Variants of GAL2 transporter and their uses

Abstract

The present invention relates to polypeptides which are Gal2 variants comprising at least one amino acid substitution at a position corresponding to M435, and optionally further amino acid substitution(s). The present invention further relates to nucleic acid molecules encoding the polypeptides and to host cells containing said nucleic acid molecules. The present invention further relates to a method for the production of bioethanol and/or other bio-based compounds, comprising the expression of said nucleic acid molecules, preferably in said host cells. The present invention also relates to the use of the polypeptides, nucleic acids molecule or host cells for the production of bioethanol and/or other bio-based compounds, and/or for the recombinant fermentation of biomaterial containing pentose(s), preferably D-xylose and/or L-arabinose.

Claims

1. A polypeptide, comprising at least one amino acid substitution at a position corresponding to M435 and at a position corresponding to N376 of the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and wherein the polypeptide has either or both of an in vitro and an in vivo pentose transport function.

2. The polypeptide of claim 1, wherein the polypeptide is Gal2 of Saccharomyces cerevisiae.

3. The polypeptide of claim 1, comprising the amino acid substitution M435I.

4. The polypeptide of claim 1, comprising an amino acid substitution that is selected from N376Y and N376F.

5. The polypeptide of claim 1, wherein the amino acid substitution at the position corresponding to M435 increases activity of either or both of the in vitro and the in vivo pentose transport function compared to said activity of the polypeptide without the amino acid substitution.

6. The polypeptide of claim 1, wherein the amino acid substitution at the position corresponding to M435 increases affinity of the polypeptide for a pentose compared to said affinity of the polypeptide without the amino acid substitution.

7. The polypeptide of claim 1, wherein the pentose for which the polypeptide has pentose transport function is either one or both of D-xylose and L-arabinose.

8. A nucleic acid molecule, comprising a nucleic acid sequence encoding the polypeptide of claim 1.

9. The nucleic acid molecule of claim 8, further comprising one or more of: (i) a vector nucleic acid sequence, (ii) an expression vector sequence, (iii) a promoter nucleic acid sequence, (iv) a terminator nucleic acid sequence, (v) a regulatory nucleic acid sequence, wherein the nucleic acid molecule comprises dsDNA, ssDNA, PNA, CNA, RNA, or mRNA, or combinations thereof.

10. A host cell containing the nucleic acid molecule of claim 8, wherein one or more of: (a) the host cell expresses said nucleic acid molecule, (b) the host cell is a fungus cell, (c) the host cell is a yeast cell, or (d) the host cell is selected from the group consisting of a Saccharomyces species, a Kluyveromyces sp., a Hansenula sp., a Pichia sp., and a Yarrowia sp.

11. The host cell of claim 10, which belongs to the species Saccharomyces cerevisiae.

12. The host cell of claim 10, which has an increased uptake rate for either or both of D-xylose and L-arabinose, compared to a cell that does not contain the nucleic acid molecule encoding the polypeptide having either or both of in vitro and in vivo pentose transport function.

13. The host cell of claim 10 which further comprises either or both of: (a) nucleic acid molecules which code for proteins of a xylose metabolic pathway, and (b) nucleic acid molecules which code for proteins of an arabinose metabolic pathway.

14. The host cell of claim 13, which has at least one of: (a) an increased rate of consumption of either or both of D-xylose and L-arabinose, or (b) a faster growth rate with either or both of D-xylose and L-arabinose present, compared to a cell that does not contain the nucleic acid molecule encoding the polypeptide having either or both of in vitro and in vivo pentose transport function.

15. The host cell of claim 13 wherein: (i) the proteins of the xylose metabolic pathway comprise at least xylose isomerase and xylulokinase, and (ii) the proteins of the arabinose metabolic pathway comprise at least rabinose isomerase, ribulokinase, and ribulose-5-P 4-epimerase.

16. A method for producing ethanol and/or other fermentation products, comprising expressing, in the host cell of claim 10, the nucleic acid molecule encoding the polypeptide having either or both of in vitro and in vivo pentose transport function.

17. The method of claim 16 which comprises recombinant fermentation by the host cell of biomaterial containing pentoses, wherein the pentoses comprise either or both of D-xylose and L-arabinose.

18. The method of claim 16 wherein the other fermentation products are selected from: 1-butanol, isobutanol, 2-butanol, other alcohols, lactic acid, acetic acid, succinic acid, malic acid, other organic acids, amino acids, alkanes, terpenes, isoprenoids, solvents, pharmaceutical compounds, and vitamins.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Growth test of Gal2_ep3.1 mutants in comparison to N376Y in EBY.VW4000.

(2) The transformants were cultivated in 5 ml SCM-ura with 20 g/l maltose at 30, washed with water and adjusted to an OD.sub.600 of 1. Thereof it followed a serial dilution. 5 l were dropped onto the respective media and it was incubated for three days at 30. As a control for the dilution SCM-ura with 20 g/l maltose was taken. The cells of the mutated transporters were dropped onto SCD-ura with 0.2% and 2% glucose, as well as SCG-ura with 0.2% and 2% D-galactose to test their functionality. Furthermore the wild type, Gal2_ep3.1 and Gal2_N376Y were used for comparison.

(3) FIG. 2: Growth test of Gal2_ep3.1 mutants in comparison to N376Y in AFY10

(4) The transformants were cultivated in 5 ml SCE-ura-leu with 2% ethanol at 30, washed with water and adjusted to an OD.sub.600 of 1. Thereof it followed a serial dilution. 5 l were dropped onto the respective media and it was incubated for five days at 30. As a control for the dilution SCE-ura-leu with 2% ethanol was taken. The cells of the mutated transporters were dropped onto SCX-ura-leu with 0.2% and 2% D-xylose, as well as SC-ura-leu with 1% D-xylose with 4% D-glucose and SC-ura-leu with 0.2% D-xylose and 2% D-glucose to test their functionality. Furthermore the wild type and Gal2_ep3.1 were used for comparison.

(5) FIG. 3: Growth test in liquid media and fermentation

(6) The strain AFY10, which was transformed with YEp181_pHXT7-opt.XI_Clos and p426H7-GAL2_wt (WT) or p426H7-GAL2_ep3.1 (ep3.1) or p426-GAL2_N376Y+M435I (M435I), was incubated in 150 ml SCE-ura-leu+G418. This was the preculture which was used to start the fermentation with an OD.sub.600 of 0.6 in 30 ml SC-ura-leu with 0.6% xylose (A) or with 0.6% xylose with 2% glucose (B). The cultures were incubated at 180 rpm and 30 for seven days. Samples were taken for growth measurements as well as metabolite analysis. The measurements of the showed xylose concentration was done with HPLC whereas the samples were diluted 1:5. The entire xylose concentration of the supernatant is showed in % (w/v). The data indicate the averages of three independent replicates whereby the error bars are not shown for reason of clarity.

(7) FIG. 4: Drop test of Gal2_M435I in EBY.VW4000 after three days at 30. Several dilutions from left to right were dropped (undiluted, 1:10, 1:100, 1:1000).

(8) FIG. 5: Drop test of Gal2_M435I in AFY10 after five days at 30. Several dilutions were dropped from left to right (undiluted, 1:10, 1:100, 1:1000).

(9) FIG. 6: Drop test of Gal2_N376F, N376F+M435I and N376Y+M435I in AFY10 after five days at 30. Several dilutions were dropped from left to right (undiluted, 1:10, 1:100, 1:1000). But on 20 g/l xylose the order was different (undiluted, 1:100, 1:1000, 1:10).

(10) FIG. 7: Xylose uptake assay of Gal2_N376Y, N376Y+M435I and Gal2 wild type in EBY.VW4000 (three independent replicates). The uptake velocity is shown in nmol xylose per minute per milligram cell dry mass.

(11) FIG. 8: Xylose uptake assay of Gal2_N376F, N376F+M435I and Gal2 wild type in EBY.VW4000 (three independent replicates). The uptake velocity is shown in nmol xylose per minute per milligram cell dray mass.

EXAMPLES

(12) Methods

(13) Strains and Media

(14) Bacteria

(15) E. coli SURE (Stratagene)

(16) Full medium LB 1% trypton, 0.5% yeast extract, 0.5% NaCl, pH 7.5 (see Sambrook and Russell, 2001)

(17) For selection on a plasmid-coded antibiotic resistance, 40 g/ml ampicillin was added to the medium after autoclaving. Solid culture media additionally contained 1.9% agar. The culture took place at 37 C.

(18) Yeast

(19) CEN.PK2-1C

(20) MATa leu2-3,112 ura3-52 trp1-289 his3-1 MAL2-8.sup.c SUC2 (EUROSCARF, Frankfurt)

(21) Strain EBY.VW4000

(22) EBY.VW4000 (Genotype: MATa leu2-3,112ura3-52 trp1-289 his3-1 MAL2-8c SUC2 hxt1-17gal2 stl::loxP agt1 ::loxP mph2::loxP mph3::loxP) (Wieczorke et al., 1999)

(23) Strain Ethanol Red

(24) available from Lesaffre, Lille, France. Described in Demeke et al. (2013).

(25) Strain HDY.GUF10

(26) Xylose and arabinose consuming industrial S. cerevisiae strain derived from Ethanol Red (Dietz 2013).

(27) Strain AFY10

(28) EBY.VW4000 glk1::loxP hxk1::loxP hxk2::loxP ylr446w::loxP pyk2::pPGK1-opt.XKS1-tPGK1 pTPI1-TAL1-tTAL1 pTDH3-TKL1-tTKL1 pPFK1-RPE1-tRPE1 pFBA-RKI1-tRKI1 loxP (Farwick et al., 2014).

(29) Strain AFY10X

(30) AFY10+YEp-kanR_optXI (Farwick et al., 2014).

(31) Synthetic Complete Selective Medium SC

(32) 0.67% yeast nitrogen base w/o amino acids and ammonium sulphate, 0.5% ammonium sulphate, 20 mM potassium dihydrogenphosphate, pH 6.3, amino acid/nucleobase solution without the corresponding amino acids for the auxotrophy markers of the plasmids used, carbon source in the respectively indicated concentration

(33) Concentration of the amino acids and nucleobases in the synthetic complete medium (Zimmermann, 1975): adenine (0.08 mM), arginine (0.22 mM), histidine (0.25 mM), isoleucine (0.44 mM), leucine (0.44 mM), lysin (0.35 mM), methionine (0.26 mM), phenylalanine (0.29 mM), threonine (0.48 mM), tryptophan (0.19 mM), tyrosin (0.34 mM), uracil (0.44 mM) and valine (0.49 mM). As carbon sources, L-arabinose, D-glucose, D-galactose, D-mannose, ethanol and maltose were used, as indicated.

(34) Solid full and selective media contained in addition 1.9% agar. The culture of the yeast cells took place at 30 C.

(35) TABLE-US-00003 Plasmids Plasmid Referenz p426H7 Becker and Boles, 2003 YEp181_pHXT7-optXI_Clos Subtil and Boles, 2012 p426H7_GAL2_WT p426H7_GAL2_ep3.1 p426H7-GAL2_EtOHred p426H7-GAL2_GUF10 p426H7-GAL2_N376Y p426H7-GAL2_N376Y + M107K p426H7-GAL2_N376Y + V239L p426H7-GAL2_N376Y + M435I p426H7-GAL2_N376Y + M558S p426H7_GAL2-M435I p426H7_GAL2-N376F p426H7_GAL2-N376F + M435I
Preparation of DNA
Isolation of Plasmid DNA from E. coli

(36) Small-scale preparations of plasmid DNA from E. coli cultures were done using the GeneJET Plasmid Miniprep Kit (Fisher Scientific) according to manufacturer's instructions. The QUIAGEN Plasmid Maxi Kit was used for large scale preparations.

(37) Isolation of Plasmid and Genomic DNA from S. cerevisiae

(38) For isolation of genomic and plasmid DNA from yeast cells 5-10 ml of stationary phase cultures were harvested by centrifugation (1 min, 2000g) and washed once in 1 ml sterile ddH.sub.2O. The cell pellet was resuspended in 400 l YP-buffer 1 by vortexing and then lysed by addition of 400 l YP-buffer 2, to Vol glass beads ( 0.25-0.5 mm) and 8 minutes of shaking on a VXR basic Vibrax (IKA) at 2000 rpm. Cell debris was pelleted by centrifugation (30 sec, 16000g) and 650 l of the supernatant transferred to a fresh eppendorf tube. 325 l of cold YP-buffer 3 were added and the sample was vortexed and then incubated on ice for 10 minutes for precipitation of proteins and other contaminants. The sample was centrifuged (10-15 min, 4 C., 16000g), 700 l of the supernatant were transferred to a fresh eppendorf tube and 700 l isopropanol were added. After mixing vigorously, the sample was incubated for 10 minutes at RT to allow precipitation of the DNA, which was then pelleted by centrifugation (30 min, RT, 16000g). The DNA-pellet was washed twice with 500 l of cold (20 C.) 70% (v/v) ethanol, with centrifugation steps of 5 minutes at RT and 16000g, then dried at RT for 10 minutes and dissolved in 15-30 l sterile ddH2O depending on the size of the DNA pellet.

(39) Determining the DNA Concentration

(40) The DNA concentration is measured by spectral photometry in a wavelength range of 240-300 nm. If the purity of the DNA, determined by the quotient E260 nm/E280 nm is 1.8, then the extinction E260 nm=1.0 corresponds to a DNA concentration of 50 g dsDNA/ml (Sambrook and Russell, 2001).

(41) DNA Purification of PCR Products

(42) The purification of the PCR products took place with the QIAquick PCR Purification Kit of the company Qiagen, according to the manufacturer's information.

(43) Digestion of DNA with Restriction Endonucleases (Restriction Digestion)

(44) For the site-specific cleavage of DNA restriction endonucleases from New England Biolabs (NEB) or Fermentas were used with the provided buffers and according to the instructions of the manufacturer. Usually 1-3 units per g DNA were used for the reaction, which was incubated for 2-12 hours. This method was used to prepare vectors for recombinational cloning, to confirm correctly assembled plasmids or to specifically degrade a certain plasmid from a mixture.

(45) Polymerase Chain Reaction (PCR)

(46) Different polymerases were used for different PCR experiments in this work. For confirmation of genomic gene deletion or integration the Crimson Taq polymerase (NEB) was used. For amplification of ORFs (for sequencing), genes for recombinational cloning or amplification of integrative cassettes for genomic gene deletion or integration the Phusion or Q5 polymerases (NEB) were used. Composition of PCR reactions and the corresponding PCR program are displayed in the Tables below. Annealing temperatures of primer pairs were calculated with the T.sub.m calculator tool on the NEB homepage. All PCRs were performed in a Mastercycler gradient (Eppendorf), Piko Thermo Cycler (Finnzymes) or Progene PCR cycler (Techne).

(47) TABLE-US-00004 Composition of PCR reactions with Crimson Taq polymerase 15 l 25 l final component reaction reaction concentration 5x Crimson Taq reaction 3 l 5 l 1x buffer 2 mM dNTP mix 1.5 l 2.5 l 200 M each 10 M primer (each) 0.3 0.5 l 0.2 M each template DNA variable variable variable Crimson Taq DNA 0.15 l 0.25 l 0.025 U/l polymerase nuclease-free water to 15 l to 25 l

(48) TABLE-US-00005 PCR program for reactions with Crimson Taq polymerase step temperature ( C.) time initial denaturation 95 1 min 30-35 cycles 95 22 sec 45-68 35 sec 68 1 min/kb final extension 68 5 min hold 4-10

(49) TABLE-US-00006 Composition of PCR reactions with Phusion or Q5 polymerase 25 l 50 l final component reaction reaction conc. 5x Phusion HF/Q5 reaction 5 l 10 l 1x buffer 2 mM dNTPs mix 2.5 l 5 l 200 M each 10 M primer (each) 0.5 l 1 l 200 M each template DNA variable variable variable Phusion/Q5 High-Fidelity 0.25 l 0.5 l 0.02 U/l DNA polymerase nuclease-free water to 25 l to 50 l

(50) TABLE-US-00007 PCR program for reactions with Phusion or Q5 polymerase step temperature ( C.) time initial denaturation 98 C. 1 min 15-35 cycles 98 C. 10 sec 50-72 C. 20 sec 72 C. 15 sec/kb (for plasmids) 30 sec/kb (for gDNA) final extension 72 C. 5 min hold 4-10 C.
Fusion PCR

(51) A fusion PCR was used for construction the ORF of HXT7-N370F for recombinational cloning. In the first step two overlapping fragments of HXT7 were amplified in two separate Q5 PCR reactions with p426H7_HXT7 as a template. The PCR reactions were separated in a 1.5% agarose gel and the correct fragments purified from the corresponding gel pieces. Equal molar amount of both fragments (20 ng minimum) were used in a Q5 PCR reaction without primers. This PCR reaction was run for 6 cycles, before 1 l of forward and reverse primer (from 10 M stocks) were added. The reaction was then run for another 20 cycles.

(52) Error-Prone PCR (epPCR)

(53) For generation of random mutagenized ORFs of GAL2 the GeneMorph II Random Mutagenesis Kit (Agilent Technologies) was applied. The manufacturer's protocol has been followed. The PCR reaction has been run for 33 cycles. The amount of template DNA has been varied to achieve different mutation rates (see Table below) The analysis of epPCR-products revealed that the desired amplification specifications have been met. The PCR fragments were purified and used as templates for a Phusion PCR reaction to extend the fragments' ends with homologous overhangs.

(54) TABLE-US-00008 Amount of template DNA used in different epPCRs and amount of template amplification mutation rate (acceptable range) desired found medium (4.5-9 mut/kb) 205 ng (100-500 ng) 10-100 45 high (9-16 mut/kb) 23 ng (0.1-100 ng) 100-10000 250
Agarose Gel-Electrophoresis for DNA or RNA Separation

(55) Fragments in DNA or RNA samples were separated by size using agarose gels with concentrations ranging from 0.7 to 2.0% (w/v) agarose (Sambrook and Russell, 2001). 1 TAE-buffer was used for preparation of gels and as running buffer. The GeneRuler 1 kb DNA Ladder (Fisher Scientific) was used for sizing of the DNA fragments. DNA samples were mixed with Vol of 6DNA loading dye before loading onto the gel. RNA samples were mixed with the same volume 2RNA loading dye, incubated at 96 C. for 10 min and stored on ice prior to loading. Gels were run at up to 6-10 V/cm for 30 to 45 minutes depending on current, gel percentage and expected fragment sizes. DNA and RNA were visualized by UV-light (254 nm) after incubation of the gel in an ethidium bromide bath.

(56) DNA-Purification and DNA-Extraction from Agarose Gels

(57) To purify DNA (e.g. from PCR reactions or after restriction digestions) and to extract DNA from agarose gels the NucleoSpin Extract II-Kit (Macherey-Nagel) was used according to manufacturer's instructions.

(58) DNA Sequencing

(59) Sequencing of DNA samples was done by GATC Biotech AG (Konstanz, Germany). The samples contained 30-100 ng/l (plasmids) or 10-50 ng/l (PCR products) of DNA. Suitable primers (10 M) were sent to GATC Biotech together with the DNA sample.

(60) Transformation of E. coli

(61) E. coli cells were transformed by electroporation according to the protocol of Dower (Dower et al., 1988) and Wirth (Wirth, 1989) using a Bio-Rad Gene Pulser. DNA (from E. coli or yeast DNA preparations) was added to the frozen competent E. coli cells and the sample was incubated and thawed for 10 min on ice. The cell suspension was then transferred to electroporation cuvettes and directly pulsed. The Bio-Rad Gene Pulser was set to a voltage of 2.5 kV per cm, a resistance of 200 and a capacity of 25 F. Immediately after the pulse the cells were mixed with 1 ml of pre-warmed SOC medium and transferred to an eppendorf tube. The cells were incubated at 37 C. for 45 min at 600-800 rpm in a Thermomixer (Eppendorf) before plating on selective LB agar plates containing kanamycin or ampicillin. In case the cells were transformed with a HXT7-coding plasmid, the incubation was performed at room temperature for 4 hours without shaking or at 20-25 C. with shaking for 2 hours.

(62) Transformation of S. cerevisiae

(63) For transformation of S. cerevisiae, two different protocols of the LiAc/SS carrier DNA/PEG method from Gietz et al. (Gietz and Schiestl, 2007a, Gietz and Schiestl, 2007b) were used with small deviations. Liquid cultures were grown in suitable medium to an OD of 0.6-1.0. Centrifugation of the culture and for the washing steps were shortened to 2 minutes at 3000g. The single-stranded carrier DNA was used as a 10 mg/ml solution, allowing a volume of 54 l or 74 l of DNA in the transformation mix, respectively. The duration of the heat-shock was 35 minutes. After transformation the whole cell suspension was directly plated on the selection medium or, in case of transformations with a dominant selection marker, transferred to 5 ml of appropriate liquid medium for regeneration. After regeneration cells were pelleted, resuspended in 50-100 l medium and plated out.

(64) DNA amounts for transformations were approx. 500 ng for single plasmids, 1000 ng each for co-transformations with multiple plasmids and 2000 ng and more for integrative DNA-cassettes (e.g. for gene deletions).

(65) Codon Optimization of Genes

(66) The ORF of some genes has been codon-optimized. The codons have been adapted to the codon-usage of S. cerevisiae as determined by the preferred codons of the glycolytic genes. Described in Wiedemann et al. (Wiedemann and Boles, 2008).

(67) Plasmid Construction by Homologous Recombination (Recombinational Cloning)

(68) Plasmids were constructed in vivo by homologous recombination of suitable DNA fragments (vector backbone and insert(s)) in S. cerevisiae. For this purpose the respective vector was linearized at the site of insertion by restriction digestion. Optionally, the resulting vector backbone was purified by agarose gel-electrophoresis and subsequent gel extraction. The inserts were designed to have flanking sequences (>30 bp) homologous to the region targeted for insertion or, in case of multiple insert fragments, to each other. Inserts were amplified by PCR and could be provided with homologous sequences by using primers with corresponding 5 ends (homologous overhangs). S. cerevisiae was transformed with the DNA fragments and transformants were plated out on selective medium. Colonies were picked to inoculate selective liquid medium. DNA was isolated from these cultures and used for transformation of E. coli for plasmid separation and proliferation. Plasmids containing the gyrase inhibitor gene ccdB are toxic to most E. coli strains so ccdB-resistant strain E. coli DB3.1 was used for these plasmids. Plasmids were isolated from E. coli single-colony cultures and verified by analytic restriction digestion and DNA sequencing. Glycerol stock cultures were set up for correct clones.

(69) Genomic Gene Deletion or Insertion by Homologous Recombination

(70) For gene deletions in the genome of S. cerevisiae, marker cassettes were integrated into the respective gene by homologous recombination (Carter and Delneri, 2010, Gilldener et al., 1996, Sauer, 1987). The marker cassettes were amplified by PCR using primers with 5 ends homologous to the target gene to enable site-directed insertion. The cassettes are composed of a dominant marker gene (kanMX4/G418, hphNT1/Hygromycin B, natNT2/clonNAT), flanked by a promoter (pTEF) and a terminator (tTEF, tCYC1 and tADH1, respectively) and loxP sites. These sites allow excision of the genome-integrated marker cassettes by the cre recombinase, which clears the marker for another round of gene deletion. After transformation of S. cerevisiae with a deletion cassette, cells were plated out on selective medium and replica plated on the same medium once. Single colonies were streaked out again to obtain single clones, which were then picked and grown in selective medium. The DNA was isolated from these cultures and the correct integration was confirmed by PCR with different primer combinations. Primers for confirmation are termed as seen in the Table below. Glycerol stock cultures were set up for correct clones.

(71) TABLE-US-00009 Nomenclature of primers used for confirmation of genomic integration by PCR name position direction A1 upstream of the integration site downstream A2 within the region that gets replaced by the upstream integration A3 within the region that gets replaced by the downstream integration A4 downstream of the integration site upstream K2 within the deletion cassette/gene that is upstream integrated K3 within the deletion cassette/gene that is downstream integrated

(72) For recycling of the marker, cells were transformed with a plasmid encoding the cre recombinase under control of the galactose-inducible GALJ-promotor (pSH47 or pNatCre). After brief induction of the recombinase, cells were selected for loss of the dominant marker by replica plating. Since full expression of the recombinase is lethal in hxt.sup.0 strains, basal expression of the recombinase under non-inducing conditions was used for these strains. Removal of the cassette was again controlled by PCR (see above). The integration of gene cassettes for overexpression of genes was done accordingly. Within these cassettes only the dominant marker is flanked by loxP sites and is excised, the rest of the cassette remains in the genome.

(73) TABLE-US-00010 Listofprimers Primer Sequence(5-3) name [SEQIDNO.] Description Gal2_M435I_ GGTGCCGGTAACTGT forwardprimerfor fw ATCATTGTCTTTACC mutagenesisofM435 TG toIinGAL2 [SEQIDNO.2] Gal2_M435I_ ACAGGTAAAGACAAT reverseprimerfor rev GATACAGTTACCGGC mutagenesisofM435 ACC toIinGAL2 [SEQIDNO.3] GAL2_for AACACAAAAACAAAA forwardprimerfor AGTTTTTTTAATTTT GAL2amplification AATCAAAAAATGGCA GTTGAGGAGAACAA [SEQIDNO.4] GAL2_rev GAATGTAAGCGTGAC reverseprimerfor ATAACTAATTACATG GAL2amplification ACTCGAGTTATTCTA GCATGGCCTTGTACC [SEQIDNO.5] Gal2_N376Y_ GTCATTGGTGTAGTC forwardprimerfor fw TACTTTGCCTCCACT mutagenesisofN376 TTCTTTAG toYinGAL2 [SEQIDNO.6] Gal2_N376Y_ GAAAGTGGAGGCAAA reverseprimerfor rev GTAGACTACACCAAT mutagenesisofN376 GACAATGG toYinGAL2 [SEQIDNO.7] GAL2T354A TTATTTTTTCTACTA forwardprimerfor fw CGGTGCCGTTATTTT mutagenesisofT354 CAAGTCAG toAinGAL2 [SEQIDNO.8] GAL2T354A GACTTGAAAATAACG reverseprimerfor rv GCACCGTAGTAGAAA mutagenesisofT354 AAATAATTG toAinGAL2 [SEQIDNO.9] GAL2V71I GTCTGAATATGTTAC forwardprimerfor fw CATTTCCTTGCTTTG mutagenesisofV71to TTTGTG IinGAL2 [SEQIDNO.10] GAL2V71I AAACAAAGCAAGGAA reverseprimerfor rv ATGGTAACATATTCA mutagenesisofV71to GACATG IinGAL2 [SEQIDNO.11] GAL2- TTGAGAAGGTTTGGT forwardprimerfor M107K_fw AAGAAACATAAGGAT mutagenesisofM107 GGTACC toKinGAL2 [SEQIDNO.12] GAL2- ACCATCCTTATGTTT reverseprimerfor M107K_rev CTTACCAAACCTTCT mutagenesisofM107 CAAAAAGTCTG toKinGAL2 [SEQIDNO.13] GAL2_V239L_ AGCTATTCGAACTCA forwardprimerfor fw CTTCAATGGAGAGTT mutagenesisofV239 CCATTAGG toLinGAL2 [SEQIDNO.14] GAL2_V239L_ TGGAACTCTCCATTG reverseprimerfor rev AAGTGAGTTCGAATA mutagenesisofV239 GCTCTTTG toLinGAL2 [SEQIDNO.15] GAL2_L558S_ GGTAATAATTACGAT forwardprimerfor fw TCAGAGGATTTACAA mutagenesisofL558 CATGACG toSinGAL2 [SEQIDNO.16] GAL2_L558S_ ATGTTGTAAATCCTC reverseprimerfor rev TGAATCGTAATTATT mutagenesisofL558 ACCTCTTC toSinGAL2 [SEQIDNO.17]
Methods for Cell Cultivation and Fermentation Experiments
Spectrophotometrical Determination of Cell Density

(74) The cell concentration in a liquid culture was quantified spectrophotometrically by measuring the optical density at 600 nm (OD.sub.600). Samples of the cell culture or dilutions thereof were placed in a polystyrene (PS) cuvette and analysed in a Ultrospec 2100 pro spectrophotometer (GE Healthcare, USA) at 600 nm.

(75) Glycerol Stock Cultures

(76) For long time storage of specific strains and plasmid-containing E. coli glycerol stock cultures were prepared. For this purpose stationary cultures of S. cerevisiae or growing cultures of E. coli were mixed 1:1 with 50% (v/v) glycerol and stored at 80 C.

(77) Semi-Solid Agar Cultivation

(78) The Semi-solid agar method of cultivation was chosen to expand the plasmid cDNA library (in E. coli). By this method representational biases that can occur during growth in liquid culture can be minimized. Incubation is done at 30 C. helping to stabilize unstable clones (Hanahan et al., 1991, Sassone-Corsi, 1991). The protocol can be found at Life technologies website. In brief, 2 concentrated LB medium is mixed with 3 g/l SeaPrep agarose while stirring, autoclaved and cooled down to 37 C. The antibiotic and 4.Math.10.sup.5 to 6.Math.10.sup.5 (per 450 ml medium) are added to the medium and mixed for 2 minutes. The bottles are then incubated in an ice-bath at 0 C. for 1 hour and then gently transferred to 30 C. for 40-45 h of incubation (without disturbance). After growth the cells can be pelleted from the semi-solid agar by centrifugation at 10400g.

(79) Serial Dilution Spot Assays (Drop Tests)

(80) For easy comparison of growth of different S. cerevisiae strains under various growth conditions a serial dilution spot assay was performed. Cells were grown in liquid culture to exponential phase in appropriate medium, collected by centrifugation (2000g, 2 min), washed twice with sterile water and then resuspended to an OD.sub.600 of 1.0 in selective medium without carbon source. From this cell suspension a ten-fold serial dilution was prepared in selective medium (four dilution steps). 6 l of each cell suspension were spotted on plates of the media to be examined and allowed to dry. Plates were incubated at 30 C.

(81) Aerobic Batch Fermentations

(82) Aerobic batch fermentations were done in shake flasks of varying sizes (volume 5-10 of the culture volume) on rotary shakers (150-180 rpm) usually at 30 C. The evolutionary engineering was done as a serial aerobic batch fermentation (details see below)

(83) Anaerobic Batch Fermentations

(84) For anaerobic batch fermentations, shake flasks were sealed with a rubber plug and a fermentation lock. The volume of the flasks was matching the culture volume of 100 ml. The cultures were stirred continuously with 120 rpm on a magnetic stirrer at 30 C. In this work the fermentations were done at OD.sub.600=10. For this purpose grown cells were harvested and set to an OD.sub.600 of 20 in 50 ml fermentation medium without carbon source. To start the experiment this cell suspension was added to the prepared flasks containing 50 ml fermentation medium supplemented with 2 concentrated carbon source. Samples for determination of cell concentration and for HPLC analysis were taken through an inserted, sterile needle and syringe.

(85) Anaerobic Fermentations in a Fermenter

(86) Some fermentations with industrial S. cerevisiae strains were conducted in an Infors Multifors fermenter (21.41) with a working volume of 800 ml and equipped with temperature, pH, O2 and CO2 sensors. The fermenter was filled with 530 ml of concentrated fermentation medium (without carbon source and supplements) and then autoclaved. Prior to the start of the fermentation, 250 ml concentrated carbon source solution, 100 l/l Antifoam 204 and the other supplements (vitamins, trace elements and antibiotics) were added to the concentrated medium. The fermenter was purged with nitrogen gas to initially create anaerobic conditions. A feed of nitrogen gas to the head space (0.4 l/min) was applied during the experiment to maintain anaerobic conditions. The gas outlet was cooled to condense and return vapor and then piped through a gas washing bottle. The culture was stirred with 300 rpm, temperature kept at 35 C. and pH kept at 5.0 by automated addition of 2M KOH or 2M H.sub.2PO.sub.4. The inoculum of cells (in 20 ml fermentation medium) was added when all set parameters were reached and constant. During the fermentations samples were taken for cell dry weight determination and HPLC analysis. Iris 5.2 Software (Infors) was used to operate the fermenter and monitor the experiment.

(87) Determination of Cell Dry Mass

(88) To determine cell dry mass 5-10 ml of a liquid culture were vacuum filtered through a pre-washed and dried (as described below) filter (nitrocellulose, pore size 0.45 m) and subsequently washed twice with ddH2O. The filters were dried in a microwave oven at 120-150 W for 15 min and left to cool and dry further in a desiccator for another 15 min. The filters were weighted prior to filtering and after drying to measure the cell dry weight. The method described has been adapted from Ask et al. (Ask et al., 2013).

(89) Evolutionary Engineering

(90) The evolutionary engineering was done as an serial aerobic batch fermentation. Transformants were first inoculated in selective SCE.sub.2 to obtain biomass and then grown in selective SC and SM medium with 20 g/l xylose to adapt the strain to xylose utilization. After these initial cultures the cells were switched to medium with 10 g/l xylose and increasing concentrations of glucose to apply an evolutionary pressure. When they were reaching late exponential to early stationary phase cells were harvested by centrifugation (2000g, 2 min) and transferred to fresh medium to an OD.sub.600 of 0.2. Glucose concentrations were increased every time an adaptation could be seen or growth was not negatively influenced by the current glucose concentration. To all media, liquid and solid, G418 was added to select for the AFY10 strain background. Liquid media additionally contained 0.5 g/l 2-deoxy-D-glucose (2-DOG) to suppress formation of suppressor mutants of the hxk.sup.0 phenotype. hxk.sup.0 strains, but not wild type strains, are resistant to 2-DOG (Subtil and Boles, 2012). Lack of glucose-consumption has been confirmed by streaking out culture samples on glucose-media-plates and also by HPLC analysis.

(91) Metabolite Analysis by HPLC

(92) For analysis of metabolites cell-free samples (5-10 min, 4 C., 16000g) were mixed with 1/9 volumes of 50% (w/v) 5-sulfosalicylic acid and centrifuged (5-10 min, 4 C., 16000g). The supernatant was analyzed in an UHPLC+ system by Thermo Scientific (Dionex UltiMate 3000) equipped with a HyperREZ XP Carbohydrate H.sup.+8 m column and a refractive index detector (Thermo Shodex RI-101). Separation was carried out at column temperature of 65 C. with 5 mM sulfuric acid as mobiles phase with a flow rate of 0.6 ml/min. Chromeleon 6.80 software was used to control the system and to analyze the data. Five standards (mixtures of D-glucose, D-xylose, xylitol, acetate, glycerol and ethanol with concentrations of 0.01-3% (w/v)) were analyzed for quantification of the different compounds.

(93) Sugar Uptake Assays

(94) Sugar uptake assays were done as described by Bisson et al. (Bisson and Fraenkel, 1983) with modifications according to Walsh et al. (Walsh et al., 1994).

(95) Transformants of strain EBY.VW4000 were grown in selective YEPE to an OD of 1.1-1.6, harvested by centrifugation and washed twice in ice-cold uptake-buffer (RT, 3 min, 3000g). Cells were kept on ice from here on. The cell pellet was resuspended in ice-cold uptake-buffer to a concentration of 60 mg.sub.ww/ml and aliquoted to 110 l. One cell suspension aliquot and one sugar solution were incubated in a water bath at 30 C. for 4-5 min. 100 l of the cell suspension were pipetted to the sugar solution (50 l), mixed briefly by pipetting and incubated for 5 (D-[U-.sup.14C]-glucose) or 20 sec (D-[1-.sup.14C]-xylose). The uptake reaction was stopped by transferring 100 l of the mixture into 10 ml ice-cold quenching-buffer, which was immediately filtered through a Durapore membrane filter (0.22 m pore size, Millipore). The filter was washed twice with 10 ml ice-cold quenching-buffer, transferred to a scintillation vial containing 4 ml scintillation cocktail (Rotiszint eco plus, Roth) and shaken thoroughly. Additionally to this filter sample (cpm.sub.filter), 10 l of each reaction were transferred directly to a scintillation vial with 4 ml scintillation cocktail for determination of the total counts in the reaction (cpm.sub.total). To determine a value for sugar that is bound unspecifically to the cell surface or the filter (cpmblank) a few samples of 33,3 l sugar solution and 66,6 l cell suspension were mixed in 10 ml icecold quenching buffer and treated as described above. Radioactivity of all vials was analysed in an Wallac 1409 liquid scintillation counter.

(96) Stocksolutions of 2M, 500 mM or 20 mM glucose or xylose (in H.sub.2O) were used to prepare the sugar solutions for the assays (threefold of the desired concentration in the uptake reaction (S, substrate concentration), 50 l aliquots). Uptake was measured at sugar concentrations 0.2, 1, 5, 25 and 100 mM for glucose and 1, 5, 25, 66, 100, 200 and 500 mM for xylose. Inhibition of xylose uptake by glucose was measured at 25, 66 and 100 mM xylose with additional 25 and 100 mM unlabelled glucose. Sugar solutions contained 0.135 to 0.608 Ci of D-[U-.sup.14C]-glucose (290-300 mCi/mmol) or D-[1-.sup.14C]-xylose (55 mCi/mmol) (American Radiolabeled Chemicals Inc., St. Louis, Mo., USA).

(97) Data of the uptake assays were used for following calculations:

(98) The amount of sugar (A.sub.sugar in nmol) taken up during the incubation time (t, in seconds) at a certain sugar concentration (S, in mM):
A.sub.sugar=((cpm.sub.samplecpm.sub.blank)/(cpm.sub.total.Math.10)).Math.S.Math.100 l

(99) Transport velocity (in nmol.Math.min.sup.1.Math.mg.sub.ww.sup.1) calculated per milligrams of cell (m, in mg.sub.ww):
V=(A.sub.sugar.Math.60 s)/(t.Math.m)

(100) Calculation of K.sub.m (Michaelis constant), V.sub.max (maximal initial uptake velocity) and K.sub.i (inhibitor constant for competitive inhibition) was done by nonlinear regression analysis and global curve fitting in Prism 5 (GraphPad Software, Inc.) with values of three independent measurements.

(101) Bioinformatic Methods

(102) DNA sequences were obtained from the Saccharomyces Genome Database (SGD, (Cherry et al., 2012)). Sequence alignments for transporter proteins were conducted using the PRALINE multiple alignment server ((Simossis and Heringa, 2005)) with standard settings plus PHOBIUS transmembrane structure prediction (KAil et al., 2004). Phylogenetic trees were calculated from PRALINE alignments with ClustalW2 phylogeny (Larkin et al., 2007) and visualized with Phylodendron software (http://iubio.bio.indiana.edu/soft/molbio/java/apps/trees/). Similarities and identities between protein sequences were calculated from PRALINE alignments using SIAS (http://imed.med.ucm.es/Tools/sias.html). Figures for sequence alignments were created with ALINE software (Bond and Schuttelkopf, 2009).

Example 1

N376Y

(103) Mutants of GAL2 were created using error-prone PCR (epPCR) and then screened in AFY10X as described in Farwick et al. (2014). This method is faster than evolutionary engineering experiments and more often generates multiple mutations per ORF that might have a unique combined effect. On the downside, there might be more silent mutations among the increased number of mutations which might aggravate the identification of the relevant mutations in each clone. The GAL2 ORF was amplified in an epPCR with short amplification primers, applying various mutation rates, and the purified fragments then again amplified for recombinational cloning in a second PCR. Functional plasmids were in vivo assembled from PCR fragments and linearized p426H7 vector directly in the screening strain AFY10X. Transformants were plated out on selective SCE.sub.2 plates and subsequently screened. Colonies were tested for improved growth on media with 1% xylose and increasing amounts of glucose (up to 5%) by replica-plating. Seven clones were selected and grown in SCE.sub.2 ura.sup. leu.sup.+G418+2-DOG and samples of these cultures streaked out on plates for comparison and to obtain single colonies. Five of these seven clones were confirmed to grow on xylose glucose media. The plasmids of two clones were successfully isolated as described (yeast DNA was digested with EcoRI) and sequenced. The sequencing revealed multiple amino acid mutations as can be seen in Table 1.

(104) TABLE-US-00011 TABLE 1 Analysis of two clones of the epPCR-screening. growth on medium (xylose + clone glucose) mutations Gal2_ep3.1 1 + 5 M107K V239L N376Y M435I L558S Gal2_ep3.3 1 + 3 T219S M255L R267C L299S N346I E528D

Example 2

M435

(105) 2.1 Investigation of Gal2_ep3.1 Mutations

(106) Based on the tests for functionality of Gal2_N376Y, it could be determined that it is not N376Y alone which is responsible for the properties of Gal2_N376Y. Therefore the further mutations of Gal2_ep3.1 should be investigated separately. N376Y is responsible for the reduced affinity for glucose and galactose. Thus the question is which amino acid is responsible for the improved uptake of xylose. For this reason the replacements M107K, V239L, M435I and L558S were made in the vector p426_Gal2_N376Y and then this vector was transformed into the screening strains EBY.VW4000 and AFY10 to test their properties with the aid of growth tests. It is expected that one or more amino acid exchanges determine the improved growth on xylose of Gal2_ep3.1. The position M435I is the only position to be examined, which is located in a transmembrane domain (TM10). The other positions are located in the loops or in the C-terminus. Apart from that the mutation N376Y can also be found in a transmembrane domain (TM8), whereas this mutation is located close to the sugar binding area. However M435I is located close to the upper membrane level.

(107) 2.2 Test for Functionality of the Ep3.1 Mutations

(108) The screening strains EBY.VW4000 and AFY10 with the vectors which contained the respective mutated GAL2-sequence, were tested for their growth behavior. Therefore the constructs were tested in EBY.VW4000 on galactose, as well as glucose with different concentrations. None of the mutants showed significant growth. Because all of the constructs got the N376Y mutation, it was supposed that the effect to reduce the affinity for hexoses, was not affected by the additional mutations (FIG. 1).

(109) The constructs were tested in AFY10 on xylose media, whereby a concentration of 0.2% was also chosen to test the transporters' xylose affinity. Furthermore the competitive inhibition by glucose was tested with media which contained xylose and glucose. It was observed that Gal2_N376Y+M435I showed similar growth like Gal2_ep3.1 on 2% xylose, as well as on 1% xylose with 4% glucose. Certainly Gal2_N376Y+M435I seems to have a higher xylose affinity, because it was observed a better growth on 0.2% xylose and 0.2% xylose with 2% glucose in comparison to Gal2_ep3.1. Apart from that the wild type showed the best growth on low xylose concentrations without glucose. The mutants with the other ep3.1 mutations were not able to grow on xylose (FIG. 2).

(110) 2.3 Growth and Fermentation of Gal2 N376Y+M435I

(111) To investigate the growth difference and the xylose consumption of the mutants in comparison to the wild type and the error prone mutant, an anaerobic growth test in liquid media was carried out. For this purpose AFY10 cells were co-transformed with the vector with the respective GAL2 sequence and YEp181_pHXT7-opt.XI_Clos and inoculated in SCE-ura-leu+G418 to have an OD.sub.600 of 0.6 in 30 ml SC-ura-leu with 0.6% xylose or with 0.6% xylose with 2% glucose. The OD.sub.600 was measured regularly during five days, the growth rate was determined and the metabolites of the samples were examined, whereby the samples were diluted 1:5 for this reason.

(112) It was shown that the transformants with Gal2_N376Y+M435I with a growth rate of 0.014 h.sup.1 in 0.6% xylose resp. 0.015 h.sup.1 in 0.6% xylose with 2% glucose had the best growth in comparison to the wild type and the error prone mutant, that had only growth rates of 0.006 to 0.007 h.sup.1. The behavior of Gal2_N376Y+M435I in both of the used media was similar, which shows that there were no impairments by glucose. The transformant with Gal2_wt showed the biggest differences, because it could grow on media without glucose much better (OD.sub.600 tend: 0,86 [Xyl] und 0,52 [Xyl+Glu]). This effect can be explained by the competitive inhibition of xylose uptake by glucose. Gal2_ep3.1 did not show significant differences regarding both media. Certainly the growth of this mutant is lower than the growth of the wild type in media with 0.6% xylose, but showed a better growth than the wild type with 2% glucose. Furthermore the growth of the transformants in general was low, because of the long generation time of AFY10, as well as xylose as the carbon source, which was very low concentrated and the required adaptation time to the new media.

(113) Furthermore several metabolites were measured, especially the concentrations of xylose and glucose. The concentration of glucose in the media with 0.6% xylose and 2% glucose did not change as expected (data not shown), because AFY10 cannot metabolize glucose. The decrease of xylose concentration in the supernatant of the different strains correlates with the growth behavior. Therefore the highest consumption of xylose could be observed for Gal2_N376Y+M435I, because it showed the highest growth rate. The xylose concentration decreased during the fermentation to 0.35% resp. 0.37%. The transformants with Gal2_wt and Gal2_ep3.1 consumed less xylose, which was analogous to their growth behavior. Because of glucose inhibition, Gal2_wt in the media with 2% glucose consumed little xylose, whereas the xylose concentration decreased only to 0.53% (FIG. 3).

Example 3

M435/N376

(114) 3.1 Combination of M435I with Various Amino Acids at Position N376

(115) In example 2 it is shown that the exchange of amino acid M435 to isoleucine could significantly improve growth of strains with the Gal2_N376Y mutation on glucose-xylose mixtures. To demonstrate whether M435 alone has an impact on sugar uptake, serial dilution drop tests were performed. It could be shown that on low xylose concentrations (2 g/L) growth of strains with a Gal2_N376F exchange was better than that of Gal2_N376Y+M435I. To find out the effect of M435I alone and in combination with N376F, corresponding mutants were constructed. To determine the kinetic properties of the various mutants, sugar uptake measurements with radioactively labelled xylose were performed.

(116) 3.2 Growth of Mutants with Gal2_M435I with Hexoses, Xylose and Sugar Mixtures

(117) To see the effect of only the exchange of M435I on the uptake of glucose and galactose, the exchange was introduced into wild-type Gal2 and characterized by serial dilution drop tests with strain EBY.VW4000. As can be seen in FIG. 4, the M435I exchange had no effect on the uptake of galactose. Uptake of glucose is strongly reduced. As well with 2 g/l as also with 20 g/l glucose growth is much slower with the M435I mutation.

(118) To test the effect of M435I on the uptake of xylose, Gal2_M435I together with construct YEp181_pHXT7-optXI_Clos was transformed into competent AFY10 cells and a drop test was performed. Due to the exchange of M435I also the uptake of xylose is reduced. No growth could be seen with 2 g/l xylose and only slow growth on 20 g/L xylose. No growth was visible on xylose-glucose mixtures (FIG. 5). These results demonstrate that M435I improves growth only in combination with other mutations, e.g. a mutation at position N376.

(119) 3.3 Growth of Mutants with Gal2_N376F+M435I with Xylose and Sugar Mixtures

(120) To characterize the different constructs which all were verified by sequencing they were transformed together with vector YEp181_pHXT7-optXI_Clos into competent AFY10 cells and drop tests were performed.

(121) Comparing growth of cells with N376Y+M435I with the other strains it becomes obvious that they grow faster on high concentrations of xylose (FIG. 6). As well with 20 g/l xylose as also with 10 g/l xylose plus 40 g/l glucose the N376Y+M435I strain shows the best growth. On low xylose concentrations cells containing the N376F+M435I combination grow the best. The additional mutation of M435I seems to improve N376F but only a little bit.

(122) 3.4 Kinetic Properties of N376/M435I Transporter Variants

(123) Xylose uptake of Gal2_N376F, N376F+M435I, N376Y und N376Y+M435I was characterized with a sugar uptake assay. The relevant plasmids were transformed in strain EBY.VW4000. Cells were cultivated in SCM-ura until they reached the exponential growth phase (OD.sub.6000.6). Then cells were harvested, concentrated to the same OD.sub.600 (OD.sub.600 35-40), separated into different aliquots and stored on ice.

(124) Three uptake assays (in tripicates) were performed with different concentrations of radioactively labelled xylose. Data were collected and fittet with GraphPad Prism according to Michaelis Menten (FIGS. 7 and 8).

(125) Mutation N376Y leads to a strong reduction in the uptake activity compared to the wild-type Gal2. However, N376Y+M435I shows a strongly improved xylose uptake rate, even significantly higher than the wild type.

(126) Surprisingly and in contrast to the growth assays (FIG. 6), N376F+M435I seems to have a reduced uptake rate compared to the wild type and to N376F mutant alone.

(127) From the fitted data the K.sub.m-values and maximal uptake rates V.sub.max were determined (Table 2).

(128) TABLE-US-00012 TABLE 2 From the uptake assays K.sub.m and V.sub.max were calculated for Gal2_variants N376F, N376Y, N376Y + M435I, N376F + M435I and Gal2 WT with standard deviations, according to Michaelis-Menten graph with GraphPad Prism. Standard Standard V.sub.max deviation K.sub.m deviation N376Y 11.92 2.777 370.4 176.7 N376Y + M435I 99.61 28.76 711.4 314.1 N376F 25.86 2.458 108.9 35.73 N376F + M435I 13.81 2.280 110.8 48.94 Gal2 WT 63.43 11.58 617.4 176.2

(129) It can be seen from Table 2 that V.sub.max of N376Y is strongly increased by the additional M435I mutation, even much higher than in the wild type. In the case of N376F V.sub.m is reduced by M345I. The K.sub.m-values of N376F and N376F+M435I look very similar. Due to the high standard deviations it cannot be concluded that N376F+M435I has a higher affinity than N376F alone. The faster growth (FIG. 6) would indicate this. It seems that at least in vivo the additional M345I mutation improves growth of cells with the N376F at low xylose concentrations (2 g/l) (FIG. 6).

(130) The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

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