Two sequences being homologous indicate a common evolutionary origin. Whether two homologous sequences are closely related or more distantly related is indicated by "percent identity" or "percent similarity", which is high or low respectively. Although disputed, to indicate "percent identity" or "percent similarity", "level of homology" or "percent homology" are frequently used interchangeably.
A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1 -44 Addison Wesley). The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm aligns amino acid sequences as well as nucleotide sequences. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. LongdenJ. and BleasbyA Trends in Genetics 16, (6) pp276— 277, http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Other matrices can be specified. The optional parameters used for alignment of amino acid sequences are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.
Global Homology Definition
The homology or identity is the percentage of identical matches between the two full sequences over the total aligned region including any gaps or extensions. The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment including the gaps. The identity defined as herein can be obtained from NEEDLE and is labelled in the output of the program as "IDENTITY".
Longest Identity Definition
The homology or identity between the two aligned sequences is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as "longest-identity".
The various embodiments of the invention described herein may be cross- combined.
The sugar composition
The sugar composition according to the invention comprises glucose, arabinose and xylose. Any sugar composition may be used in the invention that suffices those criteria. Optional sugars in the sugar composition are galactose and mannose. In a preferred embodiment, the sugar composition is a hydrolysate of one or more lignocellulosic material. Lignocelllulose herein includes hemicellulose and hemicellulose parts of biomass. Also lignocellulose includes lignocellulosic fractions of biomass. Suitable lignocellulosic materials may be found in the following list: orchard primings, chaparral, mill waste, urban wood waste, municipal waste, logging waste, forest thinnings, short-rotation woody crops, industrial waste, wheat straw, oat straw, rice straw, barley straw, rye straw, flax straw, soy hulls, rice hulls, rice straw, corn gluten feed, oat hulls, sugar cane, corn stover, corn stalks, corn cobs, corn husks, switch grass, miscanthus, sweet sorghum, canola stems, soybean stems, prairie grass, gamagrass, foxtail; sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animal wastes, lawn clippings, cotton, seaweed, trees, softwood, hardwood, poplar, pine, shrubs, grasses, wheat, wheat straw, sugar cane bagasse, corn, corn husks, corn hobs, corn kernel, fiber from kernels, products and by-products from wet or dry milling of grains, municipal solid waste, waste paper, yard waste, herbaceous material, agricultural residues, forestry residues, municipal solid waste, waste paper, pulp, paper mill residues, branches, bushes, canes, corn, corn husks, an energy crop, forest, a fruit, a flower, a grain, a grass, a herbaceous crop, a leaf, bark, a needle, a log, a root, a sapling, a shrub, switch grass, a tree, a vegetable, fruit peel, a vine, sugar beet pulp, wheat midlings, oat hulls, hard or soft wood, organic waste material generated from an agricultural process, forestry wood waste, or a combination of any two or more thereof.
An overview of some suitable sugar compositions derived from lignocellulose and the sugar composition of their hydrolysates is given in table 1 . The listed lignocelluloses include: corn cobs, corn fiber, rice hulls, melon shells, sugar beet pulp, wheat straw, sugar cane bagasse, wood, grass and olive pressings. Table 1 : Overview of sugar compositions from lignocellulosic materials.
Gal=galactose, Xyl=xylose, Ara=arabinose, Man=mannose, Glu=glucose,
Rham=rhamnose. The percentage galactose (% Gal) and literature source is given.
Lignocellulosic Rha %.
material Gal Xyl Ara Man Glu m Sum Gal.
Corn cob a 10 286 36 227 1 1 570 1 ,7
Corn cob b 131 228 160 144 663 19,8
Rice hulls a 9 122 24 18 234 10 417 2,2
Rice hulls b 8 120 28 209 12 378 2,2
Melon Shells 6 120 1 1 208 16 361 1 ,7
Sugar beet pulp 51 17 209 1 1 21 1 24 523 9,8
Wheat straw Idaho 15 249 36 396 696 2,2
Corn fiber 36 176 1 13 372 697 5,2
Cane Bagasse 14 180 24 5 391 614 2,3
Corn stover 19 209 29 370 626
Athel (wood) 5 1 18 7 3 493 625 0,7
Eucalyptus (wood) 22 105 8 3 445 583 3,8
CWR (grass) 8 165 33 340 546 1 ,4
JTW (grass) 7 169 28 31 1 515 1 ,3
MSW 4 24 5 20 440 493 0,9
Reed Canary Grass
Veg 16 1 17 30 6 209 1 379 4,2
Reed Canary Grass
Seed 13 163 28 6 265 1 476 2,7
Olive pressing residu 15 1 1 1 24 8 329 487 3,1 It is clear from table 1 that in these lignocelluloses a high amount of sugar is presence in de form of glucose, xylose, arabinose and galactose. The conversion of glucose, xylose, arabinose and galactose to fermentation product is thus of great economic importance. Also mannose is present in some lignocellulose materials be it usually in lower amounts than the previously mentioned sugars. Advantageously therefore also mannose is converted by the transformed host cell.
The transformed host cell
In an embodiment, the transformed host cell may comprise one or more copies of xylose isomerase (XI) gene (suitable Xl's are hereinafter described) and/or one or more copies of xylose reductase and/or xylitol dehydrogenase, and one or more, or two to ten copies of araA, araB and araD, genes, wherein these genes may be integrated into the cell genome.
In one embodiment, the transformed host cell comprises genes, for example the above xylose isomerase gene and/or one or more copies of xylose reductase and/or xylitol dehydrogenase, and two to ten copies of araA, araB and araD, an the genes, are integrated into the transformed host cell genome.
The number of copies may be determined by the skilled person by any known method. In the examples, a suitable method is described.
In an embodiment, the transformed host cell is able to ferment glucose, arabinose, xylose and galactose.
In an embodiment, the cell is capable of converting 90% or more glucose, xylose arabinose, galactose and mannose available, into a fermentation product. In an embodiment, cell is capable of converting 91 % or more, 92% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 100% of all glucose, xylose arabinose, galactose and mannose available, into a fermentation product.
In one embodiment of the invention the transformed host cell is able to ferment one or more additional sugar, preferably C5 and/or C6 sugar e.g. mannose or galactose. In an embodiment of the invention the transformed host cell comprises one or more of: a xylA-gene, XYL1 gene and XYL2 gene and/or XKS7-gene, to allow the transformed host cell to ferment xylose; deletion of the aldose reductase (GRE3) gene; overexpression of PPP-genes TAL1, TKL1, RPE1 and RKI1 to allow the increase of the flux through the pentose phosphate pathway in the cell. In an embodiment, the transformed host cell is an industrial cell, more preferably an industrial yeast. An industrial cell and industrial yeast cell may be defined as follows. The living environments of (yeast) cells in industrial processes are significantly different from that in the laboratory. Industrial yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include change in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of Saccharomyces cerevisiae. Under adverse industrial conditions, the environmental tolerant strains should allow robust growth and production. Industrial yeast strains are generally more robust towards these changes in environmental conditions which may occur in the applications they are used, such as in the baking industry, brewing industry, wine making and the ethanol industry. In one embodiment, the industrial transformed host cell is constructed on the basis of an industrial host cell, wherein the construction is conducted as described hereinafter. Examples of industrial yeast (S. cerevisiae) are Ethanol Red® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand).
In an embodiment the transformed host cell is inhibitor tolerant. Inhibitor tolerance is resistance to inhibiting compounds. The presence and level of inhibitory compounds in lignocellulose may vary widely with variation of feedstock, pretreatment method hydrolysis process. Examples of categories of inhibitors are carboxylic acids, furans and/or phenolic compounds. Examples of carboxylic acids are lactic acid, acetic acid or formic acid. Examples of furans are furfural and hydroxy- methylfurfural. Examples or phenolic compounds are vannilin, syringic acid, ferulic acid and coumaric acid. The typical amounts of inhibitors are for carboxylic acids: several grams per liter, up to 20 grams per liter or more, depending on the feedstock, the pretreatment and the hydrolysis conditions. For furans: several hundreds of milligrams per liter up to several grams per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions.
For phenolics: several tens of milligrams per liter, up to a gram per liter, depending on the feedstock, the pretreatment and the hydrolysis conditions.
The transformed host cells according to the invention may be inhibitor tolerant, i.e. they can withstand common inhibitors at the level that they typically have with common pretreatment and hydrolysis conditions, so that the transformed host cells can find broad application, i.e. it has high applicability for different feedstock, different pretreatment methods and different hydrolysis conditions.
In one embodiment, the industrial transformed host cell is constructed on the basis of an inhibitor tolerant host cell, wherein the construction is conducted as described hereinafter. Inhibitor tolerant host cells may be selected by screening strains for growth on inhibitors containing materials, such as illustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, 847-858, wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was selected.
In an embodiment, the transformed host cell is marker-free. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. Marker-free means that markers are essentially absent in the transformed host cell. Being marker-free is particularly advantageous when antibiotic markers have been used in construction of the transformed host cell and are removed thereafter. Removal of markers may be done using any suitable prior art technique, e.g intramolecular recombination. A suitable method of marker removal is illustrated in the examples.
A transformed host cell may be able to convert plant biomass, celluloses, hemicelluloses, pectins, starch, starch derivatives,, for example into fermentable sugars. Accordingly, a transformed host cell may express one or more enzymes such as a cellulase (an endocellulase or an exocellulase), a hemicellulase (an endo- or exo- xylanase or arabinase) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, a pectinase able to convert pectins into glucuronic acid and galacturonic acid or an amylase to convert starch into glucose monomers.
The transformed host cell further may comprise those enzymatic activities required for conversion of pyruvate to a desired fermentation product, such as ethanol, butanol, lactic acid, di-terpene, glycosylated di-terpene, 3 -hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid, itaconic acid, an amino acid, 1 ,3- propane-diol, ethylene, glycerol, a β-lactam antibiotic or a cephalosporin.
In an embodiment, the transformed host cell is a cell that is naturally capable of alcoholic fermentation, preferably, anaerobic alcoholic fermentation. A transformed host cell preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e. capable of growth at a pH lower than about 5, about 4, about 3, or about 2.5) and towards organic and/or a high tolerance to elevated temperatures.
Any of the above characteristics or activities of a transformed host cell may be naturally present in the cell or may be introduced or modified by genetic modification.
Construction of the transformed host cell
According to an embodiment, the genes may be introduced in the host cell by introduction into a host cell:
a) a cluster consisting of the genes araA, araB and araD under control of a strong constitutive promoter
b) a cluster consisting of PPP-genes TAL1, TKL1, RPE1 and RKI1, optionally under control of strong constitutive promoter; and deletion of an aldose reductase gene; c) a cluster consisting of a xylA-gene and a XKS7-gene under control of strong constitutive promoter;
d) a construct comprising a xylA gene under control of a strong constitutive promoter, which has the ability to integrate into the genome on multiple loci;
and adaptive evolution to produce the transformed host cell. The above cell may be constructed using recombinant expression techniques.
Recombinant expression
The transformed host cell is a recombinant cell. That is to say, a transformed host cell comprises, or is transformed with or is genetically modified with a nucleotide sequence that does not naturally occur in the cell in question.
Techniques for the recombinant expression of enzymes in a cell, as well as for the additional genetic modifications of a transformed host cell are well known to those skilled in the art. Typically such techniques involve transformation of a cell with nucleic acid construct comprising the relevant sequence. Such methods are, for example, known from standard handbooks, such as Sambrook and Russel (2001 ) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of host cells are known from e.g. EP-A- 0635 574, WO 98/46772, WO 99/60102, WO 00/37671 , WO90/14423, EP-A-0481008, EP-A-0635574 and US 6,265,186. Typically, the nucleic acid construct may be a plasmid, for instance a low copy plasmid or a high copy plasmid. The cell according to the present invention may comprise a single or multiple copies of the nucleotide sequence encoding a enzyme, for instance by multiple copies of a nucleotide construct or by use of construct which has multiple copies of the enzyme sequence.
The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence sequence. A suitable episomal nucleic acid construct may e.g. be based on the yeast 2μ or pKD1 plasmids (Gleer et al., 1991 , Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489). Alternatively, each nucleic acid construct may be integrated in one or more copies into the genome of the cell. Integration into the cell's genome may occur at random by non-homologous recombination but preferably, the nucleic acid construct may be integrated into the cell's genome by homologous recombination as is well known in the art (see e.g. WO90/14423, EP-A-0481008, EP-A-0635 574 and US 6,265,186).
Most episomal or 2μ plasmids are relatively unstable in yeast, being lost in approximately 10"2 or more cells after each generation. Even under conditions of selective growth, only 60% to 95% of the cells retain the episomal plasmid. The copy number of most episomal plasmids ranges from 20-100 per cell of cir+ hosts. However, the plasmids are not equally distributed among the cells, and there is a high variance in the copy number per cell in populations. Strains transformed with integrative plasmids are extremely stable, even in the absence of selective pressure. However, plasmid loss can occur at approximately 10"3 to 10"4 frequencies by homologous recombination between tandemly repeated DNA, leading to looping out of the vector sequence. Preferably, the vector design in the case of stable integration is thus, that upon loss of the selection marker genes (which also occurs by intramolecular, homologous recombination) that looping out of the integrated construct is no longer possible. Preferably the genes are thus stably integrated. Stable integration is herein defined as integration into the genome, wherein looping out of the integrated construct is no longer possible. Preferably selection markers are absent. Typically, the enzyme encoding sequence will be operably linked to one or more nucleic acid sequences, capable of providing for or aiding the transcription and/or translation of the enzyme sequence.
The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. For instance, a promoter or enhancer is operably linked to a coding sequence the said promoter or enhancer affects the transcription of the coding sequence.
As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences known to one of skilled in the art. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.
The promoter that could be used to achieve the expression of a nucleotide sequence coding for an enzyme according to the present invention, may be not native to the nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. The promoter may, however, be homologous, i.e. endogenous, to the host cell.
Promotors are widely available and known to the skilled person. Suitable examples of such promoters include e.g. promoters from glycolytic genes, such as the phosphofructokinase (PFK), triose phosphate isomerase (TPI), glyceraldehyde-3 - phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts or filamentous fungi; more details about such promoters from yeast may be found in (WO 93/03159). Other useful promoters are ribosomal protein encoding gene promoters, the lactase gene promoter (LAC4), alcohol dehydrogenase promoters (ADH1 , ADH4, and the like), and the enolase promoter (ENO). Other promoters, both constitutive and inducible, and enhancers or upstream activating sequences will be known to those of skill in the art. The promoters used in the host cells of the invention may be modified, if desired, to affect their control characteristics. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art. Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, EN01, TPI1, and AOX1. Other suitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3.
In a transformed host cell, the 3 '-end of the nucleotide acid sequence encoding enzyme preferably is operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice, such as e.g. the yeast species of choice. In any case the choice of the terminator is not critical; it may e.g. be from any yeast gene, although terminators may sometimes work if from a non-yeast, eukaryotic, gene. Usually a nucleotide sequence encoding the enzyme comprises a terminator. Preferably, such terminators are combined with mutations that prevent nonsense mediated mRNA decay in the host transformed host cell (see for example: Shirley et al., 2002, Genetics 161 :1465-1482).
The transcription termination sequence further preferably comprises a polyadenylation signal.
Optionally, a selectable marker may be present in a nucleic acid construct suitable for use in the invention. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. The marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Examples of suitable antibiotic resistance markers include e.g. dihydrofolate reductase, hygromycin-B-phosphotransferase, 3'-0- phosphotransferase II (kanamycin, neomycin and G418 resistance). Antibiotic resistance markers may be most convenient for the transformation of polyploid host cells, Also non- antibiotic resistance markers may be used, such as auxotrophic markers (URA3, TRP1, LEU2) or the S. pombe TPI gene (described by Russell P R, 1985, Gene 40: 125-130). In a preferred embodiment the host cells transformed with the nucleic acid constructs are marker gene free. Methods for constructing recombinant marker gene free microbial host cells are disclosed in EP-A-0 635 574 and are based on the use of bidirectional markers such as the A. nidulans amdS (acetamidase) gene or the yeast URA3 and LYS2 genes. Alternatively, a screenable marker such as Green Fluorescent Protein, lacL, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into the nucleic acid constructs of the invention allowing to screen for transformed cells.
Optional further elements that may be present in the nucleic acid constructs suitable for use in the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromers, telomers and/or matrix attachment (MAR) sequences. The nucleic acid constructs of the invention may further comprise a sequence for autonomous replication, such as an ARS sequence. The recombination process may thus be executed with known recombination techniques. Various means are known to those skilled in the art for expression and overexpression of enzymes in a transformed host cell. In particular, an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the host cell, e.g. by integrating additional copies of the gene in the host cell's genome, by expressing the gene from an episomal multicopy expression vector or by introducing a episomal expression vector that comprises multiple copies of the gene.
Alternatively, overexpression of enzymes in the host cells of the invention may be achieved by using a promoter that is not native to the sequence coding for the enzyme to be overexpressed, i.e. a promoter that is heterologous to the coding sequence to which it is operably linked. Although the promoter preferably is heterologous to the coding sequence to which it is operably linked, it is also preferred that the promoter is homologous, i.e. endogenous to the host cell. Preferably the heterologous promoter is capable of producing a higher steady state level of the transcript comprising the coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is the promoter that is native to the coding sequence. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters.
In an embodiment, the transformed host cell is markerfree, which means that no auxotrophic or dominant markers, in particular antibiotic resistance markers, are present in the genome or extra-chromosomally.
The coding sequence used for overexpression of the enzymes mentioned above may preferably be homologous to the host cell. However, coding sequences that are heterologous to the host may be used.
Overexpression of an enzyme, when referring to the production of the enzyme in a genetically modified cell, means that the enzyme is produced at a higher level of specific enzymatic activity as compared to the unmodified host cell under identical conditions. Usually this means that the enzymatically active protein (or proteins in case of multi-subunit enzymes) is produced in greater amounts, or rather at a higher steady state level as compared to the unmodified host cell under identical conditions. Similarly this usually means that the mRNA coding for the enzymatically active protein is produced in greater amounts, or again rather at a higher steady state level as compared to the unmodified host cell under identical conditions. Preferably in a host, an enzyme to be overexpressed is overexpressed by at least a factor of about 1 .1 , about 1 .2, about 1 .5, about 2, about 5, about 10 or about 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.
Adaptation
Adaptation is the evolutionary process whereby a population becomes better suited (adapted) to its habitat or habitats. This process takes place over several to many generations, and is one of the basic phenomena of biology.
The term adaptation may also refer to a feature which is especially important for an organism's survival. Such adaptations are produced in a variable population by the better suited forms reproducing more successfully, by natural selection.
Changes in environmental conditions alter the outcome of natural selection, affecting the selective benefits of subsequent adaptations that improve an organism's fitness under the new conditions. In the case of an extreme environmental change, the appearance and fixation of beneficial adaptations can be essential for survival. A large number of different factors, such as e.g. nutrient availability, temperature, the availability of oxygen, etcetera, can drive adaptive evolution.
Fitness
There is a clear relationship between adaptedness (the degree to which an organism is able to live and reproduce in a given set of habitats) and fitness. Fitness is an estimate and a predictor of the rate of natural selection. By the application of natural selection, the relative frequencies of alternative phenotypes will vary in time, if they are heritable.
Genetic changes
When natural selection acts on the genetic variability of the population, genetic changes are the underlying mechanism. By this means, the population adapts genetically to its circumstances. Genetic changes may result in visible structures, or may adjust the physiological activity of the organism in a way that suits the changed habitat.
It may occur that habitats frequently change. Therefore, it follows that the process of adaptation is never finally complete. In time, it may happen that the environment changes gradually, and the species comes to fit its surroundings better and better. On the other hand, it may happen that changes in the environment occur relatively rapidly, and then the species becomes less and less well adapted. Adaptation is a genetic process, which goes on all the time to some extent, also when the population does not change the habitat or environment.
The adaptive evolution
The transformed host cells may in their preparation be subjected to adaptive evolution. A transformed host cell may be adapted to sugar utilisation by selection of mutants, either spontaneous or induced (e.g. by radiation or chemicals), for growth on the desired sugar, preferably as sole carbon source, and more preferably under anaerobic conditions. Selection of mutants may be performed by techniques including serial transfer of cultures as e.g. described by Kuyper et al. (2004, FEMS Yeast Res. 4: 655-664) or by cultivation under selective pressure in a chemostat culture. E.g. in a preferred host cell at least one of the genetic modifications described above, including modifications obtained by selection of mutants, confer to the host cell the ability to grow on the xylose as carbon source, preferably as sole carbon source, and preferably under anaerobic conditions. When XI is used as gene to convert xylose, preferably the cell produce essentially no xylitol, e.g. the xylitol produced is below the detection limit or e.g. less than about 5, about 2, about 1 , about 0.5, or about 0.3 % of the carbon consumed on a molar basis.
Adaptive evolution is also described e.g. in Wisselink H.W. et al, Applied and Environmental Microbiology Aug. 2007, p. 4881-4891
In one embodiment of adaptive evolution a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in different media is applied, e.g. three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose. See Wisselink et al. (2009) Applied and Environmental Microbiology, Feb. 2009, p. 907-914.
Yeast transformation and genetic stability
Genetic engineering, i.e. transformation of yeast cells with recombinant DNA, became feasible for the first time in 1978 [Beggs, 1978; Hinnen et al., 1978]. Recombinant DNA technology in yeast has established itself since then. A multitude of different vector constructs are available. Generally, these plasmid vectors, called shuttle vectors, contain genetic material derived from E.coli vectors consisting of an origin of replication and a selectable marker (often the βΐ3θί.3ΓΠ38β gene, ampR), which enable them to be propagated in E.coli prior to transformation into yeast cells. Additionally, the shuttle vectors contain a selectable marker for selection in yeast. Markers can be genes encoding enzymes for the synthesis of a particular amino acid or nucleotide, so that cells carrying the corresponding genomic deletion (or mutation) are complemented for auxotrophy or autotrophy. Alternatively, these vectors contain heterologous dominant resistance markers, which provides recombinant yeast cells (i.e. the cells that have taken up the DNA and express the marker gene) resistance towards certain antibiotics, like g418 (Geneticin), hygromycinB or phleomycin. In addition, these vectors may contain a sequence of (combined) restriction sites (multiple cloning site or MCS) which will allow to clone foreign DNA into these sites, although alternative methods exist as well.
Traditionally, four types of shuttle vectors can be distinguished by the absence or presence of additional genetic elements:
• Integrative plasmids (Yip) which by homologous recombination are integrated into the host genome at the locus of the marker or another gene, when this is opened by restriction and the linearized DNA is used for transformation of the yeast cells. This generally results in the presence of one copy of the foreign DNA inserted at this particular site in the genome.
• Episomal plasmids (YEp) which carry part of the 2 μ plasmid DNA sequence necessary for autonomous replication in yeast cells. Multiple copies of the transformed plasmid are propagated in the yeast cell and maintained as episomes.
• Autonomously replicating plasmids (YRp) which carry a yeast origin of replication (ARS, autonomously replicated sequence) that allows the transformed plasmids to be propagated several hundred-fold.
• CEN plasmids (YCp) which carry in addition to an ARS sequence a centromeric sequence (derived from one of the nuclear chromosomes) which normally guarantees stable mitotic segregation and usually reduces the copy number of self- replicated plasmid to just one.
These plasmids are being introduced into the yeast cells by transformation. Transformation of yeast cells may be achieved by several different techniques, such as permeabilization of cells with lithium acetate (Ito et al, 1983) and electroporation methods. In commercial application of recombinant microorganisms, plasmid instability is the most important problem. Instability is the tendency of the transformed cells to lose their engineered properties because of changes to, or loss of, plasmids. This issue is discussed in detail by Zhang et al (Plasmid stability in recombinant Saccharomyces cerevisiae. Biotechnology Advances, Vol. 14, No. 4, pp. 401 -435, 1996). Strains transformed with integrative plasmids are extremely stable, even in the absence of selective pressure (Sherman, F. http://dbb.urmc. rochester.edula/bs/sherman f/yeast/9.html and references therein).
The heterologous DNA is usually introduced into the organism in the form of extra-chromosomal plasmids (YEp, YCp and YRp). Unfortunately, it has been found with both bacteria and yeasts that the new characteristics may not be retained, especially if the selection pressure is not applied continuously. This is due to the segregational instability of the hybrid plasmid when recombinant cells grow for a long period of time. This leads to population heterogeneity and clonal variability, and eventually to a cell population in which the majority of the cells has lost the properties that were introduced by transformation. If vectors with auxotrophic markers are being used, cultivation in rich media often leads to rapid loss of the vector, since the vector is only retained in minimal media. The alternative, the use of dominant antibiotic resistance markers, is often not compatible with production processes. The use of antibiotics may not be desired from a registration point of view (the possibility that trace amounts of the antibiotic end up in the end product) or for economic reasons (costs of the use of antibiotics at industrial scale).
Loss of vectors leads to problems in large scale production situations. Alternative methods for introduction of DNA do exist for yeasts, such as the use of integrating plasmids (Yip). The DNA is integrated into the host genome by recombination, resulting in high stability. (Caunt, P. Stability of recombinant plasmids in yeast. Journal of Biotechnology 9(1988) 173 - 192). We have found that an integration method using the host transposons are a good alternative. In an embodiment genes may be integrated into the transformed host cell genome. Initial introduction (i.e. before adaptive evolution) of multiple copies be executed in any way known in the art that leads to introduction of the genes. In an embodiment, this may be accomplished using a vector with parts homologous to repeated sequences (transposons), of the host cell. When the host cell is a yeast cell, suitable repeated sequences are the long terminal repeats (LTR) of the Ty element, known as delta sequence. Ty elements fall into two rather similar subfamilies called Ty1 and Ty2. These elements are about 6 kilobases (kb) in length and are bounded by long terminal repeats (LTR), sequences of about 335 base pairs (Boeke JD et al, The Saccharomyces cerevisiae Genome Contains Functional and Nonfunctional Copies of Transposon Ty1. Molecular and Cellular Biology, Apr. 1988, p. 1432-1442 Vol. 8, No. 4). In the fully sequenced S. cerevisiae strain, S288c, the most abundant transposons are Ty1 (31 copies) and Ty2 (13 copies) (Gabriel A, Dapprich J, Kunkel M, Gresham D, Pratt SC, et al. (2006) Global mapping of transposon location. PLoS Genet 2(12): e212.doi:10.1371/journal.pgen.0020212). These transposons consist of two overlapping open reading frames (ORFs), each of which encode several proteins. The coding regions are flanked by the aforementioned, nearly identical LTRs. Other, but less abundant and more distinct Ty elements in S. cereviaise comprise Ty3, Ty4 and Ty5. For each family of full-length Ty elements there are an order of magnitude more solo LTR elements dispersed through the genome. These are thought to arise by LTR-LTR recombination of full-length elements, with looping out of the internal protein encoding regions.
The retrotransposition mechanism of the Ty retrotransposon has been exploited to integrate multiple copies throughout the genome (Boeke et al., 1988; Jacobs et al., 1988). The long terminal repeats (LTR) of the Ty element, known as delta sequences, are also good targets for integration by homologous recombination as they exist in about 150-200 copies that are either Ty associated or solo sites (Boeke, 1989; Kingsman and Kingsman, 1988). (Parekh R.N. (1996). An Integrating Vector for Tunable, High Copy, Stable Integration into the Dispersed Ty DELTA Sites of Saccharomyces cerevisiae. Biotechnol. Prog. 1996, 12, 16-21 ). By adaptive evolution, the number of copies may change.
The host cell
The host cell may be any host cell suitable for production of a useful product. A host cell may be any suitable cell, such as a prokaryotic cell, such as a bacterium, or a eukaryotic cell. Typically, the cell will be a eukaryotic cell, for example a yeast or a filamentous fungus.
Yeasts are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina (Alexopoulos, C. J. ,1962, In : Introductory Mycology, John Wiley & Sons, Inc. , New York) that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism. A preferred yeast as a transformed host cell may belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia. Preferably the yeast is one capable of anaerobic fermentation, more preferably one capable of anaerobic alcoholic fermentation.
Filamentous fungi are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina. These fungi are characterized by a vegetative mycelium composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the suitable for use as a cell of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Filamentous fungal cells may be advantageously used since most fungi do not require sterile conditions for propagation and are insensitive to bacteriophage infections. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism of most filamentous fungi is obligately aerobic. Preferred filamentous fungi as a host cell may belong to the genus Aspergillus, Trichoderma, Humicola, Acremoniurra, Fusarium or Penicillium. More preferably, the filamentous fungal cell may be a Aspergillus niger, Aspergillus oryzae, a Penicillium chrysogenum, or Rhizopus oryzae cell.
In one embodiment the host cell may be yeast.
Preferably the host is an industrial host, more preferably an industrial yeast. An industrial host and industrial yeast cell may be defined as follows. The living environments of yeast cells in industrial processes are significantly different from that in the laboratory. Industrial yeast cells must be able to perform well under multiple environmental conditions which may vary during the process. Such variations include change in nutrient sources, pH, ethanol concentration, temperature, oxygen concentration, etc., which together have potential impact on the cellular growth and ethanol production of Saccharomyces cerevisiae. Under adverse industrial conditions, the environmental tolerant strains should allow robust growth and production. Industrial yeast strains are generally more robust towards these changes in environmental conditions which may occur in the applications they are used, such as in the baking industry, brewing industry, wine making and the ethanol industry. Examples of industrial yeast (S. cerevisiae) are Ethanol Red® (Fermentis) Fermiol® (DSM) and Thermosacc® (Lallemand). In an embodiment the host is inhibitor tolerant. Inhibitor tolerant host cells may be selected by screening strains for growth on inhibitors containing materials, such as illustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol. 136-140, 847-858, wherein an inhibitor tolerant S. cerevisiae strain ATCC 26602 was selected. araA, araB and araD genes
A transformed host cell is capable of using arabinose. A transformed host cell is therefore, be capable of converting L-arabinose into L-ribulose and/or xylulose 5- phosphate and/or into a desired fermentation product, for example one of those mentioned herein.
Organisms, for example S. cerevisiae strains, able to produce ethanol from L- arabinose may be produced by modifying a cell introducing the araA (L-arabinose isomerase), araB (L-ribulokinase) and araD (L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes may be introduced into a transformed host cell is order that it is capable of using arabinose. Such an approach is given is described in WO2003/095627. araA, araB and araD genes from Lactobacillus plantarum may be used and are disclosed in WO2008/041840. The araA gene from Bacillus subtilis and the araB and araD genes from Escherichia coli may be used and are disclosed in EP1499708. In another embodiment, araA, araB and araD genes may derived from of at least one of the genus Clavibacter, Arthrobacter and/or Gramella, in particular one of Clavibacter michiganensis, Arthrobacter aurescens, and/or Gramella forsetii, as disclosed in WO 200901 1591 .
PPP-genes
A transformed host cell may comprise one or more genetic modifications that increases the flux of the pentose phosphate pathway. In particular, the genetic modification(s) may lead to an increased flux through the non-oxidative part of the pentose phosphate pathway. A genetic modification that causes an increased flux of the non- oxidative part of the pentose phosphate pathway is herein understood to mean a modification that increases the flux by at least a factor of about 1.1 , about 1.2, about 1.5, about 2, about 5, about 10 or about 20 as compared to the flux in a strain which is genetically identical except for the genetic modification causing the increased flux. The flux of the non-oxidative part of the pentose phosphate pathway may be measured by growing the modified host on xylose as sole carbon source, determining the specific xylose consumption rate and subtracting the specific xylitol production rate from the specific xylose consumption rate, if any xylitol is produced. However, the flux of the non- oxidative part of the pentose phosphate pathway is proportional with the growth rate on xylose as sole carbon source, preferably with the anaerobic growth rate on xylose as sole carbon source. There is a linear relation between the growth rate on xylose as sole carbon source ^max) and the flux of the non-oxidative part of the pentose phosphate pathway. The specific xylose consumption rate (Qs) is equal to the growth rate (μ) divided by the yield of biomass on sugar (Yxs) because the yield of biomass on sugar is constant (under a given set of conditions: anaerobic, growth medium, pH, genetic background of the strain, etc.; i.e. Qs = μ/ Yxs). Therefore the increased flux of the non- oxidative part of the pentose phosphate pathway may be deduced from the increase in maximum growth rate under these conditions unless transport (uptake is limiting).
One or more genetic modifications that increase the flux of the pentose phosphate pathway may be introduced in the host cell in various ways. These including e.g. achieving higher steady state activity levels of xylulose kinase and/or one or more of the enzymes of the non-oxidative part pentose phosphate pathway and/or a reduced steady state level of unspecific aldose reductase activity. These changes in steady state activity levels may be effected by selection of mutants (spontaneous or induced by chemicals or radiation) and/or by recombinant DNA technology e.g. by overexpression or inactivation, respectively, of genes encoding the enzymes or factors regulating these genes.
In a preferred host cell, the genetic modification comprises overexpression of at least one enzyme of the (non-oxidative part) pentose phosphate pathway. Preferably the enzyme is selected from the group consisting of the enzymes encoding for ribulose-5- phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase. Various combinations of enzymes of the (non-oxidative part) pentose phosphate pathway may be overexpressed. E.g. the enzymes that are overexpressed may be at least the enzymes ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase; or at least the enzymes ribulose-5-phosphate isomerase and transketolase; or at least the enzymes ribulose-5-phosphate isomerase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase and transketolase; or at least the enzymes ribulose-5- phosphate epimerase and transaldolase; or at least the enzymes transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase, transketolase and transaldolase; or at least the enzymes ribulose-5- phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose- 5-phosphate isomerase, ribulose-5-phosphate epimerase, and transaldolase; or at least the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and transketolase. In one embodiment of the invention each of the enzymes ribulose-5- phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase are overexpressed in the host cell. More preferred is a host cell in which the genetic modification comprises at least overexpression of both the enzymes transketolase and transaldolase as such a host cell is already capable of anaerobic growth on xylose. In fact, under some conditions host cells overexpressing only the transketolase and the transaldolase already have the same anaerobic growth rate on xylose as do host cells that overexpress all four of the enzymes, i.e. the ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase. Moreover, host cells overexpressing both of the enzymes ribulose-5-phosphate isomerase and ribulose-5- phosphate epimerase are preferred over host cells overexpressing only the isomerase or only the epimerase as overexpression of only one of these enzymes may produce metabolic imbalances.
The enzyme "ribulose 5-phosphate epimerase" (EC 5.1 .3.1 ) is herein defined as an enzyme that catalyses the epimerisation of D-xylulose 5-phosphate into D-ribulose 5- phosphate and vice versa. The enzyme is also known as phosphoribulose epimerase; erythrose-4-phosphate isomerase; phosphoketopentose 3-epimerase; xylulose phosphate 3-epimerase; phosphoketopentose epimerase; ribulose 5-phosphate 3- epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphate epimerase; D- ribulose-5-P 3-epimerase; D-xylulose-5-phosphate 3-epimerase; pentose-5-phosphate 3-epimerase; or D-ribulose-5-phosphate 3-epimerase. A ribulose 5-phosphate epimerase may be further defined by its amino acid sequence. Likewise a ribulose 5- phosphate epimerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5-phosphate epimerase. The nucleotide sequence encoding for ribulose 5-phosphate epimerase is herein designated RPE1.
The enzyme "ribulose 5-phosphate isomerase" (EC 5.3.1.6) is herein defined as an enzyme that catalyses direct isomerisation of D-ribose 5-phosphate into D-ribulose 5- phosphate and vice versa. The enzyme is also known as phosphopentosisomerase; phosphoriboisomerase; ribose phosphate isomerase; 5-phosphoribose isomerase; D- ribose 5-phosphate isomerase; D-ribose-5-phosphate ketol-isomerase; or D-ribose-5- phosphate aldose-ketose-isomerase. A ribulose 5-phosphate isomerase may be further defined by its amino acid sequence. Likewise a ribulose 5-phosphate isomerase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a ribulose 5- phosphate isomerase. The nucleotide sequence encoding for ribulose 5-phosphate isomerase is herein designated RKI1.
The enzyme "transketolase" (EC 2.2.1.1 ) is herein defined as an enzyme that catalyses the reaction: D-ribose 5-phosphate + D-xylulose 5-phosphate <-> sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate and vice versa. The enzyme is also known as glycolaldehydetransferase or sedoheptulose-7-phosphate:D- glyceraldehyde-3-phosphate glycolaldehydetransferase. A transketolase may be further defined by its amino acid. Likewise a transketolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transketolase. The nucleotide sequence encoding for transketolase is herein designated TKL1.
The enzyme "transaldolase" (EC 2.2.1.2) is herein defined as an enzyme that catalyses the reaction: sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate <-> D-erythrose 4-phosphate + D-fructose 6-phosphate and vice versa. The enzyme is also known as dihydroxyacetonetransferase; dihydroxyacetone synthase; formaldehyde transketolase; or sedoheptulose-7- phosphate :D-glyceraldehyde-3 -phosphate glyceronetransferase. A transaldolase may be further defined by its amino acid sequence. Likewise a transaldolase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a transaldolase. The nucleotide sequence encoding for transketolase from is herein designated TALL
Xylose Isomerase or xylose reductase genes
According to an embodiment of the invention, one or more copies of one or more xylose isomerase gene and/or one or more xylose reductase and xylitol dehydrogenase may be introduced in the host cell, e.g. into the genome of the host cell. The presence of these genetic elements confers on the cell the ability to convert xylose by isomerisation or reduction.
In one embodiment, the one or more copies of one or more xylose isomerase gene are introduced into the genome of the host cell. A "xylose isomerase" (EC 5.3.1.5) is herein defined as an enzyme that catalyses the direct isomerisation of D-xylose into D-xylulose and/or vice versa. The enzyme is also known as a D-xylose ketoisomerase. A xylose isomerase herein may also be capable of catalysing the conversion between D-glucose and D-fructose (and accordingly may therefore be referred to as a glucose isomerase). A xylose isomerase herein may require a bivalent cation, such as magnesium, manganese or cobalt as a cofactor.
Accordingly, such a transformed host cell is capable of isomerising xylose to xylulose. The ability of isomerising xylose to xylulose is conferred on the host cell by transformation of the host cell with a nucleic acid construct comprising a nucleotide sequence encoding a defined xylose isomerase. A transformed host cell isomerises xylose into xylulose by the direct isomerisation of xylose to xylulose.
A unit (U) of xylose isomerase activity may herein be defined as the amount of enzyme producing 1 nmol of xylulose per minute, under conditions as described by Kuyper ei a/. (2003, FEMS Yeast Res. 4: 69-78).
The Xylose isomerise gene may have various origin, such as for example Piromyces sp. as disclosed in WO2006/009434. Other suitable origins are Bacteroides, in particular Bacteroides uniformis as described in PCT/EP2009/52623, Bacillus, in particular Bacillus stearothermophilus as described in PCT/EP2009/052625.
In another embodiment, one or more copies of one or more xylose reductase and xylitol dehydrogenase genes are introduced into the genome of the host cell. In this embodiment the conversion of xylose is conducted in a two step conversion of xylose into xylulose via a xylitol intermediate as catalysed by xylose reductase and xylitol dehydrogenase, respectively. In an embodiment thereof xylose reductase (XR), xylitol dehydrogenase (XDH), and xylokinase (XK) may be overexpressed, and optionally one or more of genes encoding NADPH producing enzymes are up-regulated and one or more of the genes encoding NADH consuming enzymes are up-regulated, as disclosed in WO 2004085627.
XKS1 gene
A transformed host cell may comprise one or more genetic modifications that increase the specific xylulose kinase activity. Preferably the genetic modification or modifications causes overexpression of a xylulose kinase, e.g. by overexpression of a nucleotide sequence encoding a xylulose kinase. The gene encoding the xylulose kinase may be endogenous to the host cell or may be a xylulose kinase that is heterologous to the host cell. A nucleotide sequence used for overexpression of xylulose kinase in the host cell is a nucleotide sequence encoding a polypeptide with xylulose kinase activity.
The enzyme "xylulose kinase" (EC 2.7.1 .17) is herein defined as an enzyme that catalyses the reaction ATP + D-xylulose = ADP + D-xylulose 5-phosphate. The enzyme is also known as a phosphorylating xylulokinase, D-xylulokinase or ATP :D- xylulose 5- phosphotransferase. A xylulose kinase of the invention may be further defined by its amino acid sequence. Likewise a xylulose kinase may be defined by a nucleotide sequence encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding a xylulose kinase.
In a transformed host cell, a genetic modification or modifications that increase(s) the specific xylulose kinase activity may be combined with any of the modifications increasing the flux of the pentose phosphate pathway as described above. This is not, however, essential.
Thus, a host cell may comprise only a genetic modification or modifications that increase the specific xylulose kinase activity. The various means available in the art for achieving and analysing overexpression of a xylulose kinase in the host cells of the invention are the same as described above for enzymes of the pentose phosphate pathway. Preferably in the host cells of the invention, a xylulose kinase to be overexpressed is overexpressed by at least a factor of about 1 .1 , about 1 .2, about 1 .5, about 2, about 5, about 10 or about 20 as compared to a strain which is genetically identical except for the genetic modification(s) causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.
Aldose reductase (GRE3) gene deletion
In the embodiment, where XI is used as gene to convert xylose, it may be advantageous to reduce aldose reducatase activity. A transformed host cell may therefore comprise one or more genetic modifications that reduce unspecific aldose reductase activity in the host cell. Preferably, unspecific aldose reductase activity is reduced in the host cell by one or more genetic modifications that reduce the expression of or inactivates a gene encoding an unspecific aldose reductase. Preferably, the genetic modification(s) reduce or inactivate the expression of each endogenous copy of a gene encoding an unspecific aldose reductase in the host cell (herein called GRE3 deletion). Transformed host cells may comprise multiple copies of genes encoding unspecific aldose reductases as a result of di-, poly- or aneu-ploidy, and/or the host cell may contain several different (iso)enzymes with aldose reductase activity that differ in amino acid sequence and that are each encoded by a different gene. Also in such instances preferably the expression of each gene that encodes an unspecific aldose reductase is reduced or inactivated. Preferably, the gene is inactivated by deletion of at least part of the gene or by disruption of the gene, whereby in this context the term gene also includes any non-coding sequence up- or down-stream of the coding sequence, the (partial) deletion or inactivation of which results in a reduction of expression of unspecific aldose reductase activity in the host cell.
A nucleotide sequence encoding an aldose reductase whose activity is to be reduced in the host cell is a nucleotide sequence encoding a polypeptide with aldose reductase activity.
Thus, a host cell comprising only a genetic modification or modifications that reduce(s) unspecific aldose reductase activity in the host cell is specifically included in the invention.
The enzyme "aldose reductase" (EC 1 .1 .1 .21 ) is herein defined as any enzyme that is capable of reducing xylose or xylulose to xylitol. In the context of the present invention an aldose reductase may be any unspecific aldose reductase that is native (endogenous) to a host cell of the invention and that is capable of reducing xylose or xylulose to xylitol. Unspecific aldose reductases catalyse the reaction:
aldose + NAD(P)H + H+ ^ alditol + NAD(P)+
The enzyme has a wide specificity and is also known as aldose reductase; polyol dehydrogenase (NADP+); alditokNADP oxidoreductase; alditol:NADP+ 1 - oxidoreductase; NADPH-aldopentose reductase; or NADPH-aldose reductase.
A particular example of such an unspecific aldose reductase that is endogenous to S. cerevisiae and that is encoded by the GRE3 gene (Traff et al., 2001 , Appl. Environ. Microbiol. 67: 5668-74). Thus, an aldose reductase of the invention may be further defined by its amino acid sequence. Likewise an aldose reductase may be defined by the nucleotide sequences encoding the enzyme as well as by a nucleotide sequence hybridising to a reference nucleotide sequence encoding an aldose reductase. Bioproducts production
Over the years suggestions have been made for the introduction of various organisms for the production of bio-ethanol from crop sugars. In practice, however, all major bio-ethanol production processes have continued to use the yeasts of the genus Saccharomyces as ethanol producer. This is due to the many attractive features of Saccharomyces species for industrial processes, i. e. , a high acid-, ethanol-and osmo- tolerance, capability of anaerobic growth, and of course its high alcoholic fermentative capacity. Preferred yeast species as host cells include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K fragilis.
A transformed host cell may be a cell suitable for the production of ethanol. A transformed host cell may, however, be suitable for the production of fermentation products other than ethanol
Such non-ethanolic fermentation products include in principle any bulk or fine chemical that is producible by a eukaryotic microorganism such as a yeast or a filamentous fungus.
A transformed host cell that may be used for production of non-ethanolic fermentation products is a host cell that contains a genetic modification that results in decreased alcohol dehydrogenase activity.
In an embodiment the transformed host cell may be used in a process wherein sugars originating from lignocellulose are converted into ethanol.
Lignocellulose
Lignocellulose, which may be considered as a potential renewable feedstock, generally comprises the polysaccharides cellulose (glucans) and hemicelluloses (xylans, heteroxylans and xyloglucans). In addition, some hemicellulose may be present as glucomannans, for example in wood-derived feedstocks. The enzymatic hydrolysis of these polysaccharides to soluble sugars, including both monomers and multimers, for example glucose, cellobiose, xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and other hexoses and pentoses occurs under the action of different enzymes acting in concert.
In addition, pectins and other pectic substances such as arabinans may make up considerably proportion of the dry mass of typically cell walls from non-woody plant tissues (about a quarter to half of dry mass may be pectins). Pretreatment
Before enzymatic treatment, the lignocellulosic material may be pretreated. The pretreatment may comprise exposing the lignocellulosic material to an acid, a base, a solvent, heat, a peroxide, ozone, mechanical shredding, grinding, milling or rapid depressurization, or a combination of any two or more thereof. This chemical pretreatment is often combined with heat-pretreatment, e.g. between 150-220 °C for 1 to 30 minutes.
Enzymatic hydrolysis
The pretreated material is commonly subjected to enzymatic hydrolysis to release sugars that may be fermented according to the invention. This may be executed with conventional methods, e.g. contacting with cellulases, for instance cellobiohydrolase(s), endoglucanase(s), beta-glucosidase(s) and optionally other enzymes. The conversion with the cellulases may be executed at ambient temperatures or at higher tempatures, at a reaction time to release sufficient amounts of sugar(s). The result of the enzymatic hydrolysis is hydrolysis product comprising C5/C6 sugars, herein designated as the sugar composition.
Fermentation
The fermentation process may be an aerobic or an anaerobic fermentation process. An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors. In the absence of oxygen, NADH produced in glycolysis and biomass formation, cannot be oxidised by oxidative phosphorylation. To solve this problem many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD+.
Thus, in a preferred anaerobic fermentation process pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, butanol, lactic acid, di-terpene, glycosylated di-terpene, 3 -hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1 ,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and a cephalosporin. The fermentation process is preferably run at a temperature that is optimal for the cell. Thus, for most yeasts or fungal host cells, the fermentation process is performed at a temperature which is less than about 42°C, preferably less than about 38°C. For yeast or filamentous fungal host cells, the fermentation process is preferably performed at a temperature which is lower than about 35, about 33, about 30 or about 28°C and at a temperature which is higher than about 20, about 22, or about 25°C.
The ethanol yield on xylose and/or glucose in the process preferably is at least about 50, about 60, about 70, about 80, about 90, about 95 or about 98%. The ethanol yield is herein defined as a percentage of the theoretical maximum yield.
The invention also relates to a process for producing a fermentation product.
The fermentation process according to the present invention may be run under aerobic and anaerobic conditions. In an embodiment, the process is carried out under micro-aerophilic or oxygen limited conditions.
An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than about 5, about 2.5 or about 1 mmol/L/h, and wherein organic molecules serve as both electron donor and electron acceptors.
An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used. Preferably, in a process under oxygen-limited conditions, the rate of oxygen consumption is at least about 5.5, more preferably at least about 6, such as at least 7 mmol/L/h. A process of the invention may comprise recovery of the fermentation product.
In a preferred process the cell ferments both the xylose and glucose, preferably simultaneously in which case preferably a cell is used which is insensitive to glucose repression to prevent diauxic growth. In addition to a source of xylose (and glucose) as carbon source, the fermentation medium will further comprise the appropriate ingredient required for growth of the cell. Compositions of fermentation media for growth of microorganisms such as yeasts are well known in the art
The fermentation processes may be carried out in batch, fed-batch or continuous mode. A separate hydrolysis and fermentation (SHF) process or a simultaneous saccharification and fermentation (SSF) process may also be applied. A combination of these fermentation process modes may also be possible for optimal productivity. These processes are described hereafter in more detail.
SSF mode
For Simultaneous Saccharification and Fermentation (SSF) mode, the reaction time for liquefaction/hydrolysis or presaccharification step is dependent on the time to realize a desired yield, i.e. cellulose to glucose conversion yield. Such yield is preferably as high as possible, preferably 60% or more, 65% or more, 70% or more, 75% or more 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, even 99.5% or more or 99.9% or more.
According to the invention very high sugar concentrations in SHF mode and very high product concentrations (e.g. ethanol) in SSF mode are realized. In SHF operation the glucose concentration is 25g/L or more, 30 g/L or more, 35g/L or more, 40 g/L or more, 45 g/L or more, 50 g/L or more, 55 g/L or more, 60 g/L or more, 65 g/L or more, 70 g/L or more , 75 g/L or more, 80 g/L or more, 85 g/L or more, 90 g/L or more, 95 g/L or more, 100 g/L or more, 1 10 g/L or more, 120g/L or more or may e.g. be 25g/L-250 g/L, 30gl/L-200g/L, 40g/L-200 g/L, 50g/L-200g/L, 60g/L-200g/L, 70g/L-200g/L, 80g/L-200g/L, 90 g/L , 80g/L-200g/L.
Product concentration in SSF mode
In SSF operation, the product concentration (g/L) is dependent on the amount of glucose produced, but this is not visible since sugars are converted to product in the SSF, and product concentrations can be related to underlying glucose concentration by multiplication with the theoretical maximum yield (Yps max in gr product per gram glucose)
The theoretical maximum yield (Yps max in gr product per gram glucose) of a fermentation product can be derived from textbook biochemistry. For ethanol, 1 mole of glucose (180 gr) yields according to normal glycolysis fermentation pathway in yeast 2 moles of ethanol (=2x46 = 92 gr ethanol. The theoretical maximum yield of ethanol on glucose is therefore 92/180 = 0.51 1 gr ethanol/gr glucose. For Butanol (MW 74 gr/mole) or iso butanol, the theoretical maximum yield is 1 mole of butanol per mole of glucose. So Yps max for (iso-)butanol = 74/180 = 0.41 1 gr (iso-)butanol/gr glucose.
For lactic acid the fermentation yield for homolactic fermentation is 2 moles of lactic acid (MW = 90 gr/mole) per mole of glucose. According to this stoichiometry, the Yps max = 1 gr lactic acid/gr glucose.
For other fermentation products a similar calculation may be made.
SSF mode
In SSF operation the product concentration is 25g * Yps g/L /L or more, 30 * Yps g/L or more, 35g * Yps /L or more, 40 * Yps g/L or more, 45 * Yps g/L or more, 50 * Yps g/L or more, 55 * Yps g/L or more, 60 * Yps g/L or more, 65 * Yps g/L or more, 70 * Yps g/L or more , 75 * Yps g/L or more, 80 * Yps g/L or more, 85 * Yps g/L or more, 90 * Yps g/L or more, 95 * Yps g/L or more, 100 * Yps g/L or more, 1 10 * Yps g/L or more, 120g/L
* Yps or more or may e.g. be 25 * Yps g/L-250 * Yps g/L, 30 * Yps gl/L-200 * Yps g/L, 40
* Yps g/L-200 * Yps g/L, 50 * Yps g/L-200 * Yps g/L, 60 * Yps g/L-200 * Yps g/L, 70 * Yps g/L-200 * Yps g/L, 80 * Yps g/L-200 * Yps g/L, 90 * Yps g/L , 80 * Yps g/L-200 * Yps g/L
Accordingly, the invention provides a method for the preparation of a fermentation product, which method comprises:
a. degrading lignocellulose using a method as described herein; and
b. fermenting the resulting material,
thereby to prepare a fermentation product.
Fermentation product
The fermentation product of the invention may be any useful product. In one embodiment, it is a product selected from the group consisting of ethanol, n-butanol, isobutanol, lactic acid, di-terpene, glycosylated di-terpene, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid, itaconic acid, maleic acid, citric acid, adipic acid, an amino acid, such as lysine, methionine, tryptophan, threonine, and aspartic acid, 1 ,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and a cephalosporin, vitamins, pharmaceuticals, animal feed supplements, specialty chemicals, chemical feedstocks, plastics, solvents, fuels, including biofuels and biogas or organic polymers, and an industrial enzyme, such as a protease, a cellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, an oxidoreductases, a transferase or a xylanase. For example the fermentation products may be produced by cells according to the invention, following prior art cell preparation methods and fermentation processes, which examples however should herein not be construed as limiting, n-butanol may be produced by cells as described in WO2008121701 or WO2008086124; lactic acid as described in US201 1053231 or US2010137551 ; 3-hydroxy-propionic acid as described in WO2010010291 ; acrylic acid as described in WO2009153047.
Recovery of the fermentation product
For the recovery of the fermentation product existing technologies are used. For different fermentation products different recovery processes are appropriate. Existing methods of recovering ethanol from aqueous mixtures commonly use fractionation and adsorption techniques. For example, a beer still can be used to process a fermented product, which contains ethanol in an aqueous mixture, to produce an enriched ethanol- containing mixture that is then subjected to fractionation (e.g., fractional distillation or other like techniques). Next, the fractions containing the highest concentrations of ethanol can be passed through an adsorber to remove most, if not all, of the remaining water from the ethanol.
The following examples illustrate the invention:
EXAMPLES
METHODS
Molecular biology techniques and chemicals. Restriction enzymes and T4 DNA ligase were acquired from Fermentas. Antibiotics hygromycin (HG), phleomycin (phleo) and geneticin (G418) were acquired from Invivogen. pYL16 and nourseothricin (nour) were acquired from Werner Bioagents. Ampicillin and kanamycin were acquired from Sigma-Aldrich. For PCR amplifications, Phusion® High-Fidelity DNA Polymerase was used (Finnzymes). PCR fragments were sub-cloned using the TOPO® TA Cloning® Kit or the Zero Blunt® TOPO® PCR Cloning Kit (both from Life Technologies). Oligonucleotides used for strain construction were purchased from Sigma-Aldrich.
Plasmids were amplified and maintained in chemically competent TOP10 cells (TOPO® TA Cloning® Kit, Life Techonologies) following manufacturer's instructions. Plasmids were isolated from E. coli mini cultures using the GeneJET™ Plasmid Miniprep Kit (Fermentas).
Genomic DNA was isolated from yeast using the YeaStar™ Genomic DNA Kit (ZymoResearch) following manufacturer's instructions.
Standard molecular biology and yeast genetics techniques were conducted according to textbooks including Sambrook et al. (1989, Molecular Cloning, a Laboratory Manual) and Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.).
Strains and maintenance. For storage of the strains used in this work (Table 2), shake flask cultures were performed in rich medium (YP), consisting of 10 g Γ1 yeast extract (Oxoid) and 20 g Γ1 peptone (BD Difco), supplemented with either 2% glucose (YPD), 2% maltose (YPM), or 3% xylose (YPX). Cultures were maintained at 30 °C in an orbital shaker until cultures reached stationary growth phase. After adding glycerol to 30% (v/v), samples from shake-flask cultures were stored in 2 ml aliquots at -80 °C. All microbial strains are given in Table 1.
Growth experiments. All growth experiments with DS68625- or DS69473- transformants were performed in 100 ml and 250 ml aerobic shake flasks respectively and selective Verduyn-urea according to Luttik et al. (2000, The Saccharomyces cerevisiae ICL2 gene encodes a mitochondrial 2-methylisocitrate lyase involved in propionyl-coenzyme A metabolism. J Bacteriol, 182, pp. 7007-7013) with minor alterations: as nitrogen source urea (2.3 g/liter) was used instead of (NH4)2S04. To compensate the reduced sulfate level K2S04 (6.6 g/liter) was added. As a carbon source for pre-cultures 2% maltose (for DS68625) or 3% xylose (for DS69473) was added (Verduyn-urea). Growth of non-transformed DS68625 or DS64973 was facilitated by supplementing 0.2 g Γ1 histidine (Sigma-Aldrich; to complement for the histidine auxotrophy) to the medium (Verduyn-urea-his). For the actual growth experiment, glucose or xylose at various concentrations were added (see Figure Legends). Cultivation of strains was performed at 30°C with constant shaking at 180 rpm. Yeast transformations were carried out as described by SchiestI & Gietz (1989, High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16, pp. 339-346).
Escherichia coli XL1 -blue strain was used for site-directed mutagenesis, sub- cloning and plasmid amplification, and grown in Luria-Bertani (LB) medium containing 150 mg/liter ampicillin (agar plates and liquid media). Cultivation of E. coli was performed at 37°C.
Table 1 : Strains used or prepared herein
Figure imgf000051_0001
Growth assays. Yeast transformants were pre-cultured in 100 ml shake flasks containing 10 ml of Verduyn-urea with 2% maltose (DS68625) or 3% xylose (DS69473). For the growth assays with xylose, 250 ml shake flasks with 50 ml of the same Verduyn- urea supplemented with various concentrations of this sugar (2%, 0.45% and 0.1 %) were inoculated with the corresponding pre-culture at an initial optical density (OD) at 600 nm of 0.1 . To shorten the typical long lag phase for DS68625 with xylose a small amount of maltose was given to the experiments: 0.05% maltose to growth assays with 2% xylose and 0.005% maltose to growth assays with 0.45% and with 0.1 % xylose. Cells were grown at 30°C with constant shaking at 180 rpm. All growth rates were estimated by measuring the OD at 600 nm at several time points. All measurements concerning the candidates resulting from random mutagenesis were performed in triplicates unless otherwise stated. The growth experiments for characterizing the Gal2 single and double mutants were carried out in duplicates.
Sugar consumption analysis. The concentrations of sugars were determined by high-performance liquid chromatography (HPLC) (Agilent Technologies, Boblingen, Germany) using an Aminex HPX-87H ion exclusion column (Bio-Rad Laboratories, Munich, Germany). The column was eluted with 5 mM sulfuric acid as mobile phase with a flow rate of 0.5 ml/min at 65°C. The run time was 15.5 min and for detection a refractive-index detector was used. All measurements were performed in triplicates.
Random mutagenesis and mutant library construction. Three rounds of error-prone PCR (epPCR) were performed to construct mutant libraries using the plasmid pRS313-GAL2 containing the wild type GAL2 gene (SEQ ID NO: 58; plasmid map Fig. 4C) expressed under control of the truncated S. cerevisiae (-390) HXT7 promoter and HXT7 terminator. For the first two rounds of epPCR, the restriction enzyme sites BamYW (between TM6/7) and Mun\ (between TM10/1 1 ) were used to excise the template DNA. Two oligonucleotides, SEQ ID NO: 60 and SEQ ID NO: 61 , were used as the forward and reverse primers, respectively. The PCR reaction with a total volume of 50 μΙ contained 2 mM MgCI2, 0.05-0.07 mM MnCI2, 0.4 mM dATP, 2 mM dCTP/dGTP/dTTP, 40 pmol of each oligonucleotide primer, 40 ng of template DNA, and 5 units of Taq DNA polymerase. The PCR program consisted of one cycle of 5 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 52°C, 3 min at 72°C and finally one cycle of 4 min at 72°C. The mutagenic PCR fragments were digested with BamYW and Mun\ prior to ligation into the same enzyme-digested pRS313-GAL2 vector. After ligation the mutant library was transformed directly into the S. cerevisiae strain DS68625 and screened for improved uptake of xylose.
In the first round of epPCR, plasmid pRS313-GAL2 was used as the template. The subsequently identified GAL2 variant Gal2-1 .1 served as the template for the second round of epPCR. Therefore, all further variants in the next two rounds of epPCR include the alterations in the DNA sequence from the mutant Gal2-1 .1 . The identified variant Gal2-2.1 from the second round of epPCR was chosen as the template for the third round of mutagenesis and therefore all further variants of Gal2 identified from this round include the alterations in the DNA sequence from both the variants Gal2-1 .1 and Gal2-2.1.
The silent mutation L343L in Gal2-2.1 which encodes for an additional Muni restriction site was removed and changed back into the wild-type sequence by site- directed mutagenesis prior to creation of the third mutant library. Also to expand the range for random mutagenesis between TM3 and TM1 1 , a new Eag\ restriction site located within the putative cytoplasmic loop region between TM2 and TM3 was introduced into Gal2-2.1 via site-directed mutagenesis with no change in amino acid sequence. The resulting plasmid, pRS313-GAL2-2.1/£agl, was digested with Eag\ and Mun\ to excise the DNA for a third round of random mutagenesis using epPCR. The following oligonucleotides SEQ ID NO: 62 and SEQ ID NO: 60 were used as the forward and reverse primers, respectively. The resulting mutagenic DNA was digested with the appropriate restriction enzymes and substituted for the corresponding region of pRS313- GAL2-2.1/£agl.
High-throughput screening for enhanced xylose uptake. The mutant libraries were transformed into the S. cerevisiae transporter deletion strain DS68625 for the first and second round of mutagenesis and into the hexokinase deletion strain DS69473 for the third round of mutagenesis, respectively. The transformed yeast cells of the first two mutant libraries were plated out in 100 μΙ aliquots on agar plates with Verduyn-urea containing 2% xylose and incubated for 3 days at 30°C. All plates were floated with 5 ml of Verduyn-urea without urea, vitamins and carbon source for 4 min. The cell suspension was then supplemented with the appropriate amount of urea, vitamins and 1 % xylose and incubated overnight at 30°C at 180 rpm. The cells were then washed twice with Verduyn without urea, vitamins and carbon source. The cell analysis of the grown culture was performed on a flow cytometer (BD FACSDiVa, Becton Dickinson, Heidelberg, Germany) and budding yeast cells were sorted out on agar plates with exactly 25 ml of Verduyn-urea supplemented with 0.1 % xylose as carbon source. Exactly 100 single cells were sorted per agar plate to guarantee the same growth conditions for every single yeast cell. In total 137 agar plates were incubated at 30°C for up to 4 days during the screening of the first round of epPCR. During the screening of the second round of epPCR a total of 144 agar plates were incubated at 30°C for up to 4 days. The fastest growth determined by the size of each colony over a period of 2-4 days indicated an enhanced uptake of 0.1 % xylose and marked the criteria for evolved uptake. The largest colonies were transferred to new agar plates containing the same amount of sugar and incubated at 30°C for 3 days. From the colonies that displayed the fastest growth the plasmid DNA was isolated and retransformed into a fresh strain background for validation and testing in liquid Verduyn-urea with xylose and/or glucose at various concentrations.
The third screening round of random mutagenesis was carried out in the S. cerevisiae hexokinase deletion strain DS69473. The mutant library was transformed and plated out on agar plates with Verduyn-urea containing 3% xylose. All plates were floated with 5 ml of Verduyn-urea as mentioned above. The cell analysis of the grown culture was performed on a flow cytometer. Only budding yeast cells were sorted out on agar plates with exactly 30 ml of Verduyn-urea supplemented with 2% xylose and 10% glucose as carbon sources. In total 131 agar plates were incubated at 30°C and observed for several days. After 12 days, eight colonies were transferred to new agar plates with 3% xylose for regeneration and isolation of plasmid DNA. Subsequently, the plasmid DNA was transformed into the S. cerevisiae transporter deletion strain DS68625 and analyzed for growth in liquid Verduyn-urea under different conditions. The identified mutants were termed Gal2-1 .1 (1 st-round mutant), Gal2-2.1 to 2.4 (2nd-round mutants), and Gal2-3.1 to 3.7 (3rd-round mutants), respectively (Table 2).
OD600 and HPLC analysis in shake flask culture. Shake flask cultures were sampled regularly during culture. For OD600 measurements, cultures were diluted appropriately for accurate measurement and optical density was measured at 600 nm wavelength in a Perkin Elmer Spectrophotometer K2 instrument. Remaining sample was filtrated to separate medium from yeast.
The filtrate was inserted into the appropriate vials for HPLC analysis. The concentrations of glucose, xylose, glycerol, acetic acid and ethanol in the medium were determined using a Shimadzu HPLC system. The system is equipped with column oven CTO-I OA-vp and Autoinjector SIL-10AD-vp with a guard column (Bio-Rad H cartridge, Bio-Rad) and an Aminex HPX-87H column (300 x 7.8 mm; Bio-Rad). Elution took place at 80 °C with 5 mM H2S04 at 0.6 mL/min. The eluate was monitored using a Refractive Index detector RID-10A (Shimadzu).
Microwell plate culture for growth curve profiling. For micro-well cultivation of strains, the Bioscreen C (Growth Curves Ltd.) was used. Overnight pre-cultures were pelleted, washed with demi water and diluted in demi water to twice the desired OD600 for inoculation. Medium was prepared in twice the concentration as desired. In one well of a honeycomb wellplate, 150 μΙ medium was mixed with 150 μΙ cell suspension. Measurements were conducted in triplicate. Settings for the Bioscreen C were maintained at 30°C incubation T, measurements every 15 min, shaking at type Continuous, amplitude Maximum, and speed Normal. Shaking was set to stop 5 sec before measurement.
TABLE 2 Generated Gal2 variants from three rounds of random mutagenesis
Designation Mutations Round
1.1 L31 1 R (L362L, D363D, K469K) 1 st round
Variant 1.1 was used as the template for 2nd round of random mutagenesis (further variants include these mutations)
2.1 L301 R, K310R, N314D, M435T (L343L) 2nd round
2.2 M435T, S468T (K394K) 2nd round
2.3 L301 R, K310R, N314D, Q425R, S427P, 2nd round M435T, (L343L, I356I)
2.4 M435T, (Q329Q, Q338Q, C434C) 2nd round
Variant 2.1 was used as template for 3rd round of random mutagenesis (further variants include these mutations except L343L)
3.1 T386A 3rd round
3.2 F444L (G151 G, I256I) 3rd round
3.3 (S285S, E367E) 3rd round
3.4 (D364D) 3rd round
3.5 Y176H 3rd round
3.6 Y226C, L280P (11651, 11781, A250A) 3rd round
3.7 M339V, L464Q (Y446Y) 3rd round
Silent mutations.
Example 1 - Hexose transporter gene deletions Deletion cassettes construction. Primers used in plasmid constructions are shown in Table 3; generated plasmids are shown in Table 4. Schemes with restriction sites used for cloning and sites used to release deletion constructs from the plasmid backbone are shown in Table 5.
Table 3: Primers (oligonucleotides) used in the examples
SEQ Internal Primer Sequence (5'- 3') Gene(s) Purpose ID code
NO:
5034 Kanf AAGCTTGCCTCGTCCCC kanMX Amplification
1 GCC kanMX
5035 Kanr GTCGACACTGGATGGCG kanMX Amplification
2 GCG kanMX
51 16 If2 ATTCTAGTAACGGCCGC loxP Part of SEQ Internal Primer Sequence (5'- 3') Gene(s) Purpose ID code
NO:
3 CAGTGTGCTGGAATTCG loxP flank
CCCTTAAGCTTGCCTCGT
CCCCGCCG
51 18 Ir2 CATACATTATACGAAGTT loxP Part of loxP
4 ATGCGCGCTCTAGATATC flank
GTCGACACTGGATGGCG GCG
51 15 If 1 ATCCGGACGTACGTATAA loxP Reamplification/
5 CTTCGTATAGCATACATT full loxP flank
ATACGAAGTTATTCTAGT AACGGCCGCCA
51 17 li TCATGACGTCTCGAGGC loxP Reamplification/
6 CTATAACTTCGTATAGCA full loxP flank
TAC ATTATAC G AAGTTAT GCGCGCT
1 15 Natf ACATGTAAAATGACCACT natl Amplification
10 CTTGACGACACGGC natl
1 16 Natr C AGTACTAG G G G C C AG G natl Amplification
1 1 GCATGCTC natl
28 H3f TGTACATCCGGAATTCTA HIS3
13 G ATTG GTGAG CG CTAG G
AGTCACTGCC
29 H3r CTCGAGTATTTCACACCG HIS3
14 CATATGATCCGTCG
201 Hx2uf GACTAGTACCGGTGTTTT HXT2 Upstream flank
16 C AAAAC CTAG C AAC C C C
202 Hx2ur CGTACGCGTCTTCCGGA HXT2 Upstream flank
17 AGGGTACCATCAGA I M C
A I I I GACC
203 Hx2df GAAGACACTCGAGACGT HXT2 Downstream
18 CC I I I GTCTGTGAAACCA flank SEQ Internal Primer Sequence (5'- 3') Gene(s) Purpose ID code
NO:
AGGGC
204 Hx2dr GTCGACGGGCCCTTATG HXT2 Downstream
19 TTGGTCTTG I I I AGTATG flank
GCCG
205 Hx3uf AAGCGGCCGCACTAGTA HXT3 Upstream flank
20 CCGGTGAAACAACTCAAT
AACGATGTGGGAC
206 Hx3ur ATCCGGACGTCTTCCTCA HXT3 Upstream flank
21 AGAAATCAG I I I GGGCG
ACG
210 Hx4df AGAAGACGCTCGAGACG HXT4 Downstream
22 TCCCTTATGGGAAGAAG flank
GTG I I I I GCC
21 1 Hx4dr ATGGATCCTAGGGGTTCT HXT4 Downstream
23 TGCAGAGTAAACTGCG flank
212 Hx5uf AAGCGGCCGCACTAGTA HXT5 Upstream flank
24 CATGTGAACTTGAAAACG
CTCATCAAGGC
213 Hx5ur TTCGTACGCGTCTTCCG HXT5 Upstream flank
25 GAGTAACATGAAACCAGA
GTACCACG
229 Hx7df AGAAGACCCTCGAGACG HXT7 Downstream
26 TCCGACGCTGAAGAAAT flank
GACTCACG
230 Hx7dr AGTCGACGGATCCGTAA HXT7 Downstream
27 TTTTTCTTCTTTTAAGTGA flank
CGGGCG
243 Gal2ufn AAGCGGCCGCACTAGTA GAL2 Upstream flank
28 CCGGTGATCTATATTCGA
AAGGGGCGG SEQ Internal Primer Sequence (5'- 3') Gene(s) Purpose ID code
NO:
244 Gal2ur AACGTACGTCCGGATCAT GAL2 Upstream flank
29 n TAGAATAC I I I I GAGATT
GTGCGCT
233 Ga2df AGAAGACCCTCGAGACG GAL2 Downstream
30 TCTTACCTTGGAAATCTG flank
AAGGCTGG
234 Ga2dr GTGGATCCTAGGTAAAAC GAL2 Downstream
31 GGTACGAGAAAAGCTCC flank
G
281 Hx3inr2 G CTCTTTTCACG GAG AAA HXT3-6- Integration
36 TTCGGG 7 check
323 Hx7inr1 GATGAGAATCCTTGGCAA HXT3-6- Integration
37 CCGC 7 check
299 Hx4inr2 CCATACTATTTGTCGACT HXT5-1 - Integration
38 CAAGCGC 4 check
317 Hx5inf GGGTTAATTAGTTTTAGG HXT5-1 - Integration
39 GGCACGG 4 check
324 Ga2inf1 TCAATTCGGAAAGCTTCC GAL2 Integration
40 TTCCGG check
325 Ga2inr CAGTGATAGTTTGGTTCG GAL2 Integration
41 1 AGCGG check
289 Hx2inf TCTTCGGGAACTAGATAG HXT2 Integration
42 GTGGC check
290 Hx2inr GAAGTAATCAGCCACAAT HXT2 Integration
43 ACGCC check
653 Glk1 - TATCACGTGCAGCCCAG GLK1 Hexokinase
44 psuc22 GATAATTTTCAGGACACG flank/Bipartite
7f TGTTTCGAAAGGTTTGTC cassette
GCTCCGATCGACCTCGA
GTACCGTTCG SEQ Internal Primer Sequence (5'- 3') Gene(s) Purpose ID code
NO:
651 Hxk2- CCACGAAATTACCTCCTG HXK2 Hexokinase
45 psuc22 CTGAGGCGAGCTTGCAA flank/Bipartite
7f ATATCGTGTCCAATTCCG cassette
TGATGTCTCGACCTCGA
GTACCGTTCG
645 pSUC2 GCAATTTCGGCTATACGT Bipartite
46 27r AAC cassette
654 Glk1 - ATTTAGTGAGCTGTTTCT GLK1 Hexokinase
47 psuc22 TGTCAAAACAACCAACGG flank/Bipartite
5r AAGAGGGCGAGGCTGTT cassette
TCCTCCGCGGATCCTAC
CGTTCGTATAG
652 Hxk2- TACAAAAGAAAGTACGCA HXK2 Hexokinase
48 psuc22 AGCTATCTAGAGGAAGT flank/Bipartite
5r GTAG AG AG G GTTAAAATT cassette
GGCGTGCCGGATCCTAC CGTTCGTATAG
646 pSUC2 CGTTCACTCATGGAAAAT Bipartite
49 25f AGC cassette
647 Hxk1_/ TCGGTTTCACTTCCTTGG HXK1 Hexokinase
50 oxP GAATATTCTACCGTTCCT flank /DRM
TCATCTTGTATTCCGGAT cassette C C ACTAG C ATAACTTC G
648 Hxk1_/ GACAATGCAGCAATAACA HXK1 Hexokinase
51 oxP GCAGCACCTGCACCTGA flank /DRM
ACCATCCTCAGC I I I GGG cassette CCGCCAGTGTGATGG
649 Gal1_/o TGTGCCTCGCGCCGCAC GAL1 Hexokinase
52 xPJ TGCTCCGAACAATAAAGA flank /DRM
TTCTAC AATACTAG C G G A cassette TCCACTAGCATAACTTCG SEQ Internal Primer Sequence (5'- 3') Gene(s) Purpose ID code
NO:
650 Gal1_/o AGGTATCCAAAACGCAG GAL1 Hexokinase
53 xP_r CGGTTGAAAGCATATCAA flank /DRM
GAATTTTGTCCCTGTTTG cassette
GGCCGCCAGTGTGATGG
Gal2_B CAGGGATCCTGCCGTCC GAL2 Insertion
60 amf AGG mutagenesis library
Gal2_M CTGCAATTGGAAGCAGA GAL2 Insertion
61 unr GGCC mutagenesis library
Gal2_E GTACGGCCGTAAAAAGG GAL2 Insertion
62 agf GTC I I I CG mutagenesis library
Table 4: Plasmids used in the strain construction
Number Construct Purpose SEQ ID NO: pRN201 pCR-BLU NT-loxP-kanMX-loxP DMR* 7 pRN251 pCR-BLU NJ-loxP- p MX-loxP DMR* 8 pRN365 pCR-BLU NT-loxP-natMX-loxP DMR* 9 pRN447 pCR-BLU NJ-loxP-zeoMX-loxP DMR* 12 pRN247 pCR-BLUNT-/7/s3:/oxP-/ anMX- HIS3 deletion 15 loxP construct
pRN485 pCR-BLU NJ-gal2:loxP-zeoMX- GAL2 deletion 32 loxP construct
pRN566 pCR-BLUNT-/7xf367:/oxP-/7p/7MX- HXT3-HXT6-HXT7 33 loxP cluster deletion
construct
pRN569 pCR-BLU NT- xt514 loxP-natMX- HXT5-HXT1-HXT4 34 loxP cluster deletion Number Construct Purpose SEQ ID NO:
contruct
pRN635 pCR-BLU NT-hxt2 loxP-kanMX- HXT2 deletion 35 loxP construct
pRS313- pRS313-PHXT7(-390)-GAL2-THXT7 GAL2 expression 58 GAL2 vector
pRN187 pCRE-zeoMX (based on pSH65) CRE expression 59 vector
*DMR = Dominant resistance marker
Table 5: Cloning scheme
Figure imgf000061_0001
The kanMX marker was amplified from the plasmid pFA6-kanMX4 (http://www- sequence.stanford.edu/qroup/yeast deletion project/kan mx4.txt) using primers SEQ ID NO's 1 and 2. Subsequently, the kanMX marker was floxed through adding loxP flanks by PCR amplification with primers SEQ ID NO's 3 and 4. Re-amplification was done with primers SEQ ID NO's 5 and 6. The resulting loxP-kanMX-loxP fragment was cloned in pCR-BLUNT resulting in pRN201 (SEQ ID NO: 7). For the construction of pRN251 (SEQ ID NO: 8), hphMX was isolated from pGRE3:hphMX (Kuyper et al, 2005, Metabolic engineering of a xylose-isomerase- expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res, 5, pp. 399-409). To delete a Mlu\ site as appropriate restriction site in the vicinity of hphMX, pGRE3:hphMX was cut with Eco32l and re-ligated. Subsequently, hphMX was cloned as Xho\-Mlu\ fragment into pRN201 digested with Sa/I and Mlu\ to replace kanMX.
For the construction of pRN365 (SEQ ID NO: 9), the Streptomyces noursei natl gene was PCR-amplified from pYL16 (Werner Bioagents) using primers with SEQ ID NO:'s 10 and -1 1. The Psc\-Sca\ natl fragment together with the Acc65\-Nco\ pRN201 - fragment were cloned into pRN201 , already linearized with Acc65\ and Seal, in order to replace kanR for natl.
For the construction of pRN447 (SEQ ID NO: 12), pRN201 was digested with Pml\. This served two ends. Firstly, the Streptoalloteichus hindustanus ble (zeocin or phleomycin resistance gene) ORF was isolated, and secondly, after re-ligation of the Pm/l-digested pRN201 an Λ/col site was deleted. Subsequently, ble as Nco\-Pml\ fragment and part of pRN201 as BamY\\-Nco\ vector fragment were cloned into the re- ligated pRN201 (missing ble), digested with SamHI and Seal resulting in pRN447.
For the HIS3 deletion construct, primers SEQ ID13 and-14 were used to amplify the HIS3 locus from yeast genomic DNA . Sites used to cut out the HIS3 flanks and to ligate these to the floxed kanMX marker are shown in Table 5. The ligation product was digested with Sacl and Apa\ and cloned into pCR-BLUNT digested with Sacl and Apa\. The resulting plasmid is pRN247 (SEQ ID NO: 15).
For the deletion of the eight main hexose transporters (HXT1-7 and GAL2) in S. cerevisiae, four deletion constructs were generated (see Table 4). Each deletion construct contained a different floxed dominant resistance marker. For each HXT gene 400-700 bp flanks were amplified using the primers listed in Table 3 (SEQ ID NO:'s 16- 31 ) using DS68616 genomic DNA as template. The upstream flank, the dominant resistance marker and the downstream marker were ligated using the fragments and cloning sites listed under Table 5. The ligations were amplified using the forward primers of the upstream flank and the reverse primer of the downstream flank (primer combinations SEQ ID NO:'s 16+19, SEQ ID NO:'s 20+27, SEQ ID NO:'s 24+23, and SEQ ID NO:'s 28+31 ). The fused PCR fragments were cloned into pCR-BLUNT to obtain pRN485, pRN566, pRN569, pRN635 (SEQ ID NO:'s 32-35, respectively). To obtain high yields of plasmid DNA, the plasmids were isolated from 50 mL £. coli cultures using NucleoBond® Xtra Midi kit (Bioke, Leiden, the Netherlands). Before transformation to yeast, deletion constructs were released from plasmid backbone by digestion with the release restriction sites listed in Table 5.
Strain construction. The xylose-fermenting strain DS68616 was made histidine auxotroph by the insertion of loxP-kanMX-loxP (released from pRN247; SEQ NO ID15) at the HIS3 locus. Subsequently, the marker was removed through transient expression of plasmid pRN187 (derived from pSH65 expressing galactose-inducible ere recombinase; SEQ ID NO 60). Introduction of pRN187 was selected on phleo and CRE recombinase expression was induced on YP-medium supplemented with galactose. The hexose transporters were deleted in the following order: 1 ) HXT3-HXT6-HXT7 cluster, 2) HXT5-HXT1-HXT4 cluster, 3) GAL2, 4) HXT2. The deletion constructs were linearized or released from the plasmid backbone by cutting with the enzyme combinations listed in Table 5 and these were integrated in the genome. All transformations were plated on yeast extract (10 g/L), peptone (20g/L) agar (15g/L) medium supplemented with 20g/L maltose. Maltose was added to the medium, because the uptake of this disaccharide goes via an alternative transport system than the glucose transport system (Wieczorke et al, 1999, Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett, 464, pp. 123-128). With each deletion of a (cluster of) HXT gene(s), an additional marker was inserted in the order: 1 ) hphMX, 2) natMX, 3) zeoMX, 4) kanMX. With each inserted additional marker the respective antibiotic was additionally supplemented to the medium in the following order: 1 ) HG, 2) HG and nour, 3) HG, nour and phleo, 4) HG, nour, phleo and G418. After integration of all four deletion constructs, a single colony was isolated under selection of all four antibiotics. Correct integrations were verified by PCR analysis on genomic DNA isolates. Primers outside of the integration site were used (combinations SEQ ID NO:'s 36+37, SEQ ID NO:'s 38+39, SEQ ID NO:'s 40+41 , SEQ ID NO:'s 42+43; sequences listed in Table 3).
Strain characterization. To characterize the (intermediate) hexose transporter strains, shake flask cultures were performed. Cultures were inoculated at OD600=0.1. The resulting strain, DS68625 (Ahxtl -7;Agal2-mutant; see Table 2 for exact genotype), showed a retarded growth pattern on Verduyn-urea (Verduyn-urea according to Verduyn using urea as nitrogen source; Luttik et al, 2001 ) supplemented with 0.2 g Γ1 histidine (Sigma-Aldrich; Verduyn-urea-his; to complement for the histidine auxotrophy) and 15 g Γ1 glucose and 20 g Γ1 xylose only starting to grow slowly on glucose only after 60 hours; interestingly, when glucose was present in the medium xylose was finished as well after 150 hrs (Fig. 1 ) indicating that one or more of the cryptic hexose transporter genes (HXT8-17) was induced on glucose and facilitated xylose transport (Fig. 1 ). However, on xylose as sole carbon source DS68625 did not grow on Verduyn-urea (+ 20 g 1-1 % xylose during the culturing period (Fig. 2) indicating the strain is useable as model strain for testing putative xylose transporters. DS68625 was further maintained on YPM.
Example 2 - Hexokinase gene deletions
Deletion cassettes construction. For deletion of hexokinase genes oligonucleotides were designed (SEQ ID NO:'s in Table 3) comprised of 60 nucleotide flanking sequences homologous to the hexokinase gene locus and of 20 nucleotides homologous to a floxed dominant resistance marker cassette. The oligonucleotides were used to amplify the deletion constructs. Subsequent PCR products were column filter- purified (Fermentas GeneJet Kit) and used for transformations experiments. Two types of deletion cassettes were used:
Firstly, for GLK1 and HXK2 deletions a bipartite system was used. One fragment consisted of a lox66 site, kanMX, GAL1 promoter upstream of CRE, and the 5'-part of CRE (CRE1 ) amplified from pSUC227 with one gene-specific primer (SEQ ID NO: 44 for GLK1 and SEQ ID NO: 45 for HXK2) and one pSUC227-specific primer (SEQ ID NO: 46); the second fragment consisted of the 3'-part of CRE (CRE2) with overlap on CRE1 , and a /ox71 site, amplified from pSUC225 with again one gene-specific primers (SEQ ID NO: 47 for GLK1 and SEQ ID48 for HXK2) and one pSUC225- specific primer SEQ ID NO: 49. Through homologous recombination the two fragments integrate as lox66-kanMX-CRE-lox7'\ at the hexokinase locus (pSUC225 and pSUC227 sequences and method provided in PCT/EP2013/055047).
- Secondly, for HXK1 (primers SEQ ID NO:'s 50-51 ) and GAL1 (primers SEQ ID NO:'s 52-53) deletions, a floxed dominant resistance marker (DRM) was amplified with flanking sequences homologous to the respective hexokinase to replace the coding region at the locus; as templates for the PCR amplifications of the DRM cassettes pRN774 {loxP-hphUX-loxP; SEQ ID NO: 54) and pRN775 {loxP-natMX-loxP; SEQ ID NO: 55) were used, respectively. Strain construction. For the generation of a strain incapable of hexose metabolism but capable of hexose transport, four hexokinase gene deletions were made in the xylose-fermenting strain DS68616 (Table 1 ).
Firstly, to enable selection for transporter constructs HIS3 was deleted in DS68616.The histidine-auxotroph strain (DS68616-/?/s3:.'/oxP) was the same strain, as was constructed in the DS68625 lineage. For the hexokinase deletions, as mentioned, in the case of GLK1 and HXK2, the disruption cassettes were bi-partite. Through homologous recombination the two fragments integrate as lox66-kanMX-CRE-lox71 at the hexokinase locus. The integration was selected on YPD supplemented with G418. The disruption cassettes for HXK1 and GAL1 consisted of one fragment: either loxP- natMX-loxP or loxP-hphMX-loxP, respectively. The DS68616-/?/s3.'.7oxP was transformed with the purified PCR products and the integration was selected on the appropriate antibiotic.
The hexokinase genes were deleted in the following order: 1 ) GLK1, 2) HXK1, 3) HXK2, 4) GALL After the deletion of GLK1, the kanMX marker was recycled by galactose-induced Cre-mediated recombination. After deletion of HXK2, the intermediate strain was maintained on xylose-containing rich medium (YPX). After GAL1 deletion, the integrated markers were removed by galactose-induced CRE recombination. To ensure growth of the strain, 2% xylose was added to YP 2% galactose + hygromycin (YPGX). Selection on hygromycin ensured maintenance of the hphMX marker at the GAL1 locus leaving a selection trait to be used possibly later on. After single colony isolation, the strain was verified for its deletions and delta sequence profile by colony PCR, and designated as DS69473.
Strain characterization. In Bioscreen C experiments, intermediate hexokinase mutants and the final quadruple hexokinase mutant DS69473 were screened for growth on Verduyn-urea-his supplemented with 2% glucose or xylose (Fig. 3). Cultures were inoculated at OD600=0.05. DS69473 did not grow on glucose (Fig. 3A), but did grow on xylose although somewhat hampered compared to DS68616 after deletion of HXK2 (Fig. 3B). DS69473 was further maintained on YPX for storage and handling. Example 3 - Directed evolution of Gal2 to increase its affinity for xylose: First round of random mutagenesis
To improve the affinity towards xylose a distinct part of GAL2 (SEQ ID NO: 56) was selected for mutagenesis, coding for the Gal2p protein sequence (SEQ ID NO: 57) between amino acid positions Asp292 and Ser477. This segment, which contains 185 amino acids, includes a large part of the proposed central cytoplasmic loop between the transmembrane (TM) segments 6 and 7, TM7 to TM10, and the corresponding cytoplasmic and extracellular loops (see Fig. 4A). This part of the Gal2 transport protein was chosen based on relevant amino acids concerning hexose sugar transport identified in related hexose transporter proteins in S. cerevisiae (Kasahara & Maeda, 1998. Contribution to substrate recognition of two aromatic amino acid residues in putative transmembrane segment 10 of the yeast sugar transporters Gal2 and Hxt2. J Biol Chem 273, pp. 29106-291 12; Kasahara & Kasahara, 2000, Three aromatic amino acid residues critical for galactose transport in yeast Gal2 transporter. J Biol Chem 275, pp. 422-4428; Kasahara & Kasahara, 2000, Interaction between the critical aromatic amino acid residues Tyr(352) and Phe(504) in the yeast Gal2 transporter. FEBS Lett 471 , pp. 103-107; Kasahara & Kasahara, 2003, Transmembrane segments 1 , 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter. Biochem J 372, pp. 247-252; Kasahara & Kasahara, 2010, Identification of a key residue determining substrate affinity in the yeast glucose transporter Hxt7: a two- dimensional comprehensive study. J Biol Chem, 285, pp. 26263-26268; Kasahara et al., 2004, Comprehensive chimeric analysis of amino acid residues critical for high affinity glucose transport by Hxt2 of Saccharomyces cerevisiae. J Biol Chem, 279, pp. 30274- 30278; Kasahara et al., 2007, Identification by comprehensive chimeric analysis of a key residue responsible for high affinity glucose transport by yeast HXT2. J Biol Chem, 282, pp. 13146-13150; Kasahara et al., 201 1 , Crucial effects of amino acid side chain length in transmembrane segment 5 on substrate affinity in yeast glucose transporter Hxt7. Biochemistry, 50, pp. 8674-8681 ).
After creating a random mutant library with mutagenic parameters to obtain approximately up to 4 point mutations within the 185 amino acids of Gal2, the library of mutagenic PCR fragments digested with the proper restriction zymes (see Mthods section for more details) was inserted into pRS313-GAL2 (Fig. 4C) also digested with the same restriction enzymes. Subsequently, the library of GAL2 variant plasmids was transformed into the S. cerevisiae deletion strain DS68625 lacking the hexose transporters HXT1-HXT7 and GAL2 (see Example 1 ). In addition, this strain has been engineered for xylose utilization by introduction of the xylose isomerase gene {xylA) from the anaerobic fungus Piromyces sp. E2. All transformants harboring the mutant library were collected from the selective agar plates by floating the plates, transferring all yeast cells and growing the cell suspension overnight with 1 % xylose. Prior to screening for high-affinity xylose candidates, the overnight culture was analyzed with cell flow cytometry for budding yeast cells only. Subsequently, budding yeast cells were sorted out on agar plates containing exactly 25 ml of Verduyn-urea with 0.1 % xylose as sole carbon source. On every plate 100 single cells were placed in a square grid to ensure that every grown colony on all the plates is exposed to the exactly same conditions. In total 13,700 single yeast cells were sorted in the 1st screening round. Based on the colony size after 3.5 days at 30°C, 80 large size colonies were picked and transferred to new agar plates containing the exact same medium with 0.1 % xylose. After 4 days at 30°C, 9 colonies which were significantly larger than the background colonies were tested in liquid Verduyn-urea with 2% xylose as sole carbon source. For validation of the growth results the plasmid DNA from the promising mutants were isolated and retransformed into the S. cerevisiae deletion strain DS68625. One candidate, designated as Gal2-1 .1 , facilitated a faster growth as compared to wild-type Gal2 permease (Fig. 5). DNA sequencing of this variant revealed a single amino acid substitution at position 31 1 where a leucine, located within the cytoplasmic loop region between TM6/7, was exchanged to an arginine. Additionally three silent mutations were also identified (L362L, D363D, K469K) (Table 2).
Example 4 - Directed evolution of Gal2 to increase its affinity for xylose: Second round of random mutagenesis
Using this Gal2 variant 1 .1 as a template (see Example 3), a 2nd round of random mutagenesis was performed using the same mutagenic parameters on the same segment of the transporter protein. Here, using the same high-throughput screening methods as in Example 3, 14,400 yeast cells harboring the new mutant library were sorted out on agar plates containing Verduyn-urea with 0.1 % xylose as sole carbon source. Four second round mutants were selected as faster growing colonies and extracted plasmids (Gal2-2.1 to 2.4) were sequenced (see Table 2 for mutations). DNA sequencing of the mutant Gal2-2.1 revealed beside the mutations in variant 1 .1 also one silent mutation (L343L), three new amino acid substitutions (L301 R, K310R, N314D) within the central cytoplasmic loop between TM6/7 and an additional substitution at position 435 where a methionine was changed to a threonine. This M435T mutation was also identified in all the other 2nd round mutants (2.2, 2.3, and 2.4). The mutant Gal2-2.3 revealed a similar mutation set-up as Gal2-2.1 with two further amino acid substitutions Q425R and S427P, both located within the extracellular loop between TM9 and TM10 and also one further silent mutation (I356I). Besides M435T and K394K, Gal2-2.2 showed another amino acid substitution at position 468 where a serine was substituted to a threonine within the loop region between TM10 and TM1 1. After retransformation into a fresh strain background all the candidates were tested in liquid Verduyn-urea at two different xylose concentrations for detailed characterization of the mutants: 0.45% for improved affinity and 0.1 % for high affinity (Fig. 6A and 6B). To confirm these results sugar consumption experiments were carried out in parallel. Using HPLC analysis the remaining sugar concentration in the medium was measured over time (Fig. 6C and 6D).
Compared to the wild-type and the mutant Gal2-1 .1 identified from the first round of mutagenesis all mutants from the second round of mutagenesis (Gal2-2.1 -2.4) showed an improved affinity towards 0.45% xylose (Fig. 6A). After 62 h no xylose was detectable by HPLC for the mutant 2.1 and less than 0.03% for 2.2 and 2.3 (Fig. 6C). At the same time point, 0.35% xylose and 0.42% xylose for the variant 1 .1 and the wild-type Gal2 was present in the remaining medium, respectively. Experiments with 0.1 % xylose revealed 2.1 as a high affinity transporter variant (Fig. 6B). 2.1 showed the fastest growth among all the tested variants: only 0.02% xylose was present in the remaining medium after 1 14 h of incubation whereas 1 .1 and the wild-type transporter showed no growth or consumption at this xylose concentration (Fig. 6D). The S. cerevisiae deletion strain DS68625 bearing the empty vector (pRS313) was included as a control in the experiments and showed no growth or consumption of xylose.
Example 5 - Directed evolution of Gal2 to improve the xylose uptake in the presence of glucose: Third round of random mutagenesis
The screening conditions with concentrations of 0.1 % xylose led to significantly increased affinity towards xylose due to alterations within the different loop regions of Gal2. In the subsequent screening of the mutant library the conditions were changed to improve the uptake of xylose in the presence of glucose. Due to the diauxic growth behavior of DS68625, in which glucose is first consumed, the hexokinase deletion strain DS69473 { glkl, gall, hxkl, hxk2) (see for generation strain Example 2) was used for transformation of the mutant library. Growth on glucose as the sole carbon source is completely abolished in this strain, while uptake of glucose and xylose is intact. The strain DS69473 is an ideal host for screening for transporters able to take up xylose in the presence of glucose competition for transport.
To create a new mutant library to screen for improved xylose uptake in the presence of glucose the best candidate of the 2nd round of mutagenesis, Gal2-2.1 , was chosen as the template. Prior to creating the library, an additional Mun\ restriction site caused by the silent mutation L343L in the mutant was changed back to the wild-type DNA sequence (TTG -> TTA) by site-directed mutagenesis. Subsequently, a new Eag\ restriction site between TM2/3 was introduced by site-directed mutagenesis with no change in amino acid sequence to expand the range for mutagenesis from previously 185 amino acids to 331 amino acids (see Fig. 4A).
The new mutant library was transformed into the hexokinase deletion strain DS69473. In the third screening round after flow cytometer, the mutant library was plated on Verduyn-urea agar plates containing 2% xylose and 10% glucose as carbon sources. In total 131 agar plates with single budding yeast cells were incubated at 30°C. After 12 days of incubation at 30°C only eight colonies could be identified. Out of these eight candidates DNA sequencing revealed five new Gal2 mutants with amino acid substitutions, two mutants with silent mutations and two candidates were found to be the same (Table 2). From the 3rd round mutants the plasmid DNA harboring the amino acid substitutions from the five Gal2 mutants 3.1 , 3.2, 3.5, 3.6 and 3.7 was isolated and transformed into the transporter deletion strain DS68625 to characterize each mutant more in detail concerning growth on 2% glucose and 2% xylose (Fig. 7A and 7B).
At only 2% glucose, the 2nd round mutant 2.1 which was included as a control showed a significant decreased growth compared to that of the wild-type Gal2 (Fig. 7A). Interestingly, the 3rd round mutant 3.7 which was developed from mutant 2.1 displayed an almost identical growth as the wild-type Gal2. Also mutant 3.2 showed an increased growth at this glucose concentration as compared to 2.1 but not as fast as 3.7 or the wild-type. Furthermore, the mutants 3.1 , 3.5 and 3.6 revealed a strong impaired growth on 2% glucose up to 48 h. At 2% xylose the mutant 2.1 displayed the fastest growth among all tested candidates and the wild-type (Fig. 7B). The mutants 3.2 and 3.7 showed a slightly reduced growth than 2.1 , but a clearly faster growth than the wild-type. But mutant 3.1 which showed no growth at 2% glucose for the first 48 h grew almost as fast as 3.2 and 3.7 at 2% xylose. Surprisingly, the variants 3.5 and 3.6 showed no growth at 2% xylose over the tested period of time.
Improved xylose uptake in the presence of glucose
To facilitate the simultaneous uptake of glucose and xylose from agricultural biomass the two mutants Gal2-2.1 and 3.1 were evaluated under this aspect. After retransformation into DS68625 these two evolved mutants were analyzed for growth at 2% xylose in the presence of 2% glucose (ratio 1 :1 ) to test for improved uptake at higher sugar concentrations. In parallel, the mutants were also tested for improved affinity towards xylose at 0.45% xylose in the presence of 0.45% glucose (ratio 1 :1 ). Besides measuring the optical density at 600 nm the consumption of both sugars was analyzed by HPLC by measuring the remaining sugar concentration in the medium over time (Fig. 8A-D).
The diauxic sugar consumption profile, in which glucose is consumed first, was detectable for both the Gal2 wild-type and mutant 2.1 under the growth conditions of 2% xylose with 2% glucose. The variant 2.1 showed a faster growth than the Gal2 wild-type. This was confirmed by the HPLC analyses which revealed a faster consumption of both sugars for this mutant (compare Fig. 8A and 8C). Mutant 2.1 showed also a faster cell growth than the wild-type in medium containing 0.45% xylose and 0.45% glucose (Fig. 8B). In Fig. 8D, mutant 2.1 showed an almost simultaneous consumption of xylose and glucose. After 35 h of incubation there was no glucose and only 0.07% xylose detectable for the mutant 2.1 but still 0.04% glucose and 0.38% xylose detectable for the wild-type Gal2. The variant 3.1 facilitated no consumption and no growth over the tested period of time.
Relevant amino acid for sugar transport in Gal2
We further characterized the mutant 3.1 , which showed an impaired growth at 2% glucose but not at 2% xylose (Fig. 7A and 7B), whether the mutation T386A might be involved in the transport of glucose in Gal2. Threonine at amino acid position 386 was substituted by alanine in the wild-type protein by site-directed mutagenesis. Subsequently, after transformation into the deletion strain DS68625 and growth at 2% glucose this new Gal2 variant Gal2_T386A showed a slower lag phase than the wild- type but a clearly faster growth than mutant 2.1 (Fig. 9A). The results were similar for growth at 0.1 % glucose (Fig. 9C). Mutant 3.1 which was again included as control in these experiments, showed no growth on either 2% glucose or 0.1 % glucose and confirmed our previous results of decreased growth on glucose (Fig. 9A and 9C). At 2% xylose the new variant Gal2_T386A showed a faster growth than the wild-type Gal2 but significantly slower growth than the mutants 2.1 and 3.1 (Fig. 9B). At 0.1 % xylose, both Gal2-T386A and Gal2 wild-type did not grow at this concentration (Fig. 9D). Whereas at this low xylose concentration the mutants 2.1 and 3.1 showed growth of which the variant 2.1 was the fastest. These results suggests that the mutation T386A alone is not crucial but in combination with the mutations identified from Gal2-2.1 plays a critical role in glucose transport.
Example 6 - Characterization of amino acid substitutions from different Gal2 variants
Site-directed mutagenesis to create single and double mutants
Within a random mutation library not every single change on the DNA level necessarily must have an impact on the protein level or a positive effect on a desired protein function. To get a more detailed insight on the effect of each changed amino acid position we generated single and double mutants of the variants Gal2-1 .1 , -2.1 and -2.2 identified within the first two screening rounds. All identified amino acid substitutions from these three variants were changed in wild-type Gal2 by site-directed mutagenesis and tested for growth in Verduyn-urea containing 2%, 0.45% and 0.1 % xylose as sole carbon source. The growth results of the fastest single and of the five best double mutants are shown in Fig. 10. Compared to the wild-type Gal2 the mutation at position 435, where a methionine was substituted by a threonine, showed the best growth results among all the tested single mutants under the different conditions. Within the tested double mutants 4 out of 5 of the best Gal2 variants contained also the mutation M435T: L301 R+M435T, K310R+M435T, N314D+M435T and M435T+S468T. Among these 4 double mutants the combination N314D+M435T showed the fastest growth at the three tested concentrations. In addition, the mutation K310R in combination with N314D showed also an improvement of xylose uptake. These results show that methionine at position 435 might play a relevant role in the uptake of xylose in Gal2. REFERENCES
Ausubel et al. 1995 Current Protocols in Molecular Biology, John Wiley & Sons,
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Becker J, Boles E. 2003 A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces ethanol. Appl Environ Microbiol. 69 p. 4144-4150.
Hamacher T, Becker J, Gardonyi M, Hahn-Hagerdal B, Boles E. 2002 Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology 148 p. 2783-2788.
Kasahara T, Kasahara M. 2000 Three aromatic amino acid residues critical for galactose transport in yeast Gal2 transporter. J Biol Chem. 275 p. 4422-4428.
Kasahara T, Maeda M, Boles E, Kasahara M. 2009 Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1. Biochim Biophys Acta. 1788 p.1051 -1055.
Kasahara T, Kasahara M. 2010 Identification of a key residue determining substrate affinity in the yeast glucose transporter Hxt7: a two-dimensional comprehensive study. J Biol Chem. 285 p. 26263-26268.
Kuyper M, Hartog MM, Toirkens MJ, Almering MJ, Winkler AA, van Dijken JP, Pronk JT 2005 Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Research 5 p. 399-409.
Luttik MA, Kotter P, Salomons FA, van der Klei IJ, van Dijken JP, Pronk JT 2000 The Saccharomyces cerevisiae ICL2 gene encodes a mitochondrial 2-methylisocitrate lyase involved in propionyl-coenzyme A metabolism. Journal of Bacteriology 182 p.7007-7013.
Nelissen B, De Wachter R, Goffeau A. 1997 Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae. FEMS Microbiol Rev. 21 p. 1 13-134.
Sambrook et al. 1989 Molecular Cloning, a Laboratory Manual
Schiestl RH and Gietz RD 1989 High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Current Genetics 16 p.339-346. Wieczorke R, Krampe S, Weierstall T, Freidel K, Hollenberg CP, Boles E 1999 Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters 464 p.123-128.
Young E, Poucher A, Comer A, Bailey A, Alper H. 201 1 Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host. AppI Environ Microbiol. 77 p. 331 1 -3319.

Claims

Polypeptide having one or more substitution corresponding to a substitution at position 301 , 310, 31 1 , 314, 435 and/or 468 of SEQ ID NO:57 wherein the polypeptide is member of the Major Facilitator Superfamily (MFS).
Polypeptide according claim 1 , wherein the polypeptide has one or more substitution corresponding to L301 R/M/IA /F/Q/A, K310R/N/M/Q/T/D/E, L31 1 R/M/IA /F/Q/A, N314D/H/S/K/E/T/Q/G/A, M435T/L/IA /K/R/Q/F and/or S468T/L/IA /K/R/Q/F of SEQ ID NO: 57.
Polypeptide according to claim 1 or 2, wherein the polypeptide has pentose transport activity.
Polypeptide according to claim 3, wherein the polypeptide has xylose transport activity.
Polypeptide according to any of claims 1 to 4, wherein the polypeptide has one or more substitution corresponding to L301 R, K310R, L31 1 R, N314D, M435T and/or S468T of SEQ ID NO: 57.
Polypeptide according to any of claims 1 to 5, wherein the polypeptide has one or more substitution pair corresponding to L30 1 R and M435T, L310R and M435T, N314D and M435T, M435T and S468T and/or N314D and K310R of SEQ ID NO: 57.
Polypeptide according to any of claims 1 to 6, wherein the polypeptide has reduced glucose transport activity compared to the polypeptide having SEQ ID NO: 57.
Polypeptide according to claim 7, wherein the polypeptide has increased xylose transport activity compared to the polypeptide having SEQ ID NO: 57.
Polypeptide according to any of claims 1 to 8, comprising sequences that contain one or more of the following amino acid motifs:
(a) G-R-x(3)-G-x(3)-G-x(1 1 )-E-x(5)-[LIVM]-R-G-x(12)-[GA] ; (b) R-x(14)-G-x(2)-Y-x(2)-[YF]-[YF]-[GSAL] and/or
c) V-x(15)-[GNR]-[RH]-R-x(2)-[LM]-x(2)-[GA].
10. Polypeptide according to any of claims 1 to 9 comprising a motif G-R-x(3)-G-x(3)- G-x(1 1 )-E-x(5)-[LIVM]-R-G-x(12)-[GA].
1 1 . Polynucleotide having at least 50% identity to SEQ ID NO: 56, encoding the polypeptide according to any of claims 1 to 10.
12. Nucleic acid construct comprising a polynucleotide of claim 1 1 .
13. Host cell that is transformed with a nucleic acid construct of claim 12.
14. Transformed host cell according to claim 13, which is yeast.
15. Transformed host cell according to claim 14, which belongs to the genus Saccharomyces, in particular the species Saccharomyces cerevisiae.
16. Transformed host cell comprising a heterologous nucleotide that encodes a polypeptide according to any of claims 1 to 10 .
17. Transformed host according to claim 16, wherein the polynucleotide encodes a polypeptide that is a mutant of a polypeptide that is native in the untransformed host cell.
18. Transformed host cell according to claim 17, wherein the polypeptide that is native in the untransformed host eel is a member of the the Major Facilitator Superfamily (MFS) transporters.
19. Transformed host cell according to claim 17 or 18, wherein the polypeptide that is native in the untransformed host cell is a hexose transporter polypeptide.
20. Transformed host cell according to any of claims 17 to 19, wherein the polypeptide that is native in the untransformed host cell is a hexose transporter polypeptide.
21 . Transformed host cell according to claim 20, wherein the polypeptide that is native in the untransformed host cell is a transporter polypeptide chosen from the list consisting of Gal2, Hxt1 , Hxt2, Hxt3, Hxt4, Hxt5, Hxt6, Hxt7, Hxt8, Hxt9, Hxt10, Hxt1 1 , Hxt12, Hxt13, Hxt14, Hxt15, Hxt16 and Hxt17.
22. Process for the degradation of ligno-cellulosic or hemi-cellulosic material, wherein ligno-cellulosic or hemi-cellulosic material is contacted with an enzyme composition, wherein one or more sugar is produced, and wherein the produced sugar is fermented to give a fermentation product, wherein the fermentation is conducted with a transformed host cell of any of claims 13 to 21 .
23. Process according to claim 22, wherein the fermentation product is one or more of ethanol, butanol, lactic acid, di-terpene, glycosylated di-terpene, a plastic, an organic acid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin, an amino acid, an enzyme or a chemical feedstock.
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