Patent Application: US-91872209-A

Abstract:
the invention relates to a yeast cell producing isobutanol , characterized in that the cell has an increased metabolic flow of material from pyruvate and acetolactate , 2 , 3 - dihydroxy isovalerate , 2 - ketoisovalerate , isobutyraldehyde to isobutanol , in that at least one of the genes coding the enzymes , which are involved in this conversion , is over - expressed , and without any of said genes being heterologous to said yeast cell , and to a method for the production of isobutanol using yeast cells , comprising the provision of the yeast cells according to the invention , and bringing the yeast cell into contact with a fermentable carbon source .

Description:
in a possible embodiment of the invention ( see fig1 ) the enzyme ilv5 ( acetohydroxy acid reducto - isomerase ) is modified such that it preferably uses nadh instead of nadph as co - factor . at the same time , an alcohol dehydrogenase which also uses nadh as co - factor ( e . g . adh1 or adh2 - 5 or sfa1 ) is preferably but not necessarily over - expressed . ilv5 catalyzes the reduction of acetolactate to 2 , 3 - dihydroxy isovalerate accompanied by the simultaneous oxidation of nadph + h + to nadp + . as a result of the glycolytic breakdown of sugars , however , no or only small quantities of nadph form . however , nadh does form . but nadh is not easily convertible into nadph ( boles et al ., 1993 ). for this reason , it would be desirable to modify the co - factor specificity of acetohydroxy acid reducto - isomerase such that this enzyme prefers nadh instead of nadph . this can be achieved by replacing specific amino acids of ilv5 , which are required for the exclusive use of nadph , by others which also or preferably allow a use of nadh . such amino acids are preferably but not exclusively the amino acids arg108 , gly111 , ala112 and / or ser113 of the non - processed precursor enzymes which can be derived by comparing the yeast - ilv5 enzyme with the structure of the acetohydroxy acid reducto - isomerase of spinach ( biou et al ., 1997 ). arg108 can preferably but not exclusively be converted to met , trp , phe , glu or asp , gly111 preferably but not exclusively into glu or asp , ala112 preferably but not exclusively into ser or gly and ser113 preferably but not exclusively into glu or asp . however , it is not to be ruled out that the exchange of further or different amino acids also leads to a modification of the co - factor specificity of ilv5 in favour of nadh . in another possible embodiment of the invention ( see fig2 ) the glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ) of the yeast is modified such that it prefers nadp + instead of nad + , or is replaced or supplemented by a glyceraldehyde - 3 - phosphate dehydrogenase which prefers nadp + compared with nad + . at the same time , an alcohol dehydrogenase which prefers nadph as co - factor ( e . g . adh6 or ypr1 ) is preferably but not necessarily over - expressed . gapdh is encoded e . g . in s . cerevisiae by the genes tdh1 ( seq . id . no . 41 ), tdh2 ( seq . id . no . 43 ) and tdh3 ( seq . id . no . 45 ) and catalyzes the oxidation of glyceraldehyde - 3 - phosphate accompanied by simultaneous phosphorylation to 1 , 3 - diphosphoglycerate . during the glycolytic breakdown of sugars , nad + is usually used as co - factor , and nadh + h + forms . nadh is not easily convertible into nadph ( boles et al ., 1993 ). however , as the acetohydroxy acid reducto - isomerase nadph is used as co - factor , it would be desirable to modify the co - factor specificity of gapdh such that this enzyme prefers nadp + instead of nad + . a modification of the co - factor specificity of the yeast - gapdh can be achieved by replacing specific amino acids of tdh1 ( seq . id . no . 42 ), tdh2 ( seq . id . no . 44 ) and / or tdh3 ( seq . id . no . 46 ), which are required for the exclusive use of nad + , by others which also or preferably allow a use of nadp + . such amino acids are preferably but not exclusively the amino acids asp33 and / or gly188 - pro189 , which can be derived by comparing the yeast - gapdh enzymes with the structure of nadp + - preferred gapdhs ( fillinger et al ., 2000 ). asp33 can preferably but not exclusively be converted to asn , gly , ala or ser , gly188 - pro189 preferably but not exclusively into ala - ser , val - arg , asn - pro or thr - lys . however , it is not to be ruled out that the exchange of further or different amino acids also leads to a modification of the co - factor specificity of tdh1 - 3 in favour of nadp + . alternatively , a heterologous gapdh which preferably uses nadp + , e . g . but not exclusively gdp1 ( seq . id . no . 48 ) from kluyveromyces lactis ( verho et al ., 2002 ) or gapb ( seq . id . no . 50 ) from bacillus subtilis ( fillinger et al ., 2000 ) could be over - expressed in yeast . the nadp - gapdh can be over - expressed codon - optimized in a preferred embodiment . the mutated or heterologous nadp - gapdhs can be expressed in addition to the nad - gapdhs present or in a preferred version in yeast mutants with reduced or switched - off nad - gapdh expression or activity . proteins that are transported into the mitochondrial matrix are synthesized as precursor proteins in the cytosol and then transported via translocases into the mitochondrial matrix . the n - terminal presequences are split from a mitochondrial peptidase during translocation . in a preferred embodiment , but not necessarily , the genes ilv2 , ( ilv6 ), ilv5 or ilv5 ( nadhmut .) and ilv3 are over - expressed without the mitochondrial targeting sequence of the corresponding proteins or with a broken , inactivated mitochondrial targeting sequence , with the result that the produced proteins are preferably located in the cytosol of the yeast cells ( pang and duggleby , 1999 ; omura , 2008 ). this can be carried out with the natural or codon - optimized alleles . pyruvate can be further converted by various reaction paths . the quantitatively strongest of these reaction paths is its conversion into ethanol . pyruvate is decarboxylated to acetaldehyde and further to ethanol by the pyruvate decarboxylases ( pdcs ). pyruvate is lost to the production of isobutanol . in a preferred embodiment of the invention , but not necessarily , the flow of the pyruvate to ethanol is therefore blocked or reduced by switching off or reducing the pyruvate decarboxylase expression or activities . this is carried out e . g . by deleting or reducing the expression of the genes pdc1 , pdc5 and / or pdc6 . however , as yeast requires the acetyl - coa produced in the cytosol from acetaldehyde , this must also be made available when the pyruvate decarboxylases are completely switched off . this is carried out either ( i ) by an incomplete switching - off of the expression or activity of the pyruvate decarboxylases , ( ii ) by expression of a heterologous pyruvate - formiate lyase with its activating enzyme including the over - expression of a formiate dehydrogenase , ( iii ) by heterologous expression of a reversible mitochondrial carnitine carrier or ( iv ) by the introduction of spontaneous suppressor mutations . in addition , the possibility of reducing or switching off further metabolic reactions in order to intensify the flow of the intermediate metabolites to isobutanol still remains . furthermore , the production of isobutanol as well as the resistance to toxic concentrations of isobutanol in the recombinant yeast cells can be further increased by random mutagenesis or the “ evolutionary engineering ” or “ directed evolution ” methods ( sauer , 2001 ). the present invention furthermore relates to a method for the production of isobutanol with yeast cells , comprising the provision of a yeast cell as defined above as well as bringing the yeast cell into contact with a fermentable carbon source . in a preferred embodiment , the method according to the invention is characterized in that the fermentable carbon source is a c3 - c6 carbon source . in a further preferred embodiment , the method according to the invention is characterized in that the carbon source belongs to the group consisting of monosaccharides , oligosaccharides or polysaccharides . in a further preferred embodiment , the method according to the invention is characterized in that the carbon source belongs to the group consisting of glucose , fructose , mannose , galactose , saccharose , maltose , xylose or arabinose . in a further preferred embodiment , the method according to the invention is characterized in that the host cell is brought into contact with the carbon source in culture medium . full medium lb 1 % trypton , 0 . 5 % yeast extract , 0 . 5 % nacl , ph 7 . 5 ( see maniatis , 1982 ). for the selection for a plasmid - encoded resistance to antibiotics 40 μg / ml ampicillin was added to the medium after autoclaving . solid nutrient media also contained 2 % agar . culture took place at 37 ° c . synthetic complete selective medium sc : 0 . 67 % yeast nitrogen base w / o amino acids , ph 6 . 3 , amino acid / nucleobase solution , carbon source in the respective given concentration synthetic minimal selective medium sm : 0 . 16 % yeast nitrogen base w / o amino acid and ammonium sulphate , 0 . 5 % ammonium sulphate , 20 mm potassium dihydrogen phosphate , ph6 . 3 , carbon source in the respective given concentration synthetic fermentation medium ( mineral medium ) sfm : ( verduyn et al ., 1992 ), ph 5 . 5 salts : ( nh 4 ) 2 so 4 , 5 g / l ; kh 2 po 4 , 3 g / l ; mgso 4 * 7h 2 o , 0 . 5 g / l trace elements : edta , 15 mg / l , znso 4 * 4 . 5 mg / l ; mncl 2 * 4h 2 o , 0 . 1 mg / l ; cocl 2 * 6h 2 o , 0 . 3 mg / l ; cuso 4 , 0 . 192 mg / l ; na 2 moo 4 * 2h 2 o , 0 . 4 mg / l ; cacl 2 * 2h 2 o , 4 . 5 mg / l ; feso 4 * 7h 2 o , 3 mg / l ; h 3 bo 3 , 1 mg / l ; ki , 0 . 1 mg / 1 vitamins : biotin , 0 . 05 mg / l ; p - aminobenzoic acid , 0 . 2 mg / l ; nicotinic acid , 1 mg / l ; calcium pantothenate , 1 mg / l ; pyridoxine - hcl , 1 mg / l ; thiamine - hcl , 1 mg / l ; minositol , 25 mg / l 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 ), tryptophane ( 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 carbon source . solid full and selective media also contained 1 . 8 % agar . cultivation of the yeast cells took place at 30 ° c . the synthetic mineral medium used for the fermentations contained salts , trace metals and vitamins in the concentrations listed above and l - arabinose as carbon source . a parent solution of the trace metals and vitamins was prepared . both solutions were sterile - filtered . both were stored at 4 ° c . the ph was a decisive factor for the production of the trace metal solution . the different trace elements had to be fully dissolved in water one after the other in the order given above . after every addition , the ph had to be adjusted to 6 . 0 with koh before the next trace element could be added . at the end , the ph was adjusted to 4 . 0 with hcl . to avoid foaming , 200 μl antifoam ( antifoam2004 , sigma ) was added to the medium . as the experiments were carried out under anaerobic conditions , 2 . 5 ml / l of a tween80 - ergosterol solution also had to be added to the medium after autoclaving . this consists of 16 . 8 g tween80 and 0 . 4 g ergosterol , which were made up to 50 ml with ethanol and dissolved therein . the solution was sterile - filtered . the salts and the antifoam were autoclaved jointly with the complete fermenter . the arabinose was autoclaved separately from the rest of the medium . after cooling of the medium , the trace elements as well as the vitamins were added . the transformation of the e . coli cells took place using the electroporation method according to dower et al . ( 1988 ) and wirth ( 1993 ) by means of an easyject prima device ( equibo ). the transformation of s . cerevisiae strains with plasmid dna or dna fragments took place according to the lithium acetate method of gietz and woods ( 1994 ). the isolation of plasmid dna from e . coli took place according to the alkaline lysis method of birnboim and doly ( 1979 ), modified according to maniatis et al . ( 1982 ) or alternatively with the qiagen “ qiaprep spin miniprep kit ”. high - purity plasmid dna for sequencing was prepared with the qiagen “ plasmid mini kit ” according to the manufacturer &# 39 ; s instructions . the cells of a stationary yeast culture ( 5 ml ) were harvested by centrifugation , washed and resuspended in 400 μl buffer p1 ( plasmid mini kit , qiagen ). following the addition of 400 μl buffer p2 and ⅔ volume glass beads ( ø 0 . 45 mm ), cells were broken by 5 minutes &# 39 ; shaking on a vibrax ( vibrax - vxr from janke & amp ; kunkel or ika ). ½ volume buffer p3 was added to the supernatant , and the whole mixed and incubated on ice for 10 min . after 10 minutes &# 39 ; centrifugation at 13000 rpm , the plasmid dna was precipitated at room temperature by adding 0 . 75 ml isopropanol to the supernatant . the dna pelleted by centrifugation for 30 min at 13000 rpm was washed with 70 % ethanol , dried and resuspended in 20 μl water . 1 μl of dna was used for the transformation into e . coli . the dna concentration was spectrophotometrically measured in a wavelength range of from 240 - 300 nm . if the purity of the dna , determined by the quotient e 260nm / e 280nm , is 1 . 8 , the absorbance e 260nm = 1 . 0 corresponds to a dna concentration of 50 μg dsdna / ml ( maniatis et al ., 1982 ). the polymerase chain reaction was carried out in a total volume of 50 μl with the finnzymes “ phusion ™ high fidelity pcr system ” in accordance with the manufacturer &# 39 ; s instructions . each batch consisted of 1 - 10 ng dna or 1 - 2 yeast colonies as synthesis template , 0 . 2 mm dntp - mix , 1 × buffer 2 ( contains 1 . 5 mm mgcl 2 ), 1 u polymerase and 100 pmol of each of the corresponding oligonucleotide primer . the pcr reaction was carried out in a techne thermocycler and the pcr conditions selected as follows according to the requirements : after the first denaturation 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 used or to the size of the expected product . the pcr products were examined by an agarose gel electrophoresis and then purified . the purification of the pcr products was carried out with the “ qiaquick pcr purification kit ” from qiagen in accordance with the manufacturer &# 39 ; s instructions . the separation of dna fragments measuring 0 . 15 - 20 kb was carried out in 0 . 5 - 1 % agarose gels with 0 . 5 μg / ml ethidium bromide . 1 × tae buffer ( 40 mm tris , 40 mm ethyl acetate , 2 mm edta ) was used as gel and running buffers ( maniatis et al ., 1982 ). a lambda phage dna cut with the restriction endonucleases ecori and hindiii served as marker . 1 / 10 volume blue marker ( 1 × tae buffer , 10 % glycerol , 0 . 004 % bromphenol blue ) was added to the dna samples before application and made visible after 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 qiagen “ qiaquick gel extraction kit ” in accordance with the manufacturer &# 39 ; s instructions . sequence - specific splitting of the dna with restriction endonucleases was carried out under the manufacturer &# 39 ; s recommended incubation conditions for 1 hour with 2 - 5 u enzyme per μg dna . further possible expression vectors are from the prs303x , p3rs305x and p3rs306x series . these are integrative vectors which have a dominant antibiotic marker . further details about these vectors are to be found in taxis and knop ( 2006 ). for an in - vivo cloning of dna fragments in s . cerevisiae , first the corresponding gene or dna sequence is synthesized by a pcr reaction . the therein used oligonucleotides each contain in the 5 ′ region 36 - 39 nucleotides comprising specific appendages which are homologous to the 5 ′- or 3 ′- flanking sequences of the integration region in the target vector . in the 3 ′ region , the oligonucleotides contain 20 - 22 bases homologous to the 3 ′ or 5 ′ ends of the gene to be amplified . the pcr product produced was transformed into yeast together with the vector linearized and purified by restriction in the integration region . the cells were plated out onto synthetic selective medium which lacked the corresponding amino acid or nucleotide base for the selection on the auxotrophic marker of the vector . in this way , only transformants which had again formed a stable , circular plasmid due to homologous recombination of the dna fragment in the linearized vector were obtained . the plasmids were isolated , amplified in e . coli and examined by subsequent restriction analysis , or by sequencing . this was carried out as described in becker and boles ( 2003 ) and wieczorke et al . 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