Patent Application: US-201414572137-A

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

Description:
the prokaryotic xylose isomerase ( xi ) according to the invention comes from clostridium phytofermentans . in this invention , it was achieved with a test system to express a highly functional prokaryotic xylose isomerase from clostridium phytofermentans in the yeast s . cerevisiae . it could be shown that the xylose isomerase found allows recombinant yeasts to efficiently metabolize xylose . the prokaryotic xylose isomerase ( xi ) according to the invention can be expressed in cells , particularly eukaryotic cells , in an active form . additionally , the prokaryotic xylose isomerase ( xi ) according to the invention is less sensitive to an inhibition by xylitol than the eukaryotic xylose isomerase from an anaerobic fungus known from the prior art . when the nucleic acid sequence coding for the prokaryotic xylose isomerase ( xi ) is expressed in a cell , the cell is imparted the capability to convert xylose to xylulose , which then may be metabolized further . through this , the cell is able to grow on xylose as a carbon source . the prokaryotic xylose isomerase ( xi ) according to the invention preferably comprises an amino acid sequence , which is at least 70 % identical , preferably at least 80 % identical , more preferably at least 90 % identical , even more preferably at least 95 % identical and yet more preferably 99 % identical or identical to the amino acid sequence of seq id no : 1 . the nucleic acid sequence coding for a prokaryotic xylose isomerase ( xi ) preferably comprises a nucleic acid sequence , which is at least 70 % identical , preferably at least 80 % identical , more preferably at least 90 % identical , even more preferably at least 95 % identical and yet more preferably 99 % identical or identical to the amino acid sequence of seq id no : 2 . the nucleic acid molecules according to the invention preferably comprise nucleic acid sequences , which are identical with the naturally occurring nucleic acid sequence or are codon - optimized for the use in a host cell . every amino acid is encrypted on a gene level by a codon . however , there are several different codons , which code for a single amino acid . thus , the genetic code is degenerated . the preferred choice of a codon for a corresponding amino acid differs from organism to organism . therefore , problems can arise in heterologously expressed genes if the host organism or the host cell has a very different codon usage . the gene can be expressed not at all or only slowly . even in genes from different metabolic pathways within an organism , a different codon usage can be discovered . it is known that the glycolysis genes from s . cerevisiae are expressed strongly . they have a very restrictive codon usage . it can be assumed that by adapting the codon usage of the bacterial xylose isomerase gene to the codon usage of the glycolysis genes from s . cerevisiae , an improvement of the xylose conversion in yeast is achieved . in a preferred embodiment , the nucleic acid sequence coding for a prokaryotic xylose isomerase ( xi ) comprises a nucleic acid sequence , which is codon - optimized for the use in a host cell . the codon - optimization substantially preferably consists in an adaptation of the codon usage to the codon usage of the host organism / host cell , such as yeast . the codon usage of the bacterial xylose isomerase gene is preferably adapted to the codon usage of the glycolysis gene from s . cerevisiae . for further details , see also example 2 and table 1 . the nucleic acid sequence coding for a prokaryotic xylose isomerase ( xi ) preferably comprises a nucleic acid sequence , which is at least 70 % identical , preferably at least 80 % identical , more preferably at least 90 % identical , even more preferably at least 95 % identical and yet more preferably 99 % identical or identical to the amino acid sequence of seq id no : 3 . the nucleic acid molecule used according to the invention is preferably a nucleic acid expression construct . nucleic acid expression constructs according to the invention are expression cassettes comprising a nucleic acid molecule according to the invention , or expression vectors comprising a nucleic acid molecule according to the invention or an expression cassette , for example . a nucleic acid expression construct preferably comprises promoter and terminator sequences , the promoter being operatively linked with the nucleic acid sequence coding for a prokaryotic xylose isomerase ( xi ). preferred promoter sequences are selected from hxt7 , truncated hxt7 , pfk1 , fba1 , pgk1 , adh1 and tdh3 . preferred terminator sequences are selected from cyc1 , fba1 , pgk1 , pfk1 , adh1 and tdh3 . the nucleic acid expression construct may further comprise 5 ′ and / or 3 ′ recognition sequences and / or selection markers . the selection marker is preferably selected from a leu2 marker gene , a ura3 marker gene and a dominant antibiotic - resistance marker gene . a preferred dominant antibiotic - resistance marker gene is selected from genes , which impart resistances to geneticin , hygromycin and nourseothricin . an expression vector can be selected from the group of prs303x , p3rs305x , p3rs306x , prs41 h , prs41 k , prs41 n , prs42h , prs42k , prs42n or p423hxt7 - 6his , p424hxt7 - 6his , p425hxt7 - 6his , p426hxt7 - 6his . the cell to be transformed is preferably a eukaryotic microorganism , preferably a yeast cell or a filamentous fungal cell . the yeast cell is preferably a member of a genus selected from the group of saccharomyces , kluyveromyces , candida , pichia , schizosaccharomyces , hansenula , kloeckera , schwanniomyces , arxula and yarrowia . the yeast cell is more preferably a member of a species selected from the group of s . cerevisiae , s . bulderi , s . barnetti , s . exiguus , s . uvarum , s . diastaticus , k . lactis , k . marxianus and k . fragilis . the filamentous fungal cell is preferably a member of a genus selected from the group of aspergillus , trichoderma , humicola , acremonium , fusarium and penicillium . the object is achieved according to the invention by providing cells , which are transformed with a nucleic acid expression construct coding for a prokaryotic xylose isomerase ( xi ). a cell according to the invention is preferably a eukaryotic cell . a cell according to the invention , particularly a eukaryotic cell , is transformed with a nucleic acid expression construct comprising : ( a ) a nucleic acid sequence coding for a prokaryotic xylose isomerase ( xi ), ( b ) a promoter operatively linked with the nucleic acid sequence , allowing for the expression of the prokaryotic xylose isomerase ( xi ) in the cell . in this connection , the expression of the nucleic acid expression construct imparts to the cell the capability to directly isomerize xylose into xylulose . as discussed above , the prokaryotic xylose isomerase ( xi ) according to the invention can be expressed in cells , particularly eukaryotic cells , in an active form such that the cells can thus directly isomerize xylose into xylulose ( see also fig2 ). additionally , the prokaryotic xylose isomerases ( xi ) according to the invention are less sensitive to an inhibition by xylitol than the eukaryotic xylose isomerases from an anaerobic fungus known from the prior art . the inventors have introduced a redox - neutral metabolic pathway into s . cerevisiae in which the conversion of xylose to xylulose takes place by means of a xylose isomerase ( xi ) ( fig2 ). when the nucleic acid sequence coding for the prokaryotic xylose isomerase ( xi ) is expressed in a cell , the cell is imparted the capability to convert xylose to xylulose , which then may be metabolized further . through this , the cell is able to grow on xylose as a carbon source . the prokaryotic xylose isomerase ( xi ) according to the invention preferably comes from clostridium phytofermentans . the xylose isomerase ( xi ) according to the invention preferably comprises an amino acid sequence , which is at least 70 % identical , preferably at least 80 % identical , more preferably at least 90 % identical , even more preferably at least 95 % identical and yet more preferably 99 % identical or identical to the amino acid sequence of seq id no : 1 . the promoter ( b ) is preferably selected from hxt7 , truncated hxt7 , pfk1 , fba1 , pgk1 , adh1 and tdh3 . in a preferred embodiment , the nucleic acid expression construct with which a cell according to the invention is transformed is a nucleic acid molecule according to the invention , as defined herein and above . the cell according to the invention is preferably a eukaryotic microorganism , preferably a yeast cell or a filamentous fungal cell . a yeast cell according to the invention is preferably a member of a genus selected from the group of saccharomyces , kluyveromyces , candida , pichia , schizosaccharomyces , hansenula , kloeckera , schwanniomyces , arxula and yarrowia . a yeast cell according to the invention is more preferably a member of a species selected from the group of s . cerevisiae , s . bulderi , s . barnetti , s . exiguus , s . uvarum , s . diastaticus , k . lactis , k . marxianus and k . fragilis . a yeast cell according to the invention is more preferably the strain ethanol red ™ or lallemand1 . a filamentous fungal cell according to the invention is preferably a member of a genus selected from the group of aspergillus , trichoderma , humicola , acremonium , fusarium and penicillium . the cell according to the invention is preferably a cell maintained in a cell culture or a cultured cell . the cells according to the invention are transiently or stably transformed with the nucleic acid expression construct or the nucleic acid molecule , as defined herein . in one embodiment , a cell according to the invention furthermore expresses one or more enzymes , which impart to the cell the capability to produce one or more further metabolization products . in this connection , such a further metabolization product is preferably selected from , but not limited to , the group of bio - based chemicals , such as lactic acid , acetic acid , succinic acid , malic acid , 1 - butanol , isobutanol , 2 - butanol , other alcohols , amino acids , 1 , 3 - propanediol , ethylene , glycerol , a β - lactam antibiotic or a cephalosporin , alkanes , terpenes , isoprenoids or the precursor molecule amorphadiene of the antimalarial drug artemisinin . the object is achieved according to the invention by using the cells according to the invention for the conversion / metabolization , particularly fermentation , of biomaterial containing xylose and / or for the production of bioethanol . the object is achieved according to the invention by using the corresponding cells according to the invention for the conversion / metabolization , particularly fermentation , of biomaterial containing xylose and / or for the production of a metabolization product . in this connection , the metabolization product is preferably selected from the group of bio - based chemicals ( but not limited to this group of bio - based chemicals ), such as lactic acid , acetic acid , succinic acid , malic acid , 1 - butanol , isobutanol , 2 - butanol , other alcohols , amino acids , 1 , 3 - propanediol , ethylene , glycerol , a β - lactam antibiotic or a cephalosporin , alkanes , terpenes , isoprenoids or the precursor molecule amorphadiene of the antimalarial drug artemisinin . the object is achieved according to the invention by providing a method for the production of bioethanol . ( a ) converting a medium containing a xylose source with a cell according to the invention , which converts xylose to ethanol , ( b ) optionally obtaining the bioethanol . the medium may also contain another additional carbon source , particularly glucose . the production of bioethanol preferably takes place at a rate of at least 0 . 03 g of ethanol per g of yeast dry weight and hour . the ethanol yield is preferably at least 0 . 3 g of ethanol per g of xylose . the object is achieved according to the invention by providing a method for the production of a metabolization product . in this connection , such a further metabolization product is preferably selected from , but not limited to , the group of bio - based chemicals , such as lactic acid , acetic acid , succinic acid , malic acid , 1 - butanol , isobutanol , 2 - butanol , other alcohols , amino acids , 1 , 3 - propanediol , ethylene , glycerol , a β - lactam antibiotic or a cephalosporin , alkanes , terpenes , isoprenoids or the precursor molecule amorphadiene of the antimalarial drug artemisinin . ( a ) converting / metabolizing , particularly fermenting , a medium containing a xylose source with a corresponding cell according to the invention , which converts xylose to produce the metabolization product , ( b ) optionally obtaining the metabolization product . the medium may also contain another additional carbon source , particularly glucose . the inventors have succeeded to introduce a redox - neutral metabolic pathway into s . cerevisiae in which the conversion of xylose to xylulose takes place by means of a xylose isomerase ( xi ) ( fig2 ). in this invention , it was achieved with a test system to express a highly functional prokaryotic xylose isomerase from clostridium phytofermentans in the yeast s . cerevisiae . it could be shown that the xylose isomerase found allows recombinant yeasts to efficiently metabolize xylose . furthermore , a plurality of experimental obstacles and difficulties had to be overcome in finding a functional xylose isomerase : 5 genes had to be overexpressed for the construction of the test strain mky09 . the choice of the xylose isomerases to be tested was not trivial . all the bacterial xylose isomerases hitherto tested showed no to very low activity in yeast . high expenditure in the cultivation of the organisms to be tested , which were needed for the screen . the xylose isomerase according to the invention is the first described highly active prokaryotic xylose isomerase in yeast . the xylose isomerase according to the invention is the first xylose isomerase of cluster ii ( of three clusters ) of xylose isomerases ( see fig3 ), which could be expressed functionally in yeasts . the xylose isomerase according to the invention is only slightly inhibited by xylitol . several reports about the difficulties with regard to the functional expression of xylose isomerases in yeast exist ( gárdonyi and hahn - hägerdahl , 2003 ; as well as reference cited therein ). the inventors have succeeded for the first time to express a prokaryotic xylose isomerase in functional form in yeasts such that they are enabled to metabolize xylose under physiological conditions and in significant quantities and to convert it to products ( e . g . ethanol ). as described in the prior art , this is not trivial . numerous attempts were made and all of them were so far unsuccessful ( see sarthy et al ., 1987 ; amore et al ., 1989 ; moes et al ., 1996 , u . s . pat . no . 6 , 475 , 768 ). the inventors have now succeeded to demonstrate that especially the c . phytofermentans xylose isomerase , in contrast to all the other , hitherto known prokaryotic enzymes , enable the yeast to metabolize xylose under physiological conditions and in significant quantities and to make products out of it . examples of lignocellulosic hydrolysates having a significant proportion of xylan ( hayn et al ., 1993 ): the present invention is clarified further in the following figures , sequences and examples , however , without being limited to these . the cited references are fully incorporated by reference herein . the sequences and figures show : seq id no : 1 the protein sequence of the xylose isomerase orf ( open reading frame ) of c . phytofermentans , ( see also genbank accession nos . abx41597 and cp000885 ( from 19 th nov . 2007 )), seq id no : 2 the nucleic acid sequence of the open reading frame ( orf ) of the xylose isomerase from c . phytofermentans , ( see also genbank accession no . cp000885 ( from 19 th nov . 2007 )), seq id no : 3 the nucleic acid sequence of the open reading frame ( orf ) of the xylose isomerase from c . phytofermentans in a codon - optimized form . fig1 . composition of biomass . biomass consists of cellulose , hemicellulose and lignin . the second most occurring hemicellulose is a highly branched polymer consisting of pentoses , uronic acids and hexoses . to a large proportion , the hemicellulose consists of the pentoses xylose and arabinose . fig2 . diagram of the conversion of d - xylose in recombinant s . cerevisiae by means of direct isomerization the genealogical tree of the tested xylose isomerases is depicted . comparisons with regard to the similarity of the xylose isomerases were performed with the program “ mega version 4 ”. fig4 a - 4c . used vectors . the starting plasmid for the construction of p426h7 - xi - clos ( 4b ) or p426h7 - opt . xi - clos ( 4c ) was the plasmid p426hxt7 - 6his ( 4a ). vector p426hxt7 - 6his is a 2μ expression plasmid , which has a ura3 marker . the open reading frame ( orf ) and its codon - optimized form of the xylose isomerase from c . phytofermentans according to the invention , respectively , was cloned behind the truncated strong hxt7 promoter and the cyc1 terminator of the plasmid p426hxt7 - 6his . fig5 a - 5c . growth behaviour on medium containing xylose using the different xylose isomerase genes growth tests of recombinant s . cerevisiae strains , which include the bacterial d - xylose metabolism with the xylose isomerase from c . phytofermentans . growth tests were performed on agar plates with sc medium and 2 % xylose as the only carbon source . the native ( 5b ) and the codon - optimized form ( 5c ) of the xylose isomerase from c . phytofermentans were tested . the empty vector p426hxt7 - 6his ( 5a ) served as the negative control . fig6 . xylose conversion in recombinant yeast strains using a bacterial xylose isomerase the xylose conversion of recombinant yeast cells mky09 , which contained the native and the codon - optimized form of the xylose isomerase from c . phytofermentans was tested . the empty vector p426hxt7 - 6his served as a comparison . growth curves were performed in liquid sc medium with 1 . 4 % xylose under aerobic conditions . hplc samples were taken in parallel to measure the optical density at 600 nm . see also table 2 , example 3 . eadie - hofstee plot of the xylose conversion of the native and the codon - optimized xylose isomerase from c . phytofermentans the strain cen . pk2 - 1c transformed with the plasmid p426h7 - xi - clos and p426h7 - opt . xi - clos , respectively , was grown over night in synthetic complete medium with 2 % glucose and no uracil . raw extracts were prepared and quantitative enzyme tests were performed . a representative result is shown . the values indicated in table 3 are average values from at least 3 independent measurements . 1 % tryptone , 0 . 5 % yeast extract , 0 . 5 % nacl , ph 7 . 5 ( see maniatis , 1982 ). for the selection for a plasmid - coded antibiotic resistance , 40 pg / ml of ampicillin was added to the medium after autoclaving . solid culture media additionally contained 2 % agar . the cultivation took place at 37 ° c . composition of the media and cultivation conditions , see information from the dsmz ( deutsche sammlung von mikroorganismen and zellkulturen , brunswick , germany ). mky09 is based on the strain cen . pk2 - 1c ( mata leu2 - 3 , 112 ura3 - 52 trpl - 289 his3 - δ1mal2 - 8c suc2 , promtkl1 :: loxp - prom - vkhxt7 , promrpe1 :: loxp - prom - vkhxt7 , promrki1 :: loxp - prom - vkhxt7 , prom gal2 :: loxp - prom - vkhxt7 , promxks1 :: loxp - prom - vkhxt7 ), including further unknown mutations . 0 . 67 % yeast nitrogen base w / o amino acids , ph 6 . 3 , amino acid / nucleobase solution , carbon source in the concentration respectively given 0 . 16 % yeast nitrogen base w / o amino acid and ammonium sulphate , 0 . 5 % ammonium sulphate , 20 mm of potassium dihydrogenphosphate , ph 6 . 3 , carbon source in the concentration respectively given concentration of the amino acids and nucleobases in the synthetic complete medium ( according to zimmermann , 1975 ): adenine ( 0 . 08 mm ), arginine ( 0 . 22 mm ), histidine ( 0 . 25 mm ), isoleucine ( 0 . 44 mm ), leucine ( 0 . 44 mm ), lysine ( 0 . 35 mm ), methionine ( 0 . 26 mm ), phenylalanine ( 0 . 29 mm ), tryptophan ( 0 . 19 mm ), threonine ( 0 . 48 mm ), tyrosine ( 0 . 34 mm ), uracil ( 0 . 44 mm ), valine ( 0 . 49 mm ). l - arabinose and d - glucose were used as the carbon source . cloning of the xi from b . licheniformi s in p426hxt7 - cloning of the xi from x . campestri s in p426hxt7 - 6his the transformation of e . coli cells was performed with the electroporation method according to dower et al . ( 1988 ) and wirth ( 1993 ) by means of an easyject prima instrument ( equibo ). the transformation of s . cerevisiae strains with plasmid dna or dna fragments was performed in accordance with the lithium acetate method according to gietz and woods ( 1994 ). the isolation of plasmid dna from e . coli was performed in accordance with the method of alkaline lysis according to birnboim and doly ( 1979 ), modified according to maniatis et al . ( 1982 ) or alternatively with the “ qiaprep spin miniprep kit ” from the company qiagen . high - purity plasmid dna for sequencing was prepared with the “ plasmid mini kit ” from the company qiagen according to the manufacturer &# 39 ; s instructions . the cells of a stationary yeast culture ( 5m1 ) were harvested by centrifugation , washed and resuspended in 400 μl of buffer b1 ( plasmid mini kit , company qiagen ). following the addition of 400 μl of buffer b2 and ⅔ of a volume of glass beads ( ø0 . 45 mm ), the cell disruption was performed by shaking for 5 minutes on a vibrax ( vibrax - vxr from janke & amp ; kunkel or ika ). ½ of a volume of buffer b3 was added to the supernatant , it was mixed and incubated for 10 min on ice . after centrifuging for 10 minutes at 13 , 000 rpm , the plasmid dna was precipitated at room temperature by adding 0 . 75 ml of isopropanol to the supernatant . the dna pelleted by centrifugation for 30 min at 13 , 000 rpm was washed with 70 % ethanol , dried and resuspended in 20 μl of water . 1 μl of the dna was used for the transformation in e . coli . minor amounts of cells were collected from bacterial cultures growing on a plate by means of a toothpick and transferred into a pcr reaction vessel . following the addition of h 2 o , 0 . 2 mm dntp mix , 1x pcr buffer ( contains 1 . 5 mm mgcl 2 ) and in each case 10 pmol of the corresponding oligonucleotide primer , the cell disruption was performed in a thermocycler from the company techne at 99 ° c . for 10 min . this batch was directly used in a pcr reaction as a template . by adding 1 u of polymerase , the polymerase chain reaction was started with a total volume of 50 μl . the dna concentration was measured spectrophotometrically in a wavelength range of 240 - 300 nm . if the purity of the dna , determined with the quotient e 260nm / e 280nm , is 1 . 8 , the extinction e 260nm = 1 . 0 corresponds to a dna concentration of 50 μg of dsdna / ml ( maniatis et al ., 1982 ). the polymerase chain reaction was performed in a total volume of 50 μl with the “ phusion ™ high fidelity pcr system ” from the company finnzymes according to the manufacturer &# 39 ; s instructions . each batch consisted of 1 - 10 ng of dna or 1 - 2 yeast gcolonies as the synthesis template , 0 . 2 mm of dntp mix , 1x buffer 2 ( contains 1 . 5 mm of mgcl 2 ), 1 u of polymerase and in each case 100 pmol of the corresponding oligonucleotide primer . the pcr reaction was performed in a thermocycler from the company techne and the pcr conditions were chosen as follows , as required : the polymerase was added after the first denaturation step (“ hot - start pcr ”). the number of synthesis steps , the annealing temperature and the elongation time were adapted to the specific melting temperatures of the oligonucleotides used or the size of the product to be expected , respectively . the pcr products were examined by means of an agarose gel electrophoresis and subsequently purified . the purification of the pcr products was performed with the “ qiaquick pcr purification kit ” from the company qiagen according to the manufacturer &# 39 ; s instructions . the separation of dna fragments having a size of 0 . 15 - 20 kb was performed in 0 . 5 - 1 % agarose gels with 0 . 5 μg / ml of ethidium bromide . 1x tae buffer ( 40 mm of tris , 40 mm of acetic acid , 2 mm of edta ) was used as the gel and running buffer ( maniatis et al ., 1982 ). a lambda phage dna cut with the restriction endonucleases ecori and hindiii served as a size standard . before application , 1 / 10 of a volume of blue marker ( 1x tae buffer , 10 % glycerine , 0 . 004 % bromophenol blue ) was added to the dna samples and they were visualized after the separation by irradiation with uv light ( 254 nm ). the desired dna fragment was cut out from the tae agarose gel under long - wave uv light ( 366 nm ) and isolated with the “ qiaquick gel extraction kit ” from the company qiagen according to the manufacturer &# 39 ; s instructions . sequence - specific cleavage of the dna with restriction endonucleases was performed for 1 hour with 2 - 5 u of enzyme per pg of dna under the incubation conditions recommended by the manufacturer . samples were taken at different times and centrifuged at 4 ° c . for 15 min at 13 , 000 rpm and 450 μl were collected from the supernatant . the protein precipitation was performed with 50 % sulphosalicylic acid . 1 / 10 of a volume of sulphosalicylic acid was added onto the samples , mixed and centrifuged for 20 min at 13 , 000 rpm at 4 ° c . the supernatant was collected and the samples could be used for the measurement after another dilution with water . samples with d - glucose , d - xylose , xylitol , acetate , glycerine and ethanol served as standards , which were employed in concentrations of 0 . 05 % w / w , 0 . 1 % w / v , 0 . 5 % w / v , 1 . 0 % w / v and 2 . 0 % w / v . the sugar concentration and the ethanol concentration were measured by means of biolc ( dionex ). the autosampler “ as50 ”, the column heater “ tcc - 100 ”, the ri detector “ ri - 101 ” ( shodex ) and the gradient pump “ gs50 ” were used in the measurement . the measurement of the samples was performed with the column va 300 / 7 . 7 nucleogel sugar 810 h ( macherey - nagel ). the column was eluted at a temperature of 65 ° c . with 5 mm h 2 so 4 as the eluent and at a flow rate of 0 . 6 ml . min − 1 . the evaluation of the data was performed with the program chromeleon version 6 . 50 ™ ( version 6 . 50 , dionex ). 50 ml of cultures of yeast cells were grown to the exponential phase in synthetic minimal medium with 2 % glucose . the cells were harvested , washed twice in tris - hcl buffer ( ph 7 . 5 ) and disrupted by means of glass beads ( ø = 0 . 45 nm ) for 8 min on a vibrax ( janke & amp ; kunkel , vibrax - vbr ) at 4 ° c . cell debris was removed by centrifugation for 10 min at 13 , 000 rpm . subsequently , the supernatant was collected and filled up to 2 ml with cold tris - hcl buffer ( ph 7 . 5 ) and used as a raw extract for the protein determination and for the measurement of the enzyme activities or the xylitol inhibition . the protein concentration was determined with the kit “ roti - quant ” from the company carl roth gmbh + co . according to the manufacturer &# 39 ; s instructions on the basis of bradford ( 1976 ). in this connection , bovine serum albumin ( bsa ) in concentrations of 0 - 100 μg / ml served as the standard . after an incubation time of at least 5 min at room temperature , the samples were measured in microtiter plates with a microtiter plate photometer from the company molecular devices at od 590 . to determine the xylose isomerase activity , recombinant yeast cells containing the vector p426h7 - xi - clos or p426h7 - opt . xi - clos , respectively , were grown , harvested and raw extracts were prepared . recombinant yeast cells containing the empty vector p426hxt7 - 6his served as a comparison . in a total volume of 1 ml , the conversion of 6 . 25 - 500 mm of xylose with 100 pl of raw extract , 0 . 23 mm of nadh , 10 mm of mgcl 2 , 2 u of sorbitol dehydrogenase in 100 mm of tris - hcl buffer ( ph 7 . 5 ) was continuously monitored . the acceptance of nadh as a measured variable was determined spectrophotometrically at a wave length of 340 nm . the reaction was started by adding xylose . to determine the xylitol inhibition of the xylose isomerase recombinant yeast cells containing the vector p426h7 - xi - clos were grown , harvested and raw extracts were prepared . recombinant yeast cells with the vector p426h7 - opt . xi - piro or the vector p426hxt7 - 6his , respectively , served as a comparison . in a total volume of 1 ml , the conversion of 6 . 25 - 500 mm of xylose with 100 μl of raw extract , 10 - 100 mm of xylitol , 0 . 23 mm of nadh , 10 mm of mgcl 2 , 2 u of sorbitol dehydrogenase in 100 mm of tris - hcl buffer ( ph 7 . 5 ) was continuously monitored . the acceptance of nadh as a measured variable was determined spectrophotometrically at a wave length of 340 nm . the reaction was started by adding xylose . in the yeast strain cen . pk2 - 1c , all the genes of the non - oxidative pentose phosphate pathway as well as the xylulokinase ( xks1 ) and gal2 were overexpressed . to this end , the endogenous promoters were replaced with the truncated hxt7 promoter . this strain was named mky09 and used for the screen for functional xylose isomerases . to make a selection of the xylose isomerases to be tested , protein sequences of xylose isomerases from the database ncbi blast were compared . an excerpt of the xylose isomerase obtained is depicted in fig3 . 14 xylose isomerases from different organisms were selected to be tested on their functionality in yeast . to this end , genomic dna was isolated from the organisms . the cells were grown , harvested and disrupted ( see “ isolation of plasmid dna from s . cerevisiae ” and “ colony pcr from b . licheniformis and s . degradans ”, respectively ). the open reading frame ( orf ) of xi from the mentioned organisms was amplified with primers additionally having homologous regions to the hxt7 promoter or cyc1 terminator . the obtained pcr products were together with the vector p426hxt7 - 6his linearized with ecori / bamhi transformed in yeast and cloned via in vivo recombination into the plasmid between the hxt7 promoter or cyc1 terminator , respectively ( fig4 ). the sequence of the plasmids obtained was verified by means of restriction analysis . furthermore , the functionality of the new isomerases and its effect on the xylose conversion in yeast was to be studied . however , it was not possible to amplify the desired pcr product with the xylose isomerase from the organisms streptomyces diastaticus and leifsonia xyli . both xylose isomerases thus could not be tested on functionality in yeast . out of the 12 different tested xylose isomerases , a xylose isomerase was found , which was functional in yeast strain mky09 . recombinant yeasts containing the xylose isomerase from c . phytofermentans showed good growth on plates containing xylose ( fig5 ). codon optimization of the gene for xylose degradation in yeast codon optimization of genes according to the codon usage of the glycolysis genes from s . cerevisiae the preferred codon usage of the glycolysis genes from s . cerevisiae was determined and is listed in table 1 . the orf of the gene xi from c . phytofermentans was codon - optimized . that is , the sequences of the open reading frame were adapted to the preferred codon usage indicated below . the protein sequence of the enzymes remained unchanged . the genes were synthesized by an external company and supplied in dried form in company - owned vectors . further details about the synthesis of genes can be found under www . geneart . com . to test the codon - optimized xylose isomerase gene in strain mky09 , the gene had to be subcloned into a yeast vector . to this end , the codon - optimized xi - orf was amplified with primers and cloned into the linearized vector p426hxt7 - 6his ( see “ execution of the screen ”). the sequence of the obtained plasmid p426h7 - opt . xi - clos was verified by means of restriction analysis . to test the functionality of the codon - optimized isomerase , the plasmid p426h7 - opt . xi - clos was transformed in the strain mky09 . recombinant yeast strains showed good growth on plates with medium containing xylose ( fig5 ). further characterizations of the native and the codon - optimized xi from c . phytofermentans followed . characterization of the functional prokaryotic xylose isomerase a ) growth behaviour and xylose conversion the growth of the strain mky09 with the native and the codon - optimized xylose isomerase from c . phytofermentans was investigated in growth tests on medium containing xylose under aerobic conditions . the empty vector p426hxt7 - 6his served as a comparison . the strains were grown in sc medium with 0 . 1 % glucose and 1 . 4 % xylose and inoculated with an od 600nm = 0 . 2 in 50 ml of sc medium with 0 . 1 % glucose and 1 . 4 % xylose . the incubation was performed in shaking flasks under aerobic conditions at 30 ° c . samples for the determination of the optical density and for the determination of the metabolite composition were taken several times . the growth curves showed that all the recombinant yeasts grew on glucose up to an od 600 of 2 . 5 ( table 2 ). after another 50 h , the yeast strain containing the native xylose isomerase from c . phytofermentans began to grow further on xylose and reached a final od 600 of 3 . 5 at a maximum growth rate of 0 . 0058 h − 1 on medium containing xylose . the yeast strain with the codon - optimized xylose isomerase likewise reached a final od 600 of 3 . 5 . the maximum growth rate was 0 . 0072 h − 1 . yeast transformants with the empty vector p426hxt7 - 6his showed no growth on xylose and began to die already after 150 h . the recombinant yeasts containing the native xylose isomerase from c . phytofermentans or the codon - optimized xylose isomerase , respectively , converted more than 2 . 6 g of xylose in 312 hours ( fig6 ). it could be shown with this experiment that the introduction of the native as well as the codon - optimized xylose isomerase from c . phytofermentans allows the recombinant s . cerevisiae strains growth on d - xylose and its conversion . by means of the codon optimization of the xylose isomerase , a higher max . growth rate could be achieved . enzyme tests were performed directly after the raw extract preparation . the xi activity was performed at 30 ° c . in a reaction mix ( 100 mm of tris - hcl , ph 7 , 5 ; 10 mm of mgcl 2 , 0 . 23 mm of nadh ; 2 u of sorbitol dehydrogenase ) with different raw extract concentrations . the reaction was started with 6 . 25 - 500 mm of xylose . the determination of the enzyme kinetics of the native form of the xylose isomerase resulted in a k m value of 61 . 85 ± 3 . 41 mm and for the codon - optimized form a k m value of 66 . 01 ± 1 mm ( fig7 and table 3 ). as expected , the k m values were thus the same as they do not differ significantly . v max ( μmol / min − 1 mg protein − 1 ) was 0 . 0076 for the native form of the xylose isomerase and 0 . 0344 for the codon - optimized form ( fig7 ). therefore , v max could be increased by more than 450 % by means of the codon optimization of the enzyme . the strain cen . pk2 - 1c transformed with the plasmid p426h7 - xi - clos and p426h7 - opt . xi - clos , respectively , was grown over night in synthetic complete medium with 2 % glucose and no uracil . raw extracts were prepared and quantitative enzyme tests were performed . the determination of the xylitol inhibition of the xylose isomerases was performed directly after the raw extract preparation . the xi activity was performed at 30 ° c . in a reaction mix ( 100 mm of tris - hcl , ph 7 . 5 ; 10 mm of mgcl 2 , 0 . 23 mm of nadh ; 2 u of sorbitol dehydrogenase ) with different raw extract concentrations . additionally , different concentrations of xylitol ( 10 - 100 mm ) were present in the reaction mix . the reaction was started with 6 . 25 - 500 mm of xylose . k was determined via the equation k m ′= k m ′= k m * ( 1 + i / k i ), i being the xylitol concentration used and k m ′ being the apparent k m value at the corresponding xylitol concentration . the determination of the kinetics of the xylitol inhibition of the xylose isomerase form c . phytofermentans resulted in a k i value of 14 . 24 ± 1 . 48 mm ( table 4 ). as already described several times ( yamanaka et al ., 1969 and references cited therein ), it is a competitive inhibition . the strain cen . pk2 - 1c transformed with the plasmid p426h7 - opt . xi - clos and p426h7 - opt . xi - piro , respectively , was grown over night in synthetic complete medium with 2 % glucose and no uracil . raw extracts were prepared and quantitative enzyme tests with constant xylitol concentrations of 10 - 100 mm were performed . the xylose isomerase from piromyces sp . e2 and the empty vector p426hxt7 - 6his served as a comparison . the determined k i value of the xylose isomerase from piromyces sp . e2 was 4 . 67 ± 1 . 77 mm . it can be seen from the determined k i values that the xylose isomerase from c . phytofermentans is significantly less inhibited by xylitol than the xylose isomerase from piromyces sp . e2 . the plasmid p426hxt7 - 6his was the starting plasmid for the construction of p426h7 - opt . xi - clos . the vector is a 2μp expression plasmid , which has a ura3 marker . further possible expression vectors are from the series of prs303x , p3rs305x and p3rs306x . these are integrative vectors , which have a dominant antibiotic marker . further details about these vectors can be found in taxis and knop ( 2006 ). the fermentation of xylose — an analysis of the expression of bacillus and actinoplanes xylose isomerase genes in yeast . xylose metabolism in a thermophilic mould malbranchea pulchella var . sulfurea tmd 8 . curr . microbiol . 29 : 349 - 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