Patent Application: US-49403004-A

Abstract:
the present invention relates to fungal microorganism having an increased ability to carry out biotechnological process . in particular , the invention relates to improving the regeneration of redox cofactors in biotechnological processes where useful products are produced from biomass containing pentoses . according to the invention , the microorganism is transformed with a dna sequence encoding an nadp linked glyceraldehyde 3 - phosphate dehydrogenase . the invention can be used to provide useful products for mankind from biological materials , including e . g . agricultural and forestry products , municipal waste . examples of such useful products are ethanol , lactic acid , polyhydroxyalkanoates , amino acids , fats , vitamins , nucleotides and a wide variety of enzymes and pharmaceuticals .

Description:
to look for possible proteins and their corresponding genes which could regenerate the redox cofactors nadp / nadph in catabolic reactions such as pentose fermentation the following screening method for finding nadp / nadph linked proteins and their corresponding genes can be used . in this screening method we used a saccharomyces cerevisiae strain with a deletion in the gene coding for the phosphoglucose isomerase , pgi1 . this deletion disables s . cerevisiae to grow on glucose ( boles et al ., 1993 ). it is believed that this deletion leading to a lethal phenotype on glucose is related to an overproduction of nadph in the oxidative part of the pentose phosphate pathway ( boles et al ., 1993 ). kluyveromyces lactis however can grow on glucose with a deletion in the phosphoglucose isomerase gene , i . e . it can cope with this nadph overproduction ( gonzales siso et al ., 1996 ). we therefore transformed the s . cerevisiae strain with the deletion in phosphoglucose isomerase gene with a gene library from kluyveromyces lactis and screened for growth on glucose . in this screening we found a dna fragment that contained several open reading frames . a transposon was randomly inserted into the dna fragment and those transposon insertions , which did not restore growth on glucose , were analysed . with this technique we identified the open reading frame which could restore growth on glucose . this open reading frame had high homology to nad - gapdh . we further investigated this open reading frame . for that purpose we overexpressed it and analysed the enzyme activity and found that it has activity with nadp . we further purified the enzyme after adding a histidine tag and found that the open reading frame codes for a protein which has a preference for nadp over nad , i . e . it is not an nad - gapdh ( ec 1 . 2 . 1 . 12 ) but an nadp - gapdh ( ec 1 . 2 . 1 . 13 ). this is surprising since there are no reports in the literature about nadp - gapdh in eukaryotic organisms except plants , and there they are involved in photosynthesis , a reaction not carried out by yeasts . the nadp - gapdh is encoded by the dna sequence comprising seq id no . 1 . glyceraldehyde 3 - phosphate dehydrogenases ( gapdh ) are known as non - phosphorylating enzymes ( gapn , ec 1 . 2 . 1 . 8 ) and phosphorylating enzymes . for the phosphorylating enzymes nicotinamide dinucleotide ( nad ) dependent enzymes ( nad - gapdh , ec 1 . 2 . 1 . 12 ) and nicotinamide dinucleotide phosphate ( nadp ) dependent enzymes ( nadp - gapdh , ec 1 . 2 . 1 . 13 ) are known . the nad - gapdh is a glycolytic enzyme , which is highly conserved in prokaryotes and eukaryotes . nadp - gapdh is known in bacteria ( e . g . koksharova et al . 1998 , fillinger et al . 2000 ). for plants an nadp - gapdh , which is involved in the photosynthetic co 2 assimilation and located in the chloroplasts , is known ( cerff 1982 ). the nadp - gapdh of chloroplasts has the two subunits a and b ( shih et al . 1991 , baalmann et al . 1996 ). other eukaryotic nadp - gapdh are not known . in the present invention we have two oxidoreductases with a common substrate but opposite coenzyme specificities , i . e . nad - gapdh ( ec 1 . 2 . 1 . 12 ) and nadp - gapdh ( 1 . 2 . 1 . 13 ). this however does not lead necessarily to cyclic oxidation - reduction reactions . as illustrated in the fig3 for d - xylose fermentation , 3 moles of nadph , which are used for the reduction of 3 moles of d - xylose , can be regenerated through the nadp - gapdh . the nad which is used by xylitol dehydrogenase and the nad - gapdh are regenerated by the alcohol dehydrogenase . the fermentation of 3 moles of d - xylose to 5 moles of ethanol and 5 moles of co 2 is done without a cyclic transhydrogenase reaction , which makes the present invention different from the patent publication u . s . pat . no . 5 , 830 , 716 and from the application wo 99 / 46363 . an nadp - gapdh can be beneficial in processes where it is not desired to have the reduction of nadp to nadph coupled to co 2 production . one example is hexose fermentation . because the microorganism grows during the fermentation it produces excesses of both nadh and nadp ( oura , 1972 ). ethanol production is accompanied by glycerol production , which is required to reoxidise the excess nadh , and by the production of more than one mole of co 2 per mole of ethanol , which is required to reduce the excess nadp . these reactions decrease the yield of ethanol on fermentable carbohydrate . with an nadp - gapdh nadp can be reduced without extra to co 2 production and by reducing nadp by using the glyceraldehyde 3 - phosphate pool , less nadh is produced through the nad - gapdh and consequently less glycerol is produced , i . e . the introduction of nadp - gapdh can increase the ethanol yield in hexose fermentation and decrease the formation of undesired sideproducts , glycerol and co 2 . the invention in this way makes the environmentally friendly production of fuel alcohol from hexose carbohydrates still more efficient and less polluting . an nadp - gapdh can also be beneficial in pentose fermentation . by the invention d - xylose and l - arabinose can be fermented to ethanol in a redox neutral way without creating a redox cofactor imbalance . in examples 3 and 5 we show that d - xylose is fermented more efficiently to ethanol . ethanol is produced from d - xylose with a higher yield and with less unwanted side products such as xylitol and co 2 . this is shown in example 3 where we show the effect of an nadp - gapdh on anaerobic xylose fermentation . the strain overexpressing nadp - gapdh produces , in molar ratios , about 30 % less xylitol and about 40 % less co 2 . as a consequence the ethanol is produced at a higher yield , i . e . from the same amount of d - xylose about 30 % more ethanol is produced . in addition to yield improvement and reduction of by - product formation , increased ability of the recombinant strain to carry out biotechnical processes may also be seen as improved rate of product formation , prolonged metabolic activity in process conditions or decreased demand for oxygen , all these factors increasing the efficiency of the process . to further increase the ethanol yield , and decrease the co 2 and xylitol yield , additional improvement strategies can be used . these include ( 1 ) decreasing the reactions competing for nadp with the nadp - linked gapdh of our invention and ( 2 ) increasing the capacity or affinity of the nadp - gapdh for nadp . nadph regeneration through an nadp - gapdh is not the only way to regenerate nadph . other pathways like through the oxidative part of the pentose phosphate pathway compete for the nadp . this nadph regeneration is coupled to co 2 production . it can be of further benefit to inhibit or delete this or similar pathways . we show in example 5 that glucose 6 - phosphate dehydrogenase competes for nadp and that the deletion of the corresponding gene , the zwf1 , together with the overexpression of the nadp - gapdh has a further beneficial effect on ethanol production , i . e . ethanol is produced at a higher yield at the expense of unwanted side products such as xylitol or co 2 . in example 5 we demonstrate that decreasing the competing reactions for nadp we can further decrease the production of unwanted side products and thereby increase the ethanol yield . by deleting the gene for the glucose 6 - phosphate . dehydrogenase , a reaction competing for nadp , and simultaneously overexpressing the nadp - gapdh , we could decrease the production of unwanted xylitol by another 20 %. other reactions competing for nadp include the nadp dependent acetaldehyde dehydrogenase ald6 and isocitrate dehydrogenases idp1 - 3 . these and other reactions competing for nadp can be suppressed in a variety of ways . a gene encoding an enzyme catalysing the reaction can be deleted , as described in example 5 for glucose 6 - phosphate dehydrogenase . such a gene can also be disrupted , so that it no longer produces a functional dehydrogenase . the promoter of the gene can also be altered ( for example , by deletion of parts of the sequence upstream of the open reading frame ) so that the expression level of the enzyme is decreased but not abolished . this can be advantageous if the reaction catalysed is beneficial to the microorganism so that e . g ., complete suppression prevents growth of the microorganism . in practice , little experimentation is required , because if complete suppression prevents growth , this is immediately apparent and milder methods can be used with an obvious advantage . similarly , mutations can be introduced to the active site of the competing enzyme , so that its catalytic efficiency is decreased , but not abolished . for example , a mutation that increased the km for nadp of the competing enzyme would suffice , but it is not necessary to characterise the kinetic effects of such a mutation . the active site sequences of dehydrogenases are recognisable by those familiar with art to increase the capacity or affinity of the nadp - gapdh the expression level can be increased or an nadp - gapdh with a higher affinity towards nadph can be used . decreasing competing reactions or increasing the capacity of the nadp - gapdh are not only beneficial for pentose fermentation but also for the other examples where the nadp - gapdh has a positive effect . we describe here the introduction of an nadp - gapdh from k . lactis to a strain of s . cerevisiae that contains the d - xylose pathway . for somebody knowledgeable in the art it is easy to find a similar enzyme from another fungi or other eukaryotic source . the introduction of an nadp - gapdh can be beneficial independent of its source , whether it is bacterial , fungal or from another eukaryotic organism . nadp - gapdh are known from bacteria and from plants . in this invention we describe an nadp - gapdh from fungi . an nadp - gapdh can be generated e . g . through modification of the amino acid sequence of an nad - gapdh . for example with the sequence of nadp - gapdh disclosed herein comparison to the sequences of other dehydrogenases of known nad and nadp specificity and some degree of amino acid identity , and in the best case to those for which the 3 - d structure is known allows a person skilled in the art to predict the amino acids in the protein sequence which are responsible for the cofactor specificity . with this knowledge and using site directed mutagenesis the cofactor specificity can be changed , i . e . an nadp - gapdh can be made by site directed mutagenesis from an nad - gapdh . it can be advantageous to create an nadp - gapdh through mutagenesis in cases where the expression of a heterologous nadp - gapdh is difficult . the desired change can also be done with random approaches . one example how one can find in the sequence amino acids important for cofactor specificity of the enzyme is the following . aligning the amino acid sequence of the nadp - gapdh with those of glyceraldehyde 3 - phosphate dehydrogenases from different organisms with different specificities and comparing this with the known structural information suggests that the amino acid 46 asparagine can be of importance ( see also fillinger et al ., 2000 ). in all nad - gapdh the corresponding amino acid is the negatively charged aspartic acid . from the available structural information one would expect that the negatively charged phosphate of the nadp is in this area when nadp binds to the active site , i . e . nad - gapdh do not use nadp because of the unfavorable interaction between negative charges . by changing the negatively charged aspartic acid to a neutral residue , such as the asparagine disclosed here for the nadp - gapdh of k . lactis , or to a positively charged amino acid one could change the specificity of an nad - gapdh so that it could also use nadp . an nadp - gapdh can also be beneficial in l - arabinose fermentation since the l - arabinose pathway creates a cofactor imbalance similar to the d - xylose pathway . polyhydroxyalkanoates ( phas ) are commercially produced to make biodegradable plastics , but prices are too high for widespread use except where this is enforced by legislation ( e . g . in germany ). it is therefore desirable to improve the efficiency of the microbial processes producing phas . in the biosynthesis of phas , glucose is metabolised to acetyl - coa , producing 2 nadh molecules / acetyl - coa molecule , and the acetyl - coa is then condensed to acetoacetyl - coa which is reduced by nadph to 3 - hydroxybutyrylcoa . synthesis of each molecule of 3 - hydroxybutyrylcoa therefore produces 4 molecules of nadh and requires 1 molecule of nadph . the 3 - hydroxybutyrylcoa is then polymerised to polyhydroxybutyrate ( phb ) or copolymerised with other acyl - coas such as propionyl - coa to form mixed phas . the requirement for one nadph molecule and production of 4 nadh molecules per monomer unit means that microorganisms synthesising phas need to divert part of their carbon flux through reactions such as glucose - 6 - phosphate dehydrogenase or isocitrate dehydrogenase in order to generate nadph , with consequent excess production of co 2 and waste of carbon source , as explained above . at the same time , nadh must be reoxidised , causing either further carbon losses or increased oxygen demand or both . by using a production microorganism transformed according to the present invention , so providing it with a novel mechanism that produces the nadph at the expense of nadh ( for reviews , see e . g . anderson and dawes [ 1990 ]; poirier et al . [ 1995 ]). the waste of biomass as co 2 is decreased , and so is the oxygen requirement , with consequent decreases the aeration costs . the introduction of an nadp - gapdh is not only beneficial in a strain of s . cerevisiae but also in other fungi , such as yeast species that naturally use pentoses . in any fungal species it is beneficial in d - xylose fermentation and in l - arabinose fermentation or in any biotechnological process where an imbalance of the redox cofactors imposes a hindrance . the fermentation products can be ethanol , lactate / lactic acid or other products . it is well known to somebody skilled in the art that the amino acid sequence of an enzyme can be deliberately or accidentally ( e . g . in pcr cloning ) changed ( e . g . parts deleted or added or amino acid changes introduced ) so that the changed enzyme can still catalyse the same reaction as the original enzyme . the present invention can also be practised using recombinant dna sequences that encode such ‘ functionally active ’ variants of nadp - gapdh . the present invention can also be practised by transforming a microorganism with a recombinant dna molecule with a promoter different from the promoters used in the examples . it is not necessary that the transforming dna molecule contains a nucleotide sequence encoding a complete functional enzyme . for example the beneficial effect can be obtained by transforming the natural host of an nadp - gapdh with a dna molecule that modifies the natural promoter , and so leads to an elevated expression level of the nadp - gapdh . any method known in the art for transducing or transforming genes into the host is suitable for this invention and various types of vectors can be used , including autonomously replicating plasmid vectors or artificial chromosomes . methods described in the art to integrate single or multiple copies of transforming genes into chromosomes in functional , expressible forms are also suitable for this invention . it is envisaged in the invention that it can be advantageous in some cases to cause expression of the transformed genes only under specific culture conditions . for example it can be useful to first grow the organism to a certain cell density , and then cause expression of the transforming gene . promoters are known that can be induced by changes in temperature or ph , by particular carbon or nitrogen sources or by the presence or absence in the medium of certain organic or inorganic substances such as phosphate or copper . the present invention is further illustrated by the following examples , which are meant for illustration only and do not in any way limit the invention . if not otherwise indicated , all biotechnological procedures are carried out using methods conventional in the art . we used a screening system for nadp ( h ) related redox enzymes that is based on a deletion of the phosphoglucose isomerase gene in s . cerevisiae . a strain ( δpgil ) with such a deletion is unable to grow on glucose , which is related to a lethal overproduction of nadph ( boles et al ., 1993 ). in kluyveromyces lactis such a deletion does not lead to a similar phenotype ( gonzales siso et al . 1996 ). we used a s . cerevisiae with a phosphoglucose isomerase deletion and screened a k . lactis genomic library for growth on glucose to find k . lactis genes that would allow the δpgil mutant to grow on glucose . we found a gene for nadp linked gapdh , as described above . thus , this screening method provides genes suitable for practising the present invention . constructing the host strain for the library screening : deleting the pgi1 gene in s . cerevisiae : the pgi1 gene of the s . cerevisiae haploid strain cen . pk2 was deleted . a s . cerevisiae pgi1 fragment was obtained by pcr using the primers 3645 and 3646 . the primer 3646 ( 5 ′- cgaccg gtcgac taccagcctaaaaatgtc - 3 ) had a sali digestion site ( underlined ) to facilitate the cloning and the primer 3645 ( 5 ′- ggcacg ctgcag agagcgatttgttcacat - 3 ′) had a psti digestion site . the pgi1 fragment was digested with sali and psti and ligated into the pbluescript sk - vector ( stratagene ). the resulting plasmid ( b1186 ) was digested with ecori and bstbi to remove a 715 bp fragment from the middle of the pgi1 gene . the his3 gene was obtained by drdi digestion from the yeast expression vector prs423 . the his3 fragment was blunted with t4 dna polymerase and ligated to the pbluescript sk - ecorv site . this plasmid ( b1185 ) was digested with ecori and clai and the 1 . 5 kb fragment carrying the his3 gene was ligated into ecori and bstbi digested b 1186 plasmid . the resulting plasmid was named b1187 . the pgi1 + his3 - fragment was released from the b1187 plasmid with sali and muni digestion and the s . cerevisiae strain cen . pk2 was transformed with the fragment . the li - acetate method ( hill et al ., 1991 ; gietz al ., 1992 ) was used for the yeast transformation . the yeast transformants were confirmed by southern blot - analysis using a fragment from the s . cerevisiae pgi1 gene as the probe . the resulting strain , cen . pk2 δpgil , was then used for the screening . the k . lactis genomic library was constructed into a yeast multicopy vector carrying the leu2 marker gene as described by brummer et al ., 2001 . the library was transformed into the cen . pk2 δpgil yeast strain . transformants were plated on medium containing sc - leu + 2 % fructose + 0 . 1 % glucose . after 2 days cultivation 1 . 3 * 10 6 transformants from the plates were pooled into 0 . 9 % nacl . for the yeast library screening 6000 independent clones from the library were plated on medium containing sc - leu + 2 % fructose + 0 . 1 % glucose . after 3 days cultivation the colonies in the plates were replicated to sc - leu + 0 . 1 % glucose plates . the replica plates were cultivated for 9 days . 72 slowly growing colonies were streaked on sc - leu + 0 . 1 % glucose plates . pcr - analysis was made to determine if the clones growing on glucose carried the k . lactis rag2 gene coding for phosphoglucose isomerase . the pcr was made with specific primers 4719 and 4720 for the k . lactis rag2 . 5 ′- primer 4719 is 320 bp downstream from the atg ( 5 ′- cactgaaggacgtgctgtgt - 3 ′) and 3 ′- primer 4720 is 1150 bp downstream from the atg ( 5 ′- agctgggaatctgtgcaagt - 3 ′). the pcr - analysis was made for 18 colonies . six clones were found that did not carry the k . lactis rag2 gene according to the pcr - analysis . plasmid - dna was extracted from these 6 clones and transformed into e . coli for further analysis . the plasmids were retransformed to the cen . pk2 δpgil yeast strain and the transformants tested for growth on glucose . 2 clones were able to restore growth on glucose . partial sequencing of the insert suggested that the two clones were identical . one of the plasmids was called b1513 . the recovered plasmid had an insert of estimated 10 kb . a transposon was randomly inserted into the plasmid with the ‘ template generation system ’ ( finnzymes ). 10 different transposon insertions ( as judged by pcr with primers from the transposon and the vector ) were selected . they were then retransformed to the cen . pk2 δpgil strain tested for growth on 0 . 1 % d - glucose . from strains , which were maintained on 2 % d - fructose + 0 . 05 % d - glucose , but showed no growth on 0 . 1 % d - glucose the plasmids were recovered and sequenced with primers of the transposon sequence . a plasmid that could not restore growth on d - glucose had a transposon inserted into an open reading frame with high homology to glyceraldehyde 3 - phosphate dehydrogenase ( gapdh ). the amino acid sequence of the enzyme which later turned out to be an nadp - gapdh is presented by the seq id no . 2 . it is a protein with 356 amino acids having a molecular mass of 39030 da . it is encoded by the open reading frame in the nucleotide sequence between nucleotides 384 and 1451 of the nucleotide sequence seq id no . 1 . cloning and expression of the gapdh homologue , testing for nadph - gapdh activity cloning the k . lactis gapdh homologue to the yeast expression vector pyes2 : the gapdh homologue was amplified by pcr from the plasmid b1513 from example 1 by using the following primers : gapbamh : aa ggatcc aagcgtctccttaaacaccagc and gaphind : ata aagctt aagatgcccgatatgacaaacgaatcttc . the annealing temperature in the pcr was 65 ° c . the pcr product was digested with bamhi and hindiii and ligated to the corresponding sites in the multiple cloning site of the pyes2 vector ( invitrogen ). the pyes2 is a yeast expression vector with a multiple cloning site between a galactose inducible promoter and terminator . the resulting vector was called b1612 . expression of the k . lactis gapdh homologue in s . cerevisiae the plasmid b1612 from above and as a control the plasmid pyes2 were transformed to the s . cerevisiae strain cen . pk2 . the resulting strains were grown on selective medium with 20 g / l d - glucose and 20 g / l d - galactose . cells were harvested at an optical density of 1 and a cell extract prepared . the cell extract was prepared by vortexing 0 . 5 g cells ( fresh weight ) 500 mg glass beads ( 0 . 4 mm diameter ) and 1 ml buffer ( 10 mm sodium phosphate ph 7 . 0 plus protease inhibitors ). the extract was then used for an enzyme activity assay . the nadp - gapdh enzyme activity was measured in a buffer containing 500 mm triethanol amine ph 7 . 8 , 1 mm atp , 2 mm mgcl 2 , 0 . 2 mm nadph , 3 - phosphoglycerate kinase . to start the reaction , glycerate 3 - phosphate was added at a final concentration of 5 mm . the activity was calculated from the decrease in nadph absorbance at 340 nm . we found an nadph - gapdh activity of 0 . 05 nkat per mg of extracted protein . in the control , where the empty pyes2 plasmid was transformed we found 0 . 006 nkat per mg . effect of k . lactis gapdh homologue on d - xylose fermentation in an s . cerevisiae strain for the d - xylose fermentation the nadp - gapdh gene was ligated to a yeast expression vector with adh1 promoter . therefore the nadp - gapdh was amplified by pcr as described in the example 2 except that the following primers , each of them containing a bamhi restriction site , were used : ( bamhi sites are underlined ) aa ggatcc aagatgcccgatatgacaaacgaatcttc and aa ggatcc aagcgtctccttaaacaccagc . the pcr product was then cloned to a topo vector ( invitrogen ) and the 1 kb bamhi fragment from the resulting vector ligated to the bamhi site of the pvt102u ( vernet et al 1987 ). the resulting vector ( b1731 ) was then transformed to a s . cerevisiae strain ( h2217 , aristidou et al 1999 ), which overexpressed the enzymes of the xylose pathway , i . e . xylose reductase ( xr ), xylitol dehydrogenase ( xdh ) and xylulokinase ( xk ) were integrated into the genome . as a control we used the same strain , except that it lacked the nadp - gapdh . instead it contained the empty vector pvt102u . with both strains pure d - xylose was fermented under anaerobic conditions . the cells were first grown in a medium with yeast nitrogen base ( difco ) and all amino acids except uracil and 30 g / l d - glucose as a carbon source in a volume of 1 . 6 l at 30 ° c ., ph 5 . 0 and an airflow rate of 2 l / min . after 48 hours the biomass was 3 to 4 g / l and the ethanol concentration between 0 . 5 and 1 g / l when 0 . 4 l of a d - xylose solution was added so that the final concentration of d - xylose was 50 g / l . the gas flow was changed to nitrogen at a flow rate of 0 . 1 l / min . liquid samples were taken and analysed for dry weight and by hplc for ethanol , xylose and xylitol and other components . the outlet gas was analysed by mass spectroscopy . the results are in the fig3 and 4 . the main products of such a fermentation are xylitol , ethanol and co 2 . when introducing the nadp - gapdh the molar ratio of produced ethanol to xylitol was increased . without the nadp - gapdh the molar concentrations of xylitol and ethanol are similar . with the introduction of the nadp - gapdh the production of xylitol is decreased by about 30 % ( fig4 ). the ethanol yield on d - xylose is also affected . the maximal theoretical yield is 1 . 67 mol ethanol per mol d - xylose . in the control we find a molar ratio of ethanol produce to d - xylose consumed of 0 . 44 which is 26 % of the theoretical yield , with the nadp - gapdh the molar ratio of d - xylose consumed to ethanol produced is 0 . 57 which is 34 % of the theoretical yield ( fig4 ). also the co 2 yield is affected . we analysed the co 2 production in the period from 30 to about 90 hours and compared it with the ethanol production in the same period . with the nadp - gapdh the molar ratio of co 2 over ethanol is 1 . 15 as compared to 1 . 91 in the control . to purify the nadph - gapdh we overexpressed it in yeast with an additional tag of 6 histidines at the n - terminus of the protein . it was cloned by pcr similar to the example 2 and 3 . the primers were aa ggatcc aagatgcccgatatgacaaacgaatcttc for the start of the gene with a bamhi restriction site and aa ggatcc ttaatgatgatgatgatgatgaacaccagcttcgaagtcct tttgagcc for the end of the gene with the introduction of 6 histidines and a bamhi restriction site underlined . the pcr product was first cloned to a topo vector ( invitrogen ) and the bamhi fragment from the topo vector then ligated to the bglii site of a yeast expression vector with a pg1 promoter ( b1181 ). this yeast expression vector was made by digesting the yeast expression vector pma91 ( mellor et al ., 1983 ) with hindiii and ligating the resulting 1 . 8 kb fragment , containing the pgk1 promoter / terminator with a bglii cloning site , to the hindiii site of the yeplac195 vector ( gietz and sugino , 1988 ). the plasmid was then transformed to a yeast strain with a mutation in the phosphoglucose isomerase gene . the plasmid could restore growth on glucose showing that the histidine tag did not affect the enzyme activity . the his - tagged protein was then purified with a ninta column ( qiagen ). the so purified protein was then applied to a sds - page as shown in fig6 . the enzyme is almost pure . an estimated 80 to 90 % of the protein in the sds - page is in a single band of about 40 kda . the activity was measured as described in the example 2 with 200 μm nadph or 200 μm nadh . with nadph we found an activity of 140 nkat / mg , with nadh an activity of 47 nkat / mg . effect of deletion of the glucose 6 - phosphate dehydrogenase in the presence of nadp - gapdh on d - xylose fermentation the zwf1 gene coding for the glucose 6 - phosphate dehydrogenase ( g6pdh ) was obtained by pcr using s . cerevisiae genomic dna as a template . specific primers 3994 ( 5 ′- gctatc ggatccaagctt aggcaagatgagtgaaggtt - 3 ′) and 4006 ( 5 ′- gctatc ggatccaagctt agtgacttagccgataaatg - 3 ′) were used . both the primers had bamhi and hindiii sites to facilitate the cloning . the restriction sites are underlined . the zwf1 fragment obtained from the pcr was digested with bamhi and ligated into the pbluescript sk - plasmid ( stratagene ). the resulting plasmid b1768 was digested with bglii . in the digestion a 1063 bp fragment was released from the middle of the zwf1 gene . the digested vector was blunted with mung bean nuclease . the his3 marker gene was obtained from the prs423 plasmid ( christianson et al ., 1992 ) by bsmbi and draiii digestion . the 1591 bp fragment containing the his3gene was blunted with mung bean nuclease and ligated into the bglii digested and blunted b 1768 vector . the resulting plasmid was named b1769 . the zwf1 deletion cassette was released from the b1769 plasmid with bamhi digestion and the s . cerevisiae strain h2217 ( see example 3 ) was transformed with the fragment by li - acetate method . the deletion of the zwf1 gene was confirmed by pcr - analysis , by southern blot - analysis and by g6pdh enzyme activity assay . the cell extracts for the g6pdh enzyme activity measurement were prepared by disrupting the yeast cells in 10 mm na - phosphate ph 7 . 0 buffer using glass beads . the protease inhibitors pmsf ( final concentration 1 mm ) and pepstatin a ( 0 . 01 mg / ml ) were added into the extraction buffer . the activity was measured with cobas mira analyser ( roche ). the activity was measured in buffer containing 10 mm na - phosphate ph 7 . 0 and 1 mm nadp and 10 mm g6pdh was used as start reagent . no g6pdh activity was found in the δwf1 deletion strain . the zwf1 gene coding for the glucose 6 - phosphate dehydrogenase was deleted in a s . cerevisiae strain in which the genes for xylose reductase , xyhtol dehydrogenase and xylulokinase were integrated into the genome as described in the example 3 . the resulting strain was then transformed with a multicopy expression vector with the nadp - gapdh under the pgk1 promoter . to make this expression vector the 1 kb bamhi fragment with the nadp - gapdh as described in the example 3 was ligated to the bglii site of the b1181 vector as described in the example 4 . a control strain was made with the empty vector b1181 in the zwf1 deletion strain . four strains were compared strain 1 : gdp1 , the strain expressing the gene for the gapdh ; strain 2 : control , the strain with the empty vector ; strain 3 : gdp1 δzwf1 , the strain expressing the the gene for the gapdh in the background of a zwf1 deletion and strain 4 : δzwf1 , the strain with the zwf1 deletion and an empty plasmid . all strains have also the genes coding for d - xylose reductase , xylitol dehydrogenase and xylulokinase integrated into the genome . these strain were then used to ferment d - xylose under anaerobic conditions as described in the example 3 . the result is summariesed in the table 1 and 2 and fig8 and 10 . anaerobic shake flask cultivation on d - glucose d - xylose mixtures with 14 c - labelled d - xylose . the initial biomass was 0 . 365 g / l dry weight . 100 ml of yeast suspension in a d - glucose , d - xylose mixture as indicated , was stirred with a magnetic stirrer in a 100 ml erlenmeyer with a waterlock to ensure anaerobiosis at 30 ° c . fermentation was 75 hours . after the fermentation the ethanol was distilled from 50 ml of medium and filled up to a volume of 50 ml . the total ethanol was then measured by measuring the density of the distillate with the anton - paar dma58 density meter . the ethanol derived from d - xylose was estimated from the radioactivity of the distillate . all strains are derived from a strain with xr , xdh and xk integrated and are described in the previous example aristidou , a ., londesborough , j ., penttilä , m ., richard , p ., ruohonen , l ., söderlund , h ., teleman , a . and toivari , m ., transformed microorganisms with improved properties pct / f199 / 00185 . wo 99 / 46363 ( 1999 ) anderson a j and dawes e a ( 1990 ). occurrence , metabolism , metabolic role , and industrial uses of bacterial polyhydroxyalkanoates . microbiol . reviews 54 : 450 - 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