Patent Grant
8063194
This application is a National Stage Application of International Application Number
PCT/EP2007/010668, filed Dec. 7, 2007; which claims priority to German Application No. 10 2006 060 381.8, filed Dec. 20, 2006, which are incorporated herein by reference in their entirety.
The beer, wine and baking yeast Saccharomyces cerevisiae has already been used for centuries for the production of bread, wine and beer owing to its characteristic of fermenting sugar to ethanol and carbon dioxide. In biotechnology, S. cerevisiae is used particularly in ethanol production for industrial purposes, in addition to the production of heterologous proteins. Ethanol is used in numerous branches of industry as an initial substrate for syntheses. Ethanol is gaining increasing importance as an alternative fuel, due to the increasingly scarce presence of oil, the rising oil prices and continuously increasing need for petrol worldwide.
In order to make possible a favourably-priced and efficient bioethanol production, the use of lignocellulose-containing biomass, such as for example straw, waste from the timber industry and agriculture and the organic component of everyday household waste, presents itself as an initial substrate. Firstly, said biomass is very convenient and secondly is present in large quantities. The three major components of lignocellulose are lignin, cellulose and hemicellulose. Hemicellulose, which is the second most frequently occurring polymer after cellulose, is a highly branched heteropolymer. It consists of pentoses (L-arabinose, D-xylose), uronic acids (4-O-methyl-D-glucuronic acid, D-galacturonic acid) and hexoses (D-mannose, D-galactose, L-rhamnose, D-glucose) (see
In order to be able to use pentoses for fermentations, these must firstly enter the cell through the plasma membrane. Although S. cerevisiae is not able to metabolize D-xylose, it can uptake D-xylose into the cell. However, S. cerevisiae does not have a specific transporter. The transport takes place by means of the numerous hexosetransporters. The affinity of the transporters to D-xylose is, however, distinctly lower than to D-glucose (Kotter and Ciriacy, 1993). In yeasts which are able to metabolize D-xylose, such as for example P. stipitis, C. shehatae or P. tannophilus (Du Preez et al., 1986), there are both unspecific low-affinity transporters, which transport D-glucose, and also specific high-affinity proton symporters only for D-xylose (Hahn-Hagerdal et al., 2001).
In earlier experiments, some yeasts were found, such as for example Candida tropicalis, Pachysolen tannophilus, Pichia stipitis, Candida shehatae, which by nature ferment L-arabinose or can at least assimilate it. However, these yeast lack entirely the capability of fermenting L-arabinose to ethanol, or they only have a very low ethanol yield (Dien et al., 1996). Moreover, very little is yet known about the uptake of L-arabinose. In the yeast C. shehatae one assumes a proton symport (Lucas and Uden, 1986). In S. cerevisiae, it is known from the galactose permease Gal2 that it also transports L-arabinose, which is very similar in structure to D-galactose. (Kou et al., 1970).
Alcoholic fermentation of pentoses in biotechnologically modified yeast strains of S. cerevisiae, wherein inter alia various genes of the yeast strain Pichia stipitis were used for the genetic modification of S. cerevisiae, was described in recent years particularly in connection with the fermentation of xylose. The engineering concentrated here particularly on the introduction of the genes for the initial xylose assimilation from Pichia stipitis, a xylose-fermenting yeast, into S. cerevisiae, i.e. into a yeast which is traditionally used in the ethanol production from hexose (Jin et al. 2004).
Jeppson et al. (2006) describe xylose fermentation by S. cerevisiae by means of the introduction of a xylose metabolic pathway which is either similar to that in the yeasts Pichia stipitis and Candida shehatae, which naturally use xylose, or is similar to the bacterial metabolic pathway.
Katahira et al. (2006) describe sulphuric acid hydrolysates of lignocellulose biomass such as wood chips, as an important material for the production of fuel bioethanol. In this study, a recombinant yeast strain was constructed, which is able to ferment xylose and cellooligosaccharides. For this, various genes were integrated into this yeast strain and namely for the inter-cellular expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae and for the presentation of beta-glucosidase from Aspergillus acleatus on the cell surface. In the fermentation of sulphuric acid hydrolysates of wood chips, xylose and cellooligosaccharides were fully fermented by the recombinant strain after 36 hours.
Pitkanen et al. (2005) describe the obtaining and characterizing of xylose chemostat isolates of a S. cervisiae strain, which over-expresses genes of Pichia stipitis coding for xylose reductase and xylitol dehydrogenase and the gene which codes endogenous xylulokinase. The isolates were obtained from aerobic chemostat cultures on xylose as the single or major carbon source. Under aerobic conditions on minimal medium with 30 g/l xylose, the growth rate of the chemostat isolates was 3 times higher than that of the original strain (0.15 h−1 compared with 0.05 h−1). The xylose uptake rate was increased almost two-fold. The activities of the key enzymes of the pentose phosphate metabolic pathway (transketolase, transaldolase) were increased two-fold, whilst the concentrations of their substrates (pentose-5-phosphates, sedoheptulose-7-phosphate) were lowered accordingly.
Becker and Boles (2003) describe the engineering and the selection of a laboratory strain of S. cerevisiae which is able to use L-arabinose for growth and for fermenting it to ethanol. This was possible due to the over-expression of a bacterial L-arabinose metabolic pathway, consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous over-expression of yeast galactose permease transporting L-arabinose in the yeast strain. Molecular analysis of the selected strain showed that the predetermining precondition for a use of L-arabinose is a lower activity of L-ribulokinase. However, inter alia, a very slow growth is reported from this yeast strain (see
Therefore, a need exists in the art for specific pentose transporters, in particular L-arabinose transporters, which allow to specifically take up pentoses, in particular L-arabinose, into cells, such as yeast cells, and therefore to promote a utilization and fermentation of pentoses, in particular L-arabinose.
It is therefore an object of the present invention to provide specific pentose transporters, such as arabinose transporters.
The problem is solved according to the invention by providing polypeptides which have an in vitro and/or in vivo pentose transport function, and variants and fragments thereof.
In particular, the polypeptide according to the invention is selected from the group of
Preferably, the polypeptide according to the invention comprises a fragment of at least 200 or 300 continuous amino acids according to SEQ ID NO:1. Here, such a fragment is characterized in that it has an in vitro and/or in vivo pentose transport function.
In a preferred embodiment, a polypeptide according to the invention comprises a fragment of 502 amino acids which corresponds to the first 502 amino acids of SEQ ID NO:1. Such a fragment is characterized in that it has an in vitro and/or in vivo pentose transport function.
The polypeptide according to the invention preferably comprises a polypeptide which is at least 90%, preferably 95%, more preferably 99% identical to the amino acid sequence according to SEQ ID NO:1 and has an in vitro and/or in vivo pentose transport function.
Variants of the polypeptides according to the invention can also be those which have conservative amino acid substitutions or smaller deletions and/or insertions as long as these modifications do not substantially affect the in vitro and/or in vivo pentose transport function.
Polypeptides according to the invention can further comprise heterologous amino acid sequences. The skilled artisan can select suitable heterologous amino acid sequences depending on the application or use.
Preferably, the pentose is arabinose, in particular L-arabinose, so that a polypeptide according to the invention preferably has an in vitro and/or in vivo arabinose transport function, in particular an L-arabinose transport function.
The polypeptide according to the invention preferably originates from a yeast, preferably from Pichia, in particular Pichia stipitis.
The problem is further solved according to the invention by providing isolated nucleic acid molecules which code for a polypeptide according to the invention.
Preferably, a nucleic acid molecule according to the invention is at least 90%, preferably 95% and more preferably 99% identical to the nucleic acid sequence according to SEQ ID NO:2 or 3.
A nucleic acid molecule according to the invention further comprises vector nucleic acid sequences, preferably expression vector sequences. Vector nucleic acid sequences are preferably selected from sequences which are comprised from the vectors of the group consisting of YEp24, p426HXT7-6HIS, p426Met25, pYES260, pYES263, pVTU260, pVTU263, pVTL260, pVTL263. For further embodiments, see
Nucleic acid molecules according to the invention can furthermore comprise nucleic acid sequences which code for further heterologous polypeptides. The skilled artisan can select suitable heterologous nucleic acid sequences which code for the further heterologous polypeptides himself, depending on the application or use. These include for example antibiotic resistance marker sequences.
Nucleic acid molecules according to the invention preferably comprise dsDNA, ssDNA, PNA, CNA, RNA or mRNA or combinations thereof.
The problem is further solved according to the invention by providing host cells which contain at least one nucleic acid molecule according to the invention. Host cells according to the invention preferably also express said at least one nucleic acid molecule according to the invention.
A host cell according to the invention is, in particular, a fungal cell and preferably a yeast cell, such as Saccharomyces species, e.g. S. cerevisiae, Kluyveromyces sp., e.g. K. lactis, Hansenula sp., e.g. H. polymorpha, Pichia sp., e.g. P. pastoris, Yarrowia sp., e.g. Y. lipolytica.
Preferably, host cells according to the invention further contain nucleic acid molecules which code for proteins of the arabinose metabolic pathway, in particular for L-ribulokinase, L-ribulose-5-P 4-epimerase, L-arabinose-isomerase.
Preferably, these concern proteins of the bacterial arabinose metabolic pathway, in particular E. coli araB L-ribulokinase, E. coli araB L-ribulose-5-P 4-epimerase and B. subtilis araA L-arabinose-isomerase. See also
Particularly preferred host cells of this invention are cells of the strain MKY06-4P, which was deposited, under the terms of the Budapest Treaty, on 23 Aug. 2006 at the German Collection of Microorganisms and Cell Cultures, located at Inhoffenstraβe 7 B, 38124 Braunschweig, Germany, under accession number DSM 18544. See also
Further, the subject MKY06-4P deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which ma issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. During pendency of this application, access to the deposit will be afforded to one determined by the Commissioner to be entitled thereto. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it.
A preferred host cell according to this invention is a yeast cell which was modified by the introduction and expression of the genes araA (L-arabinose-isomerase), araB (L-ribulokinase) and araD (L-ribulose-5-P-4-epimerase) and in addition over-expresses a TAL1 (transaldolase) gene, as described for example by the inventors in EP 1 499 708 B1, and in addition to this contains at least one nucleic acid molecule according to the invention.
The problem is further solved according to the invention by providing antibodies or antibody fragments, comprising an immunologically active part, which binds selectively to a polypeptide according to the invention. Methods for the generation of antibodies or antibody fragments are known in the art.
The problem is further solved according to the invention by methods for the production of a polypeptide according to the invention. Such a method comprises the cultivating of a host cell according to the invention under conditions by which a nucleic acid molecule according to the invention is expressed. General methods for the generation of polypeptides by means of cell culture are known in the art.
The problem is further solved according to the invention by a kit comprising a compound which selectively binds to a polypeptide according to the invention, if applicable with further additives and instructions for use.
The compound is preferably a pentose, such as for example arabinose, and in particular L-arabinose, or a derivative of such a pentose.
The problem is further solved according to the invention by methods for identifying a compound which binds to a polypeptide according to the invention and/or modulates its activity. Such a method comprises the following steps:
Contacting a polypeptide or a cell, which expresses a polypeptide according to the invention, with a test compound, and
Determining whether the polypeptide binds to the test compound and, if applicable
Determining whether the test compound modulates the activity of the polypeptide.
The compound is preferably a pentose, such as for example arabinose, and in particular L-arabinose, or a derivative of such a pentose.
The problem is further solved according to the invention by methods for modulating the activity of a polypeptide according to the invention. Such a method comprises contacting a polypeptide or a cell, which expresses a polypeptide according to the invention, with a compound which binds to the polypeptide in a concentration which is sufficient to modulate the activity of the polypeptide.
The compound is preferably a pentose, such as for example arabinose, and in particular L-arabinose, or a derivative of such a pentose.
The problem is further solved according to the invention by methods for the production of bioethanol. Such a method according to the invention comprises the expression of a nucleic acid molecule according to the invention in a host cell according to the invention.
The polypeptides, nucleic acid molecules and host cells according to the invention are particularly preferably used for the production of bioethanol. For preferred embodiments, reference is made to
The polypeptides, nucleic acid molecules and host cells according to the invention are further particularly preferably used for the recombinant fermentation of pentose-containing biomaterial.
Specific genes of Pichia stipitis, which specifically increase the uptake of the pentose L-arabinose in S. cerevisiae, were isolated using a gene bank and integrated into the yeast strain MKY06-3P, which is then able to ferment the L-arabinose to ethanol. The screening of the relevant genes led to a novel specific L-arabinose transporter, the nucleotide- and protein sequence of which is available (see SEQ ID NOs: 1-4). For this, reference is also made to the examples and figures.
Due to the specificity of this novel transporter, after expression in existing ethanol-producing systems the uptake rate for L-arabinose can be improved, because on the one hand the competitive situation with respect to glucose is improved at high L-arabinose concentrations, and on the other hand the transport of L-arabinose becomes more efficient at low L-arabinose concentrations due to a high affinity.
Uptake of L-arabinose
In order that the pentose L-arabinose can be metabolized by S. cerevisiae, it must firstly be taken up by the cell. Only little is known with regard to this uptake. Hitherto, no genes are known in eukaryontes, which code for specific L-arabinose transporters. All hexose transporters tested for the pentose D-xylose have a much higher affinity to D-glucose than to D-xylose. For L-arabinose, a similar situation is assumed. Of all strains constructed hitherto, which can utilize pentoses (D-xylose or L-arabinose), a relatively slow growth is reported. Above all, the slow and poor uptake of the pentoses is named as a reason for this (Becker and Boles, 2003; Richard et al., 2002). In fermentations in a sugar mixture, consisting of D-glucose and D-xylose or D-glucose and L-arabinose, the sugars are not converted simultaneously. Due to the high affinity of the transporters for D-glucose, D-glucose is metabolized at first. A so-called Diauxic shift occurs. Only after the D-glucose is exhausted is the pentose converted in a second, distinctly slower growth phase (Kuyper et al., 2005a; Kuyper et al., 2005b). The absence of specific transporters for pentoses is given as an explanation.
Novel Specific L-arabinose Transporter from P. stipitis
For industrial applications, it would be ideal if the microorganism which was used could convert all the sugars present in the medium as far as possible simultaneously (Zaldivar et al., 2001). In order to achieve this, specific transporters for each sugar type would be of great benefit. None were known hitherto particularly for L-arabinose.
In this invention, the inventots succeeded in finding a specific L-arabinose transporter gene from the genome of P. stipitis with a test system (see examples). Genome fragments from P. stipitis are localized on the plasmids pAraT1 and pAraT7, which are responsible for a specific growth on L-arabinose but not on D-glucose, D-mannose or D-galactose medium. The observed low growth on D-galactose was not caused by the plasmids pAraT1 or pAraT7. This concerned only the weak growth of EBY.VW4000, the initial strain of MKY06, which was already reported by Wieczorke et al. (1999). The possibility that the obtained growth was caused by a genomic mutation in MKY06 was ruled out. After a selection for the loss of the plasmid of the P. stipitis gene bank by twice streaking on FOA medium, no further growth was established after again streaking on L-arabinose medium. Therefore, the growth originated from the plasmids of the P. stipitis gene bank (see examples). It was shown that the plasmids found code a transporter.
In a BLAST search with the recently published genome of Pichia stipitis, a 100% conformity with HGT2 was found. Due to its high homology to the high-affinity glucose transporter HGT1 of Candida albicans, HGT2 was annotated as putative high-affinity glucose transporter. When the sequence is examined with regard to the possible transmembrane domains, 12 transmembrane domains are obtained, which is typical for transporters. It is therefore surprising that it is a pentose transporter (arabinose transporter) and not a hexose transporter.
Furthermore, a multitude of experimental obstacles and difficulties had to be overcome in locating and providing the transporter according to the invention, which can also be seen in greater detail from the examples and figures.
Some reports exist with regard to the difficulties concerning heterologously expressed transporters, see on this subject Chapter 2 in the book “Transmembrane Transporters” (Boles, 2002) and the article by Wieczorke et al., 2003.
Further biomass with significant amounts of arabinose:
The arabinose transporter according to the invention is also of great importance for their utilization.
Possibilities for use of a functional and at the same time specific arabinose transporter in the yeast S. cerevisiae are on the one hand the production of bioethanol and the production of high-grade precursor products for further chemical syntheses.
The following list originates from the study “Top Value Added Chemicals From Biomass”. Here, 30 chemicals were categorized as being particularly valuable, which can be produced from biomass.
As soon as these chemicals are produced from lignocelluloses by bioconversion (e.g. fermentations with yeasts), it is important to have a specific transporter for the hemicellulose arabinose.
The present invention is further clarified in the following figures, sequences and examples, without however being restricted thereto. The cited references are fully included herewith by reference. In the sequences and figures there are shown:
The second most frequently occurring hemicellulose is a highly branched polymer consisting of pentoses, uronic acids and hexoses. The hemicellulose consists in a large proportion of the pentoses xylose and arabinose.
The initial strain for the construction of MKY06-4P was the yeast strain EBY.VW4000, in which all hexose transporter genes (HCTs) were deleted. In this strain, the endogenous transaldolase TAL1 was over-expressed by the exchange of the native promoter for the shortened HXT7 promoter (HXT7-Prom). This led to the strain MKY06. Into this strain, the plasmids p423H7araABsre (araA), p424H7araBre (araB) and p425H7araDre (araD) were transformed for the arabinose metabolism (=MKY06-3P). In addition, the plasmid p426H7-araT (araT), which codes the arabinose transporter according to the invention from Pichia stipitis was also transformed into this strain and, thus, the strain MKY06-4P was obtained. The transporter is expressed and is functionally incorporated into the plasma membrane (AraT).
Each streak concerned the MKY06-3P and in addition a plasmid from the gene bank YEpTW. As a negative control (−), instead of a plasmid of the gene bank, p426HXT7-6HIS was transformed, and as positive control (+)pHL125 was transformed.
All SC medium plates were incubated at 30° C. The L-arabinose plate (A) shows growth after 9 days and all other plates (B) after 2 days. The colonies 1 and 7 grew on L-arabinose but not on D-glucose, D-mannose and only weakly on D-galactose.
The complete open reading frame of the transporter according to the invention was sequenced double-strand with overlapping regions. The promoter and terminator region was sequenced single-strand. The arrows indicate the regions of individual sequencings.
The initial plasmid for the production of the P. stipitis gene bank was the plasmid YEp24 (A). Both the plasmid pAraT1 (B) and the plasmid pAraT7 are therefore based on YEp24 and only differ in the size of the insert. The open reading frame (ORF) of the arabinose transporter according to the invention was amplified by the pAraT1 and was cloned after the shortened strong HXT7 promoter of the plasmid p426HXT7-6HIS (C). With this, the plasmid p426H7-AraT (D) was produced, which has a uracil marker. Another possible expression plasmid is p426Met25 (E).
Growth of MKY06-3P, which additionally also contains the plasmid pHL125re or the plasmid p426H7-AraT (=MKY06-4P), in SM medium with A) 0.5%, B) 1% and C) 2% L-arabinose under aerobic conditions. The strains with the various plasmids were adducted in SM medium with 1% L-arabinose and inoculated with an OD600nm=0.2 in 30 ml SM medium with A) 0.5%, B) 1% and C) 2% L-arabinose. The incubation took place in 300 ml shaking flasks under aerobic conditions at 30° C. Samples were taken several times in the day to determine the optical density.
Shown are the results of HPLC analyses of the strain BWY1 with the plasmids p423H7araABsre, p424H7araBre, p425H7araDre and p426H7-AraT in SFM medium with 1.7% L-arabinose under semi-anaerobic conditions.
1. Strains and Media
Bacteria
E. coli SURE (Stratagene)
Full medium LB 1% trypton, 0.5% yeast extract, 0.5% NaCl, pH 7.5 (see Maniatis, 1982)
For selection on a plasmid-coded antibiotic resistance, 40 μg/ml ampicillin was added to the medium after autoclaving. Solid culture media additionally contained 1.9% agar. The culture took place at 37° C.
Yeast
Strain EBY.VW4000
EBY.VW4000 (Genotype: MATa leu2-3, 112ura3-52 trp1-289 his3-Δ1 MAL2-8c SUC2 Δhxt1-17Δgal2 stlΔ::loxP agt1Δ::loxP mph2Δ::loxP mph3Δ::loxP) (Wieczorke et al., 1999)
Strain MKY06
MKY06 (Genotype: MATa leu2-3,112 ura3-52 trp1-289 his3-MAL2-8c SUC2 hxt1-17 gal2 stl1::loxP agt1::loxP mph2::loxP mph3::loxP PromTAL1::loxP-Prom-vkHXT7, description: EBY.VW4000 PromTAL1::loxP-Prom-vkHXT7)
Strain MKY06-3P
MKY06-3P (Genotype: MATa leu2-3,112 ura3-52 trp-289 h is 3-1 MAL2-8c SUC2 hxt1-17 gal2 stl1::loxP agt1::loxP mph2::loxP mph3::loxP PromTAL1::loxP-Prom-vkHXT7, description: EBY.VW4000 PromTAL1::loxP-Prom-vkHXT7); contains the plasmids p423H7araABsre, p424H7araBre and p425H7araDre.
Strain with Accession Number DSM 18544
MKY06-4P (Genotype: MATa leu2-3,112 ura3-52 trp1-289 his3-1 MAL2-8c SUC2 hx1-17 gal2 stl1::loxP agt1::loxP mph2::loxP mph3::loxP PromTAL1::loxP-Prom-vkHXT7, description: EBY.VW4000 PromTAL1::loxP-Prom-vkHXT7); contains the plasmids p423H7araABsre, p424H7araBre, p4251-17araDre and p426H7-AraT.
Strain BWY1:
BWY1 is based on the strain JBY25 (Genotype: MATa leu2-3,112 ura3-52 trp1-289 his3-Δ1MAL2-8c SUC2+unknown mutations for better growth on L-arabinose) (Becker and Boles, 2003); the strain JBY25 was further selected and has still further mutations for improved growth on L-arabinose under oxygen-limited conditions (Wiedemann, 2005).
Full Medium YEP
1% yeast extract, 2% bacteriological peptone, carbon source in the respectively indicated concentration
Synthetic Complete Selective Medium SC
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
Synthetic Minimal Selective Medium SM:
0.67% yeast nitrogen base w/o amino acids and ammonium sulphate, 0.5% ammonium sulphate, 20 mM potassium-dihydrogenphosphate, pH 6.3, carbon source in the respectively indicated concentration
Synthetic Fermentation Medium (Mineral Medium) SFM (Verduyn at al., 1992), pH5.0
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 and maltose were used. For the selection on loss of a plasmid with URA3 selection marker gene, synthetic complete medium plates were used, which in addition to uracil contained 1 mg/ml 5-FOA, which was added after autoclaving (Boeke et al., 1984).
Solid full and selective media contained in addition 1.9% agar. The culture of the yeast cells took place at 30° C. The synthetic mineral medium used for the fermentation contained salts, trace metals and vitamins in the concentrations listed above and the indicated carbon source. A stock solution was prepared from the trace metals and the vitamins. The trace metal solution was autoclaved (20 min, 121° C.) and the vitamin solution was sterile-filtered. Both were stored at 4° C. The pH value had a decisive role for the production of the trace metal solution and prevented the precipitation of individual components. The various trace metals had to be completely dissolved in the above sequence in succession in water. After each addition, the pH value had to be adjusted with KOH to 6.0 before the next trace metal was able to be added. At the end, the pH value was adjusted with HCl to 4.0. In order to avoid foaming, 50 μl/l antifoam was added to the medium (Antifoam204, Sigma). In anaerobic experiments, in addition 2.5 ml/l of a Tween80-ergosterol solution was added to the medium after autoclaving. This consists of 16.8 g Tween80 and 0.4 g ergosterol, which were filled up to 50 ml with ethanol and dissolved therein. The solution was sterile-filtered. The salts, the corresponding quantities of trace metal solution and the antifoam were autoclaved together with the complete fermenter. The carbon source was autoclaved separately from the remaining medium. Before autoclaving, the pH was set to 5.0 in all. The sterile vitamin solution was added to the medium after cooling.
2. Plasmids
B. subtilis araA in p423HXT7-His,
E. coli araB in p423HXT7-His;
E. coli araD in p425HXT7-His;
3. Pichia stipitis Gene Bank
YEpTW Pichia stipitis: Gene bank with chromosomal fragments of Pichia stipitis in the over-expression plasmid YEp24, URA3 marker gene (Weierstall et al., 1999)
4. Transformation
Transformation of S. cerevisiae
The transformation of S. cerevisiae was carried out by the lithium-acetate method (Gietz and Woods, 2002). For the selection on a geneticin resistance, the cells were incubated after the transformation for 4 h at 30° C. in full medium and subsequently plated on medium plates containing G418.
Transformation of E. coli
The transformation of the E. coli cells took place by the electroporation method (Dower et al., 1988; Wirth, 1993) by means of an Easyject prima apparatus from EQUIBO.
5. Preparation of DNA
Isolation of Plasmid-DNA from S. cerevisiae
The cells of a stationary yeast culture (5 ml) were harvested, washed and re-suspended in 100 μl buffer 1 (taken from the “Plasmid Mini Kit”). After the addition of 200 μl buffer 2 and ⅔ volume glass beads (diameter=0.45 mm), the cells were solubilised for 8 min on a Vibrax (Janke and Kunkel, Vibrax-VXR) at 4° C. The supernatant was mixed with 150 μl buffer 3 and incubated for 10 min on ice. After centrifuging for 15 minutes at 10000 R/min, the supernatant was used and the plasmid-DNA was precipitated with 400 μl isopropanol (−20° C., 10 min). The DNA, which was pelleted through centrifuging (30 min, 13000 rpm) was washed with 70% cold ethanol and held in 20 μl water. The DNA was then used for a transformation in E. coli or a DNA amplification by means of PCR.
Isolation of Plasmid-DNA from E. coli
The isolation of plasmid-DNA from E. coli took place with the “Plasmid Mini Kit” of the company Qiagen, according to the manufacturer's information.
Determining the DNA Concentration
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 μg dsDNA/ml (Maniatis, 1982).
6. DNA Amplification by Means of PCR
Use of the Expand™ High Fidelity System
The polymerase chain reaction (PCR) took place with the “Expand™ High Fidelity PCR System” of the company Roche, according to the manufacturer's information. 0.2 mM dNTP-mix, 1× buffer 2 (contains 1.5 mM MgC12), 1 U polymerase and 100 μmol each of the corresponding oligonucleotide primers were added together to the plasmid- or genomic DNA to be amplified. The PCR reaction was carried out in a thermocycler (Techne) or mastercycler (Eppendorf).
For the amplification of the DNA, the following temperature cycles were selected.
After the first step, the polymerase was added (“hot start PCR”). The number of synthesis steps, the annealing temperature and the elongation time were adapted to the specific melting temperatures of the oligonucleotides which were used or to the size of the product which was to be expected. The PCR products were checked by a subsequent agarose gel electrophoresis and then purified.
DNA Purification of PCR Products
The purification of the PCR products took place with the “QIAquick PCR Purification Kit” of the company Qiagen, according to the manufacturer's information.
Gel Electrophoretic Separation of DNA Fragments
The separation of DNA fragments with a size of 0.15-20 kb took place in 1-4% agarose gels. 1×TAE buffer (40 mM Tris, 40 mM acetic acid, 2 mM EDTA) was used as gel- and running buffer (Maniatis, 1982). Serving as marker was either a lambda phage DNA cut with the restriction endonucleases EcoRI and HindIII, or the 2-log DNA ladder (NEB). Before application, the DNA samples were mixed with 1/10 volume blue marker (1×TAE buffer, 10% glycerine, 0.004% bromophenol blue). After the separation, the gels were incubated in an ethidium bromide bath and the DNA fragments were made visible by irradiation with UV light (254 nm).
Isolation of DNA Fragments from Agarose Gels
The desired DNA fragment was cut out from the TAE agarose gel under longwave UV light (366 nm) and isolated with the “QIAex II Gel Extraction Kit” or the “QIA-quick Gel Extraction Kit” of the company Qiagen, according to the manufacturer's information.
7. Enzymatic Modification of DNA
DNA restriction
Sequence-specific splittings of the DNA with restriction endonucleases were carried out under the incubation conditions recommended by the manufacturer for 2-3 hours with 2-5U enzyme per μg DNA.
8. HPLC Analyses
The samples taken in the tests were centrifuged for 10 min at 3000 R/min, in order to pellet the yeast cells. The supernatant was removed and immediately frozen at −20° C. For the protein precipitation, subsequently 50% sulphosalicylic acid was added, mixed, and centrifuged off for 30 min at 13000 R/min and 4° C. The supernatant was removed, a 1/10 dilution with water was produced therefrom and used for the HPLC analyses. Serving as standards for the measurements were samples with D-glucose, L-arabinose, and ethanol, which were used in concentrations of 0.1% w/w, 0.5% w/w and 1.0% w/w. The sugar- and ethanol concentrations were measured by means of BioLC (Dionex). The autosampler “AS50”, the column oven “TCC-100”, the gradient pump “GS50” (all Dionex) and the RI detector “RI-101” (Shodex) were used in the measurement. As a column, the VA 300/7.7 nucleogel sugar 810H (Machery-Nagel) was used with 20% sulphuric acid as eluent (0.6 ml/min). For the evaluation of the analysis data, the Chromeleon™ program (Version 6.50, Dionex) was used.
A) Construction of the MKY06
In the yeast strain EBY.VW4000 all the genes of the hexose transporter family and in addition three genes of the maltose transporter family were deleted. This strain grew on maltose medium unchanged, but was no longer able to grow on glucose, fructose and mannose and only very weakly on galactose (Wieczorke et al., 1999). As all hexose transporters are deleted, it can be assumed that the strain also can no longer receive any L-arabinose and is therefore suitable for arabinose transport investigations.
In preceding tests (see Becker and Boles, 2003), it had been found that in addition to a functional L-arabinose metabolic pathway, also an increased activity of transaldolase was necessary for the use of L-arabinose. For this reason, by exchange of the endogenous promoter of TAL1 in EBY.VW4000 for the shortened HXT7 promoter TAL1 was over-expressed. This strain was named MKY06 and is provided, with the plasmids for the L-arabinose metabolism and a transporter which can transport L-arabinose, to grow on this carbon source.
B) Introduction of the L-arabinose Metabolic Pathway
The strain MKY06 was transformed with the plasmids p423H7araABsre, p424H7araBre and p425H7araDre (=MKY06-3P), so that it obtains the capability of L-arabinose use. The transformation with the three plasmids took place simultaneously. The transformants were plated on medium with 2% maltose. In a further transformation, as positive control in addition the transporter Gal2, known as L-arabinose, was transformed in and as negative control the empty plasmid p426HXT7-6HIS and plated again on medium plates containing maltose. The positive control, which contains an L-arabinose transporter and the three plasmids for the L-arabinose use and over-expresses transaldolase, should be able to grow on L-arabinose. The negative control should show no growth owing to the absent transporter. This was investigated.
C) Checking the Test System
In order to be able to use the constructed test system further, firstly the positive and negative controls had to be investigated with regard to their growth. Several colonies of the transformants obtained on the SC plates with 2% maltose were removed with a sterile inoculating loop and smeared on SC plates with 2% L-arabinose and incubated at 30° C. for ten days. After this time, the positive control showed a distinct growth and the negative control, as expected, showed no growth.
The growth behaviour was likewise investigated in liquid medium with 2% maltose or 2% L-arabinose. For this, precultures were adducted with the corresponding carbon sources (maltose or L-arabinose) under aerobic conditions at 30° C. After reaching the late exponential phase, these precultures were used in order to inoculate 30 ml of the same medium with an initial OD600nm=0.2. As the negative control did not grow on medium plates containing L-arabinose, starting from the preculture with 2% maltose 30 ml SC medium was inoculated with 2% L-arabinose. The growth behaviour was followed over several days by measurement of the optical density at 600 nm. In the maltose medium, the positive and negative control showed identical growth, as expected, with a growth rate of 0.197 h−1. In L-arabinose growth, precisely as already in the tests with the L-arabinose medium plates, growth was only found in the positive control (μ=0.01 h−1), which still had the transporter Gal2 compared with the negative control. The low growth at the start in the negative control can be explained in that before the reinoculation of maltose the cells were not grown on medium containing L-arabinose. In addition, the glycogen stores of the yeast make still make a slight growth possible.
This test system was therefore functional and was able to be used for the investigation of L-arabinose transporters. The positive and negative control mentioned here always served as a comparison in this.
The test system was now used in order to seek in a gene bank of Pichia stipitis one of the yeasts which can use L-arabinose, for possible L-arabinose transporter genes.
A) Carrying Out the Screen
The gene bank YEpTW which was used here was produced form the Pichia stipitis strain CBS5774. Chromosomal DNA was partially digested with the restriction endonuclease Sau3A and ligated into the vector Yep24 linearised with BamHI (Weierstall et al., 1999).
The gene bank, exactly like the plasmid pHL125re, had a uracil auxotrophy marker. The gene bank YEpTW was transformed into the strain MKY06-3P and smeared on SC medium with 2% maltose. The colonies obtained after three days at 30° C. were replica-plated on SC medium plates with 2% L-arabinose. After 10 days, colonies were sought which showed growth on L-arabinose. Growth was only possible when the plasmid of the gene bank coded a transporter which was able to transport L-arabinose.
In order to be able to rule out genomic mutations which could be responsible for the growth, the colonies which were found were smeared on complete medium with 5-FOA. Thereby, selection was carried out on a loss of the plasmid of the gene bank. With renewed smearing on L-arabinose medium, these colonies of the 5-FOA plate were no longer able to grow hereon. Therefore, a genomic mutation was able to be ruled out as the cause of the growth. The colonies which were found were also smeared on other carbon sources, in order to test the substrate spectrum.
B) Growth Behaviour
Of the over 30000 replica-plated colonies, the eleven colonies found here which showed slight growth were smeared again on L-arabinose plates. Here, however, only five colonies showed growth again on L-arabinose. This was the strain MKY06-3P, which additionally contained the plasmid pAra1, pAraT6, pAraT7, pAraT8 or pAraT11 from the gene bank YEpTW (see
The colonies with pAraT6, pAraT7 and pAraT11 were adducted in liquid SC medium with 2% L-arabinose and glycerine cultures were prepared herefrom, which are stored for later investigations at −70° C. Further work was carried out with the plasmids pAraT1 and pAraT7 and the growth behaviour of the strains was investigated in liquid medium. In SC medium with 2% maltose, the MKY06-3P, which additionally contained pAraT1 or pAraT7, behaved identically to the positive- (additionally also pHL125re) and the negative control (additionally also p426HXT7-6HIS). The growth rates were 0.197 h−1.
Differences were found on SC medium with 2% L-arabinose. The negative control (p426HXT7-6HIS) showed no growth on this medium. The slight growth at the start resulted from the absent washing step in the reinoculation from the preculture on maltose medium to the main culture in L-arabinose medium. Both L-arabinose transporters which were found behaved similarly. In the growth rate (μ=0.087 h−1) and also in the maximum OD600nm there were no differences between the MKY06-3P with the plasmid pAraT1 or the pAraT7. If one compares the growth behaviour of the two new transporters with the positive control (pHL125re, μ=0.1 h−1, then a somewhat lower growth rate and a lower maximum OD600nm are found.
C) Isolation of the L-arabinose Transporters which were Found
In order to be able to analyse the transporters which were found on a genomic level, firstly the gene bank plasmids had to be isolated again from the yeast. It was to be noted here that the strain MKY06 not only contains the plasmid from the gene bank YEpTW, which codes the sought transporter, but at the same time also contains the three plasmids for the L-arabinose metabolism (p423H7araABsre, p424H7araBre, p425H7araDre) and that the plasmid p423H7araABsre is present in a much higher number in the cells than the other three plasmids. As the plasmids of the gene bank YEpTW had a uracil auxotrophy marker, the cells were inoculated starting from the L-arabinose plates in maltose medium without uracil (ura-). After reaching the stationary phase, these were reinoculated into fresh maltose-ura medium and adducted again. Here, the cells were adducted with the four plasmids five times in maltose-ura liquid medium up to the stationary phase. The aim of this was an enrichment of the plasmid pAraT1 or pAraT7. From these two cultures, separation smears were prepared on maltose-ura medium plates. The colonies which were produced were replica-plated onto further maltose plates and incubated for two days at 30° C. These plates did not contain the corresponding amino acid for the auxotrophy marker of one of the other three plasmids (histidine for p423H7araABsre, tryptophan for p424H7araBre or leucine for p425H7araDre). These replica plates were compared with the maltose-ura plates. Colonies which only grew on the maltose-ura plate were selected. These only had the plasmid pAraT1 or pAraT7. The plasmids were isolated from yeast. Thereafter, the plasmids were amplified in E. coli, and after the isolation from E. coli were characterized by means of a restriction analysis. NcoI and NheI were used as restriction enzymes. NcoI only cuts in the URA3 marker gene. When the sought plasmid from the gene bank is concerned, then a 934 bp large fragment occurs.
A) Sequencing
The chromosomal fragments from P. stipitis localized on the plasmids pAraT1 and pAraT7 found in Example 2 were sequenced.
The complete ORF of the transporter which was found was double-strand sequenced with overlapping regions. The promoter and terminator region was single-strand sequenced (cf.
In the sequencing it was found that the two plasmids pAraT1 and pAraT7 contain overlapping fragments of the same gene. This concerns one and the same transporter and not two different transporter genes. The plasmid pAraT1 has an insert with approximately 5 kb; it contains the complete open reading frame (ORF) of AraT, which consists of 1629 bases and consequently 542 amino acids (plus the STOP codon). Additionally also promoter- and terminator sequences. The plasmid pAraT7 has an insert which is approximately 3 kb in size; it does not contain the complete ORF of AraT, but rather only the first 1507 bases. Nevertheless, this fragment was, however, functional.
In a BLAST search with the recently published genome of Pichia stipitis a 100% conformity with HGT2 was found. HGT2 was annotated as putative high-affinity glucose transporter owing to its high degree of homology to the high-affinity glucose transporter HGT1 of Candida albicans. When one examines the sequence with regard to the possible transmembrane domains, one obtains 12 transmembrane domains, which is typical for transporters.
B) Examples for Vectors for AraT
The initial plasmid for the production of the gene bank was the plasmid YEp24. The plasmid pAraT1 and also the plasmid pAraT7 are therefore based on YEp24 and differ only in the size of the insert. The vector YEp24 is an episomal plasmid.
The open reading frame (ORE) of the arabinose transporter which was found was amplified by pA-raT1 and cloned behind the shortened strong HXT7 promoter of the plasmid p426HXT7-6HIS. With this, the plasmid p426H7-AraT was produced, which has a uracil marker.
Another possible expression plasmid is p426Met25. For vector maps, see
Further possible expression vectors are pYES260, pYES263, pVTU260, pVTU263, pVTL260 and pVTL263.
C) Growth in Dependence on the L-arabinose Concentration in the Medium
The growth of the strain MKY06-4P was investigated under aerobic conditions as a function of the L-arabinose concentration in the medium. As a comparison, the strain MKY06-3P was used, which additionally also contained the plasmid pHL125re or p426HXT7-6HIS.
The strains with the various plasmids were adducted in SM medium with 1% L-arabinose and inoculated with an OD600nm=0.2 in 30 ml SM medium with 0.5%, 1% or 2% L-arabinose. The incubation took place in 300 ml shaking flasks under aerobic conditions at 30° C. Samples were taken several times in the day to determine the optical density.
The results are shown in
It is therefore shown that the L-arabinose uptake system according to the invention makes it possible for the recombinant S. cerevisiae cells to use L-arabinose substantially more efficiently.
In
The strains were adducted aerobically in the same medium to a high optical density. The cells were centrifuged off and used for the inoculation of the semi-anaerobic fermentation tests. Already after approximately 25 hours, ethanol production begins in both strains. In the strain which contains the plasmid p426H7-AraT a higher ethanol production is able to be established at the start. On the other hand, the arabinose concentration at the end of the test decreases more strongly in the case of cells with p426H7-AraT than with pHL125re, which indicates that here a higher affinity of AraT leads to an improved fermentation of low arabinose concentrations.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2006 060 381 | Dec 2006 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/010668 | 12/7/2007 | WO | 00 | 1/27/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/080505 | 7/10/2008 | WO | A |
| Number | Date | Country |
|---|---|---|
| WO 2007143245 | Dec 2007 | WO |
| WO 2007143247 | Dec 2007 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20100143936 A1 | Jun 2010 | US |
