Patent Application: US-201314652133-A

Abstract:
described herein is a method to increase ethanol yield during alcoholic fermentation by expression a truncated versions of the saccharomyces cerevisiae pho8 gene coding for vacuolar or cytosolic form of alkaline phosphatase , which expression lowers biomass accumulation while diverting greater carbon to ethanol production . also described are as nucleic acid sequences and vectors for expression of the pho8 gene and strains carrying the same . strains containing intact pho8 gene encoding vacuolar form of alkaline phosphatase , had slightly lower intracellular atp levels with insignificant changes in biomass accumulation and up to 13 % increase in ethanol production .

Description:
the saccharomyces cerevisiae strain by4742 ( matα , his3δ1 , leu2δ0 , lys2δ0 , ura3δ0 ; ( giaever et al ., 2002 ) and an industrial strain designated as400 were used for the expression of the intact or truncated versions of pho8 orf coding for the alkaline phosphatase , escherichia coli dh5α ( φ80dlaczδm15 , reca1 , enda1 , gyra96 , thi - 1 , hsdr17 ( r k + , m k + ), supe44 , rela1 , deor , δ ( laczya - argf ) u169 ) was used for general purposes and routine subcloning . for the isolation of plasmid dna e . coli strains were grown in lb media at 37 ° c . for 18 hours as described ( sambrook and rusell 2001 ). s . cerevisiae strains were incubated at 30 ° c . for routine application , yeast strains were maintained in a rich ypd medium ( 1 % yeast extract , 1 % peptone and 2 % glucose ) or in a minimal medium of ynb ( 0 . 67 %, yeast nitrogen base without amino acids , difco , 0 . 5 % ammonium sulfate , 2 % glucose ) media . for ethanol fermentation , ynb medium was supplemented with 10 % glucose or corn steep liqour ( csl ) medium supplemented with hydrolyzed corn maltodextrin were used . when antibiotic selection was needed , strains were incubated with ampicillin ( 100 μg ml − 1 ) or geneticin ( 200 mg l − 1 ). when required , histidine ( 20 mg l − 1 ), leucine ( 60 mg l − 1 ), lysine ( 20 mg l − 1 ), or uracil ( 20 mg l − 1 ) were added . chromogenic substrates x - gal and iptg ( fermentas , vilnius , lithuania ) were used according to the manufacturer specifications . genomic dna from s . cerevisiae strains was isolated using the wizard ® genomic dna purification kit ( promega , madison , wis . usa ). plasmid dna from e . coli was isolated using the wizard ® plus sv minipreps dna purification system ( promega ). taq and high fidelity polymerase mix , t4 dna ligase , t4 dna polymerase and restriction enzymes were used according to recommendation of supplier ( fermentas ). s . cerevisiae transformation was performed by standard protocol ( sambrook and russell 2001 ). 154 bp part of s . cerevisiae yjrwdelta12 sequence was amplified from genomic dna of s . cerevisiae strain by4742 using the primers sm16 ( ccg gaa ttc gac ggg cag tc t gtt gga ata gaa atc aac tat c ) and sm17 ( cat cat ttt ata tgt tta tat tca tct aga ccc ggg gtc gac ttg atc cta tta cat tat caa tcc ) and the other 180 bp part of this sequence was amplified from the same template using the primers sm18 ( gga ttg ata atg taa tag gat caa gtc gac ccc ggg tct aga tga ata taa aca tat aaa atg atg ) and sm19 ( ccc aag ctt gac ggg cag tc t gag aaa tat gtg aat gtt gag ). then these two parts containing delta sequences were fused via overlap pcr using primers sm16 and sm19 , digested with ecori and hindiii and cloned into ecori / hindiii - linearized plasmid puc57 . the resulted plasmid was named puc57 - delta1_2 , 807 bp dna fragment corresponding to adh1 gene ( encoding alcohol dehydrogenase ) promoter was amplified from genomic dna of s . cerevisiae strain by4742 using primers ko419 ( cgc gtc gac tta att aaa gtc caa tgc tag ) and ko420 ( gat atc gac aaa gga aaa ggg gcg gcc gcg gat cc c tcg agt gta tat gag ata gtt gat tg ). a 269 bp dna fragment corresponding to cyc1 gene ( encoding cytochrome c ) terminator was amplified from genomic dna of by4742 strain using primers then promoter and terminator were fused via overlap pcr using printers ko419 and ko454 . the obtained dna fragment was digested with sali and xmai endonucleases and cloned into corresponding sites of the plasmid puc57 - delta1_2 giving the plasmid puc57 - delta1_2 - adhpr - cyct . a 1470 bp dna fragment corresponding to the selective marker kanmx providing resistance to geneticin was cut out from the plasmid prs303k with restriction endonucleases saci and smai , blunt - ended and cloned into xbai - digested and blunted plasmid puc57 - delta1_2 - adhpr - cyct yielding the plasmid puc57 - delta1_2 - adhpr - cyct - kanmx ( fig1 a ). a 1701 bp dna fragment bearing the orf of pho8 gene coding for unspecific alkaline phosphatase was amplified from genomic dna of s . cerevisiae strain by4742 using primers this fragment was digested with bamhi and noti and subcloned into bamhi / noti digested plasmid puc57 - delta1_2 - adhpr - cyct - kanmx . the resulted plasmid was designated puc57 - delta1_2 - adhpr - pho8 - cyct - kanmx ( fig1 b ). it was decided to create several truncated forms of alkaline phosphatase . the first form lacks the 60 initial amino acids that are responsible for transmembrane protein delivery ; and 22 terminal amino acids , that compose the c - terminal propeptide that is normally cleaved from the protein in vacuole . a 1452 bp dna fragment corresponding to this truncated form , was amplified from genomic dna of by4742 strain using primers digested with endonucleases bamhi and noti and cloned into bamhi / noti digested plasmid puc57 - delta1_2 - adhpr - pho8 - cyct - kanmx . the obtained plasmid was designated puc57 - delta1_2 - adhpr - pho8_trunc - cyct - kanmx ( fig2 a ). the second form of alkaline phosphatase was constructed that has extracellular export signal from mating pheromone a - factor ( mf1α ) instead of its natural delivery signal . to construct such a form , the 258 bp dna fragment corresponding to mf1α export signal , was amplified from genomic dna of by4742 strain using primers sm31 ( cgc gga tcc atg aga ttt cct tca att ttt act gca g ) and sm32 ( gac att ctt ctt ctt gtg tga tgc aga ctc tct ttt atc caa aga tac ccc ), and the 1452 bp dna fragment corresponding to pho8 truncated form , was amplified from the same template using primers and sm29 . then both fragments were fused by overlap - pcr using primers sm31 / sm29 , digested with endonucleases bamhi and noti and cloned into bamhi / noti digested plasmid puc57 - delta1_2 - adhpr - pho8 - cyct - kanmx . the constructed plasmid was designated puc57 - delta1_2 - adhpr - matαsign .- pho8_trunc - cyct - kanmx ( fig2 b ). the third form of alkaline phosphatase has the extracellular export signal from acid phosphatase ( pho5 ). to construct such a form , a 1503 bp dna fragment corresponding to pho8 truncated form was fused with pho5 delivery signal , and amplified from genomic dna of by4742 strain using primers sm30 ( cgc ggatcc atg ttt aaa tct gtt gtt tat tca att tta gcc gct tct ttg gcc aat gca tct gca tca cac aag aag aag aat gtc ) and sm29 ( ttt gcg gcc gc t caa tct gat gtg tgt ttg gtg tcc cta atc ), digested with endonucleases bamhi and noti and cloned into bamhi / noti digested plasmid puc57 - delta1_2 - adhpr - pho8 - cyct - kanmx . the constructed plasmid was designated as puc57 - delta1_2 - adhpr - pho5sign .- pho8_trunc - cyct - kanmx ( fig2 c ). the vectors containing full or truncated versions of the pho8 gene were digested with the ahdi restriction endonuclease . after that dna fragments containing expression cassette and selective marker flanked by two parts of delta sequences , were eluted from an agarose gel and used for transformation of s . cerevisiae strains by4742 and as400 . the transformants were selected on a solid ypd medium supplemented with 200 mg / l of geneticin . the selected transformants were stabilized by alternating cultivation in non - selective and selective media and examined by diagnostic pcr using a forward primer specific to the adh1 promoter ( ko419 ) and a reverse one specific to pho8 gene ( ko509 ). alkaline phophatase activity was assayed by using p - nitrophenylphosphate as the substrate with cell extracts as the enzyme source , as described elsewhere ( kaneko et al ., 1982 ). one unit of alkaline phosphatase activity was defined as the amount of enzyme which liberates 1 μmol of p - nitrophenol per min under the assay conditions . atp extraction from the s . cerevisiae cells was performed using the method of metabolites extraction with boiling ethanol and subsequent evaporation ( entian et al . 1983 ). to optimize atp assay via ethanol extraction , a wide range of yeast biomass from 2 to 15 mg was tested . reproducible results were obtained during atp extraction with 8 - 10 mg of s . cerevisiae cells using 1 ml of boiling ethanol . the measurement of extracted atp level was determined using a mixture of hexokinase and glucose - 6 - phosphate dehydrogenase ( ano y . et al . 2005 ). strains were grown overnight in ypd media followed by washing of cells with sterile water and an equal amounts of each culture were used to inoculate test media . for s . cerevisiae by4742 strain and its pho8 - expressing derivatives , ynb supplemented with 10 % glucose was used . strains were grown using a rotary shaker inkubator 1000 heidolph ( schwabach , germany ) under semi - anaerobic ( 120 revolutions / min ) conditions at 30 ° c . and samples were taken every 24 hours . for s . cerevisiae as400 strain and its pho8 - expressing derivatives , a corn steep liquor ( csl ) medium supplemented with hydrolyzed maltodextrin was used . strains were incubated under semi - anaerobic condition at 34 ° c . for 2 days and samples were taken every 24 hours . ethanol and glucose concentrations were measured by protocols described elsewhere ( gonchar et al ., 2001 , gonchar , 1998 ). to prepare solution 1 , 200 - 280 g of maltodextrin was mixed with 800 ml of water and the ph adjusted to 6 . 0 . alpha - amylase ( at a rate of 0 . 1 unit of liquizyme sc ds per grant of maltodextrin ) was added for liquefaction with the mixture heated to 80 ° c . and sample held for 30 minutes . to prepare solution 2 , 63 ml of csl concentrate ( containing ˜ 50 % dry solids ) was mixed with 137 ml of deionized water . following that solutions 1 and 2 were autoclaved , cooled , combined and mixed . an aliquot of glucoamylase ( at a rate of 0 . 2 units of glucoamylase per gram of maltodextrin ) was added to sterile flasks and the flasks were incubated at 28 ° c . glucose concentration was determined before yeast inoculation . s . cerevisiae unspecific alkaline phosphatise , encoded by gene pho8 , catalyzes the hydrolysis of the diphosphate bonds in different compounds including atp ( kaneko et al ., 1982 ). therefore this enzyme may operate as other atpases . in order to carry out the hydrolysis of diphosphate bonds in cells the activity of this enzyme need to be quite high . the activity of this cloned enzyme in s . cerevisiae cells depends on many factors , among them strength of chosen promoter , stability of produced mrna , absence of posttranslational enzyme inhibition , and the number of copies of the gene integrated into the host genome . we have shown that adh1 promoter of the gene encoding alcohol dehydrogenase provides inducible expression of target genes , under the conditions of alcoholic fermentation ( sibirny et al , 2011 ). we decided to use this promoter for pho8 gene expression . in order to get higher copy number of pho8 gene integrated into the yeast genome , an integrative plasmid containing δ sequences was constructed . the s . cerevisiae δ sequences are the long terminal repeats of the retro - transposons ty1 and ty2 . a total of approximately 425 δ sequences are typically dispersed throughout the yeast genome . it was shown that usage of δ sequences - based vectors , can provide tandem multicopy integration in one or several sites of yeast genomic dna via homologous recombination ( lee and da silva , 1997 ). the δ sequences - based plasmid harboring pho8 gene under the control of adh promoter was constructed as described in materials and methods and subsequently transformed into s . cerevisiae laboratory strain by4742 . the specific alkaline phosphatase activity of the selected recombinant strains was assayed . among 11 tested transformants , six strains were characterized by considerably higher specific phospatase activity ( fig3 , strains 1 - 5 and 11 ). in these strains , the specific activity of alkaline phospatase was 30 - 40 - fold higher when compared to the initial host strain by4742 . thus , this constructed expression vector can be used for efficient multi - copy integration into the genome of a saccharomyces host . genomic dna preparations isolated from the recombinant strains , was subjected to dot - blot hybridization to estimate plasmid copy number which harbour the target gene , integrated into the genome . gene pho8 was used as a probe ( fig4 ). it was revealed that recombinant strains contained from 1 to 7 - 9 additional copies of pho8 gene . a good correlation between copies number of pho8 gene and specific alkaline phospatase activity was observed . the southern - hybridization was performed to analyse the vector integration pattern of the tested strains ( fig5 , a ). total genomic dna from wt and recombinant strains were hindiii digested and hybridized with a labelled pho8 gene . it was shown that constructed δ sequences - based vector provide tandem multi - copy integration in up to three sites of yeast genomic dna in so called “ head - to - tale ” conformation ( fig5 , b ), which is consistent with published results ( lee and da silva , 1997 ). surprisingly , several of the selected recombinant strains revealed impaired biomass accumulation , which does not correlate with the increased alkaline phosphatise activity level ( fig6 ). the plasmid puc57 - delta1_2 - adhpr - pho8 - cyct - kanmx was used for transformation of industrial strain as400 . selected transformants possess at the most 5 . 5 - fold increase of specific activity of alkaline phospatase as compared with parental strain as400 ( fig7 ). recombinant strains with the highest specific activity of alkaline phospatase were subjected to tested for the efficiency of alcoholic fermentation on a csl medium supplemented with hydrolyzed corn meal . strains as400 +- pho8 - 6 , as400 + pho8 - 8 , as400 + pho8 - 3b , as400 + pho8 - 12 produced increased amount of ethanol ( 84 . 4 , 86 . 5 , 84 . 4 , 90 . 1 g / l ) as compared with the parental strain ( 79 . 1 g / l ) ( table 1 ). some of the analyzed recombinant strains possessed slightly lower intracellular atp level than that of wild type strain as400 ( fig8 ). as described herein , some of the recombinant strains bearing plasmid puc57 - delta1_2 - adhpr - pho8 - cyct - kanmx had up to 30 fold increase in alkaline phosphatase activity , but their growth level did not differ substantially from that of the wt strain . this result can be explained by the vacuolar localization of the alkaline phosphatase that may hinder atp hydrolysis . in order to test we decided to construct strains containing gene encoding truncated version of alkaline phosphatase that will remain in the cytoplasm instead of being delivered to the vacuole . however there are several drawbacks that prevent that obstruct the generation of an active cytosolic form of alkaline phosphatase . first , it was shown that alkaline phosphatase is synthesized as an inactive precursor containing a c - terminal propeptide that is afterwards cleaved from the protein in vacuole in a pep4 - dependent manner ( klionsky d . and emr s . d . 1989 ). during vacuolar delivery , which shares the same early stages with the secretory pathway , the precursor form of this enzyme is glycosylated in the endoplasmic reticulum . it has also been shown that the active form of alkaline phosphatase receives its metal cofactor zinc in the vacuole rather than in earlier compartments of the secretory pathway ( qiao w . et al . 1985 ). to estimate how these factors influence this enzyme &# 39 ; s activity , we decided to produce several truncated forms of alkaline phosphatase . the first form lacks 60 initial amino acids that are responsible for transmembrane protein delivery ; and 22 terminal amino acids , that composite the c - terminal propeptide that is normally cleaved from the protein in the vacuole . the second form has an extracellular export signal from mating pheromone a - factor ( mf1α ) instead of its natural delivery signal , and , finally , the third form constructed has an extracellular export signal from acid phosphatase ( gene pho5 ). the dna fragments containing the expression cassette of three mentioned pho8 modified forms in part with selective marker flanked by δ sequences were used to transform s . cerevisiae strain by4742 . the presence of these expression cassettes was confirmed after transformation by pcr . the specific alkaline phosphatase activity of the obtained recombinant strains was assayed and compared with the activity of transformants with intact form of pho8 . the intracellular alkaline phosphatase activity in strains containing plasmid puc57 - delta1_2 - adhpr - pho8_trunc - cyct - kanmx was at the same level as in strains with intact form of pho8 . intracellular alkaline phosphatase activity in strains containing plasmid puc57 - delta1_2 - adhpr - matαsign .- pho8_trunc - cyct - kanmx was very low , almost in the same level as in wt strain . most likely this form of alkaline phosphatase is secreted from the cells . the intracellular alkaline phosphatase activity of strains containing plasmid puc57 - delta1_2 - adhpr - pho5sign .- pho8_trunc - cyct - kanmx was high on the first day of cultivation . this activity decreased on the second day of cultivation and this can be explained by the delay in the export of the secreted form of alkaline phosphatase . ( fig9 a , b ). growth kinetics of the constructed recombinants was tested . almost all strains with different truncated pho8 forms had impaired growth on the first and , to a lesser degree by the second day of cultivation ( fig1 ) these recombinant strains especially those that had a truncated form of alkaline phospatase that remains in the cell &# 39 ; s cytoplasm produced significantly less ethanol , also ( fig1 ). this indicates that the cytosolic form of alkaline phosphatase also strongly influences the level of other cellular phosphorylated compounds , that affect all of the metabolic pathways , in particular , the reactions responsible for alcoholic fermentation . in conclusion and on the basis of this work , the intact vacuolar form of alkaline phosphatase is suitable for achieving higher ethanol in s . cerevisiae cells . ano y ., hattori t ., kato n ., sakai y . 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