Patent Application: US-95245798-A

Abstract:
an isolated dna encoding phenyl acetyl - coa - ligase and a process of increasing the production of penicillin g in a strain of penicillium chrysogenum by transforming the strain with the isolated dna . also vectors and host organisms having the isolated dna .

Description:
1 . isolation of the gene which codes for the enzyme phenylacetyl - coa ligase in pseudomonas putida u the strain of pseudomonas putida u , which had phenylacetyl - coa ligase activity when grown in the mm described in ref . 9 , was mutated by the insertion of the transposon tn5 ( ref . 11 ), as is detailed in the protocol shown in fig2 . the strains which were unable to break down phenylacetic acid were selected , which suggested that the insertion had occurred in one of the genes , or intergenic regions , corresponding to the catabolic pathway of this aromatic compound . in all the mutants pcl activity was assayed as described in spanish patent p8902421 and in the corresponding publication ( ref . 9 ). for this purpose the various mutants were grown in the same mm , but it now contained , as carbon sources , 4 - hydroxyphenylacetic acid ( 4 - ohpa ), which does not induce pcl , and phenylacetic acid ( pa ), which , although it cannot be broken down , could induce pcl ( ref . 12 ). in this mm the 4 - ohpa is used by the bacteria to sustain cell growth whereas the pa acts as an inducer of the enzyme phenylacetyl - coa ligase . by this simple procedure the various mutants were characterized in such a way that two groups could be established : a ) those which possessed functional pcl ( called pcl +) and in which the transposon tn5 had thus inserted itself into a gene on the pathway ( or into an intergenic region ) after the gene coding for pcl , and b ) the others in which this activity could not be detected ( called pcl −). the absence of pcl in this second group of mutants could be due to two reasons : 1 ) it could be due to the fact that the transposon had inserted itself in front of the gene coding for the ligase ( pcl ) ( if , as was suspected , all the catabolic pathway responsible for the breakdown of pa is under the control of one promoter ), or it could be due to the fact that 2 ) the tn5 had incorporated itself into the pcl gene itself , or into a regulator gene or sequence . from one of the mutants in which no pcl activity was detected , called e 1 , the insertion of tn5 was identified by the use of oligonucleotide sequences which were exactly the same as the ends of tn5 ( 5 ′ d 3 ′: act tgt gta taa gag tca g seq id no : 13 ) and which had been radioactively labelled . the zone of the e 1 mutant genome linked to the transposon was cloned in the plasmid puc 18 and the escherichia coli strain d5α ′ was transformed in accordance with the conventional protocols ( ref . 13 ). the insert was then sequenced and the gene which hybridized with the gene sequence isolated from the mutant e 1 , and which corresponded to the adjacent zone of the transposon , was searched for in a pseudomonas putida u dna library produced in the phage λ embl4 . of all the phages which gave positive hybridization , three which contained 13 , 15 and 18 kb fragments of the genomic dna of pseudomonas putida were selected . from one of these , the one which contained a 13 kb insert , dna was extracted ( ref . 13 ), it was cleaved with the restriction enzyme ecori , and a 10 kb fragment was selected . when this fragment of escherichia coli dh5α ′ was introduced , using the plasmid puc 18 as transformation vector , the presence of the insert gave the latter bacterium the ability to grow in mm which contained pa as the sole carbon source . growth was slower , however , than that observed in pseudomonas putida u — which suggested that although it contained genes which made catabolism of this compound possible , its breakdown rate in escherichia coli was much slower . this effect could be due either to the fact that ( i ) a gene ( or genes ) needed for total breakdown of pa is ( are ) missing or incomplete in the fragment — which would force the bacterium to replace it ( them ) with another one ( or others ) present in its own genome , so as to allow it to use the accumulated catabolite , albeit more slowly ; or to the fact that ( ii ) despite all the required genes being present in the fragment , the toxicity caused by pa ( or by one of its breakdown products ) prevented more effective utilization of this compound in escherichia coli . in addition , it was shown that those bacteria ( escherichia coli dh5α ′) which did not contain the plasmid puc 18 + insert , or others which only had the plasmid puc 18 without the 10 kb insert , were unable to grow in mm + pa as sole carbon source — which clearly demonstrates that ( i ) the catabolism of phenylacetic acid was essentially due to the expression of the genes included in the 10 kb fragment and ( ii ) the escherichia coli strain used ( dh5α ′) did not have any functional enzymes enabling it to grow in mms which contained pa as the sole carbon source . it was subsequently confirmed that this fragment contained the pcl gene , as considerable phenylacetyl - coa ligase activity was detected in cell - free extracts of escherichia coli dh5α ′, whereas not even basal levels of the said activity were detected in the same bacteria without puc 18 + insert , or in others which only had puc 18 . subsequent studies allowed a more discrete fragment ( 2090 base pairs ) to be obtained which , cloned in puc 18 , coded for a protein with pcl activity , and the restriction analysis of which is given in detail in fig3 . the nucleotide sequence of the pcl gene was called seq id no : 1 . the amino acid sequence of the protein coded for by this gene was called seq id no : 2 . the determination of ligase activity was carried out as described in ref . 9 , but now starting with cell - free extracts of escherichia coli which had grown in lb medium ( see luria - bertani in ref . 13 ), supplemented with 100 μg / ml of the antibiotic ampicillin ( in those cases where cells containing the gene which coded for the β - lactamase present in the plasmid puc 18 were being analyzed ) or in the absence of the antibiotic , if the cells did not contain the plasmid . in all cases the bacteria were collected when the absorbance ( abs 540 nm ) of the culture diluted 1 / 10 was 0 . 2 . under these conditions the proportion of pcl present in the extracts free of those cells which contained the insert carrying the pcl gene was 23 % relative to total protein . all the oligopeptides obtained by analysis of the amino terminal of the protein previously purified from pseudomonas putida u were found in this sequence , as well as the others obtained by tryptic digestion of the same enzyme . this protein presents a consensus sequence ( ssgttgkp seq id no : 14 ) which corresponds to an amp binding site ( ref . 14 - 15 ). in addition , the enzyme was purified from the escherichia coli dh5α ′ strain which had been transformed with puc 18 + the insert indicated in fig3 it being possible to show that : ( i ) the protein expressed had the same molecular weight as that obtained from pseudomonas putida u ; and ( ii ) that this enzyme could also be linked to the ipns and at of penicillium chrysogenum , giving in vitro synthesis of penicillin g . when expression in escherichia coli dh5α ′ using the plasmid puc 19 as vector was employed , however , pcl activity was not detected — which suggests either that the pseudomonas putida u promoters are not expressed in escherichia coli or that there are no promoter sequences in the dna fragment available . subsequent studies carried out to shorten the fragment shown in fig3 by digesting it with the enzymes exonuclease iii / nuclease s1 ( using the erase - a - base system supplied by the promega company ) ( ref . 16 ) allowed a clone bal116 ( seq id no : 8 ) to be obtained which had lost a sequence fragment ( in front of the one coding for pcl ) but which , however , kept the same amino terminal sequence as the pcl purified from pseudomonas putida u ( mnmyh ). analysis of the different gene sequences obtained when the fragment of 2090 base pairs was digested with exonuclease iii / nuclease si ( erase - a - base system ) ( ref . 16 ) for different periods of time suggested that the expression of these dna fragments in escherichia coli could lead to different pcls in which only the amino terminal sequence changed . analysis of the pcl activity expressed in escherichia coli ( table i ) revealed that the amino terminal end of the native protein ( mnmyh ), corresponding both to that purified from pseudomonas putida u and to that encoded by the clone bal116 , could change without any appreciable variation in enzymatic activity . thus , the clone bal112 ( seq id no : 7 ), which codes for a pcl with a longer amino terminal end ( mtmitnssnsseamnm seq id no : 15 ), kept the same activity as that expressed from the clone bal116 . the same happened when a study was made of the activity of the proteins expressed by the clones bal142 ( seq id no : 10 ) and bal101 ( seq id no : 3 ), the amino terminals of which had been considerably reduced ( mtmitnsryh ( seq id no : 16 ) and mtmitnssda ( seq id no : 17 ), respectively ). from the clone bal110 ( seq id no : 6 ) a protein was obtained the amino terminal of which ( mtmitnsswraayknnsseamnmyh seq id no : 18 ) was longer than that encoded by the construct corresponding to bal112 due to the presence of an internal wraaykn ( seq id no : 19 ) sequence . this protein did not exhibit any pcl activity — which shows that although it is possible to introduce certain modifications into the amino terminal end of the protein without changing the activity , others , such as the one described , lead to non - functional enzymes in spite of keeping the complete sequence of the native protein . this result is particularly interesting as it shows that , provided that the mtm sequence belonging to the puc 18 polylinker exists , the pcl cloned in escherichia coil is not expressed from its own atg . with the aim of establishing the importance of the two methionines present in the polylinker ( mtm ) use was made of a variant of puc 18 in which a deletion of one of the two cytosines located between the two atgs of the plasmid had occurred and which consequently gave rise to a stop signal . the sequence in this mutated plasmid is called seq id no : 11 . the loss of this c produces a reading frame shift such that the protein would only be able to start in the second methionine . using this vector a study was made of the expression of a construct which did not have any of the nucleotide sequences coding for the two methionines present in the amino terminal end of the native pcl , which is the case with the construct ball42 . analysis of the pcl expressed revealed that a functional protein was produced , showing that the second atg of the polylinker marks the beginning of the pcl cloned . in those other constructs , created in the original plasmid zuc 18 , in which there were stop signals in the three reading frames , the different pcls start to synthesize from the amino terminal of the native protein ( this is the case with the clones bal106 — seq id no : 4 -, bal107 — seq id no : 5 -, bal116 and bal117 — seq id no ; 9 -). a ) the gene cloned corresponds to the one which codes for the enzyme pcl in pseudomonas putida u b ) the expression of this protein in escherichia coli dh5α ′ is governed by the promoter of β - galactosidase present in the plasmid puc 18 , and that c ) discrete modifications to the amino terminal sequence lead to functional pcls , whereas introduction of the amino acid sequence wraaykn ( seq id no : 19 ( bal112 ) causes total loss of phenylacetyl - coa ligase activity . 2 . expression of the pseudomonas putida u gene in the mold penicillium chrysogenum once the pcl gene of pseudomonas putida u had been characterized , the next objective was to introduce it into penicillium chrysogenum with the aim of determining whether its expression in this mold resulted directly in the production of a greater quantity of penicillin g . penicillium chrysogenum wis 54 - 1255 was chosen as the control strain . usng the procedure described by sanchez et al . ( ref . 17 - 18 ), protoplasts were obtained from mycelium that had grown in minimal medium . the protoplasts were transformed ( 17 - 18 ) with a plasmid derived from pbc ( stratagene ), which contained a gene for resistance to the antibiotic phleomycin ( ref . 19 ), the promoter of the gene pcb ab , the acvs of penicillium chrysogenum ( ref . 19 ), the pcl gene of pseudomonas putida u ( starting from the construct indicated as bal101 ) and the terminator of the trpc gene of penicillium chrysogenum . the construct , called palps9 ( fig4 ), was produced as follows : a 1 . 2 kb fragment ncoi , the ends of which were filled with flenow ( ref . 13 ), was obtained from the plasmid palp498 , carrying a 2316 pb fragment bamhi which includes the bidirectional promoter pcbab - pcbc ( p pcbab , see ref . 19 ). this fragment was bound with bal101 , which had previously been digested with ecori , filled with klenow and dephosphorylated , giving rise to insertions in both directions which were called palps1 and palps2 . the clone palpsi contains the pcl gene under the control of the promoter pcbab . by digestion of this plasmid with xbai , filling of the ends with klenow and subsequent digestion with hindiii , a 2 . 8 kb fragment with romo - hindiii ends was obtained which contained the pcbab promoter - pcl gene complex . this fragment was subcloned in the fungal transformation vector palfleo digested with xhoi , filled with klenow and finally digested with hindiii . the plasmid obtained , which had the pcbab promoter - pcl complex inserted in the same direction as the phleomycin resistance cassette ( ble r ), was called palps8 . finally , a 725 pb fragment was introduced which includes the terminator of the trpc gene ( ttrpc ) of penicillium chrysogenum in the ecorv site of palps8 , located between the pcl gene and the phleomycin resistance cassette . the construct bearing the trpc terminator , in the correct orientation , was called palps9 and used for expression of the pcl gene in different strains of penicillium chrysogenum . this construct is shown in fig4 . the transformant strains of penicillium chrysogenum , which expressed the phleomycin resistance gene ( ble r ), were selected ( ref . 19 ) and analyzed . all the transformants selected carried the pcl gene , as is shown by the fact that when amplification by pcr ( polymerase chain reaction ) was carried out , using the dna of the different transformants and 2 internal oligonucleotides corresponding to the pcl gene , the respective sequences of which were : 5 ′ d 3 ′: atc tgg gec ggg aac ac ( seq id no : 20 ) and ggc gca agg geg aca a ( seq id no : 21 ), amplified fragments of a size equivalent to that expected ( 651 base pairs ) were obtained in all cases ( fig5 ). amplification was not observed , however , either in the untransformed control strain or in that transformed with a construct in which the pcl gene had been eliminated ( fig5 ), which shows that ( i ) this same gene does not exist in the genome of penicillium chrysogenum and ( ii ) there are not any similar sequences which could be amplified . 98 % of the phleomycin - resistant transformants analyzed contained the pcl insert . in order to complete this study , four transformants and an untransformed control were selected and both the expression of the gene ( appearance of phenylacetyl - coa ligase activity in cell - free extracts ) and its effect on benzylpenicillin production were analyzed . for studies of this type the control and the transformants selected were inoculated in plates of complete production medium , the composition of which , in g / l , is as follows : corn steep solid 30 , lactose 30 , phenylacetic acid 1 , agar 20 g and distilled water up to 1 l . the ph was adjusted to 6 . 5 with naoh ( 30 % w / v ) and 10 g of caco 3 were then added . after it had been prepared , the medium was sterilized at 121 ° c . for 30 min and divided up at the rate of 30 ml per petri dish of 9 cm diameter . the dishes were inoculated with the spores collected from the different colonies and were incubated at 25 ° c . for 9 days . at this point in time 10 ml of sterile h 2 o per dish were added and the spores of each of the transformants were collected . these strains were used to inoculate an inoculation medium which contained , in corn steep solid 20 , sucrose 20 and soluble maize distillates ( dds ) 20 . the ph of the medium was adjusted to 5 . 7 and 5 g of caco 3 were then added . the medium was distributed into 250 - mi erlenmeyer flasks containing 50 ml of the medium described . the flasks were sterilized in an autoclave at 121 ° c . for 30 min , and after cooling they were inoculated with 1 ml of a spore suspension containing 10 9 spores / ml . the flasks were incubated at 25 ° c . in a gallenkamp orbital shaker at 230 rpm for 24 h . 2 . 5 - ml aliquots of this culture were then used to inoculate each 250 - ml erlenmeyer flask which contained 30 ml of complete production medium ( without agar ), prepared as described earlier . the fermentations were carried out for 56 h , taking aliquots at different times in order to determine the amount of penicillin produced , the dry weight ( mg / ml ) and the presence or absence of pcl in the different strains studied . the mycelium was collected by filtration , washed with 2000 volumes of sterile distilled h 2 o and dried with filter paper , and 1 . 5 - g aliquots of wet mycelium ( equivalent to 600 mg of dry weight ) were resuspended in 0 . 5 m phosphate buffer solution , ph 8 . 0 , containing 1 mm phenylmethylsulphonylfluoride ( pmsf ), and it was immediately disrupted by sonication . the extracts were centrifuged to eliminate the unbroken cells and the cell walls , and the supernatant was used to estimate phenylacetyl - coa ligase activity . the procedure followed was the one employed for analysis of the enzyme in pseudomonas putida , as we described earlier ( see ref . 9 and spanish patent p8902421 ). the formation of phenylacetyl - coa ( pa - coa ) was monitored by hplc , using a high - pressure liquid chromatograph consisting of a waters 600 pump , a waters 481 detector , a 10 μm nucleosil c18 column ( 250 × 4 . 6 mm ), a wisp 717 automatic injector and a waters computer system ( millenium 2010 ). the injection volume was 50 μl . 0 . 2 m potassium phosphate , ph 4 . 5 / isopropanol ( 90 : 10 vol / vol ) was used as the mobile phase . the flow rate was 1 ml / min and the wavelength selected was 254 nm . under these conditions pa - coa has a retention time of 19 . 30 min and phenylacetic acid ( pa ) a retention time of 10 . 8 min . analysis of the various transformants revealed that phenylacetyl - coa ligase activity was detected in all of them , whereas this did not appear in the control ( see table ii ), which indicated that the pcl gene of pseudomonas putida u was being expressed in penicillium chrysogenum . the transformants thus obtained were deposited in the spanish standard cultures collection , where they were given the registration number cect 20192 . the amount of penicillin g accumulated in the culture media was estimated by hplc , using equipment consisting of a waters 510 pump , a varian 2050 detector , a 5 - μm hypersil ods column ( 100 × 4 . 6 mm ) and a wisp 717 automatic injector . the injection volume was 20 μl . 0 . 05 m ammonium acetate , ph 6 . 8 / methanol ( 60 : 40 vol / vol ) was used as the mobile phase . the flow rate was 0 . 8 ml / min and the wavelength chosen was 235 nm . under these conditions the retention time of penicillin g is 4 . 5 min . as can be seen in table ii , all the transformants produced between 84 % and 121 % more penicillin g than the control strain , which irrefutably demons - rates that the expression of the pcl of pseudomonas putida i in penicillium chrysogenum helps to increase penicillin g synthesis considerably in this mould . fig1 pathway for biosynthesis of penicillin g ( benzylpenicillin ) in penicillium chrysogenum fig2 diagram of the protocol of mutagenesis with the transposon tn5 . rif : rifampicin 20 μg / ml ; km : kanamycin 25 μg / ml ; pa : phenylacetic acid ; fru : fructose ; m9 : minimal medium with a composition as described in reference 13 . 3 precipitate of escherichia coli + pseudomonas putida , 40 μl of which is collected and deposited in a filter on a dish with lb medium ( reference 13 ) fig4 pcl gene of pseudomonas expressed under the control of the promoter of the pcbab gene of penicillium chrysogenum fig5 amplification of part of the sequence of the pcl gene , using the oligonucleotides indicated in the report as initiators . 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( 1990 ). j . biol . chem . 265 : 16358 - 16365 . gaagcc atg aac atg tac cat gat gcc gac cgt gcc ctg ttg gac ccg 108 atg gaa acc gcc agt gtc gac gcc ctg cgc cag cac cag ctg gag cgc 156 met glu thr ala ser val asp ala leu arg gln his gln leu glu arg ctg cgc tgg agc ctg aag cac gcc tac gac aat gtg ccg ctg tac cgc 204 leu arg trp ser leu lys his ala tyr asp asn val pro leu tyr arg cag cgc ttt gcc gaa tgc ggc gcc cac ccc gac gac ctc acg tgc ctg 252 gln arg phe ala glu cys gly ala his pro asp asp leu thr cys leu gaa gac ctg gcg aag ttc ccc ttc acc ggc aag aac gac ctg cgc gac 300 glu asp leu ala lys phe pro phe thr gly lys asn asp leu arg asp aac tac ccc tac ggg atg ttc gcc gtc ccc cag gaa gag gtg gtg cgc 348 asn tyr pro tyr gly met phe ala val pro gln glu glu val val arg ctg cat gct tcc agc ggc acc acc ggc aag ccg acg gtg gtc ggt tac 396 acc cag aat gac atc aac acc tgg gcc aat gtc gtg gcg cgc tcg atc 444 cgt gcg gcc ggc ggg cgc aag ggt gac aaa gtg cat gtt tcc tac ggc 492 tat ggg ctt ttc act ggc ggg ctt ggt cgg cac tac ggc gcc gag cgc 540 ctg ggc tgt acg gta atc ccg atg tcg ggt ggc cag acc gag aag cag 588 leu gly cys thr val ile pro met ser gly gly gln thr glu lys gln gtg cag ctg atc cgc gac ttt cag ccc gac atc atc atg gtc aca ccg 636 tcc tac atg ctc aac ctg gcc gac gag atc gag cgc cag ggc atc gac 684 ser tyr met leu asn leu ala asp glu ile glu arg gln gly ile asp ccg cat gac ctc aag cta cgc ctg ggc att ttc ggt gcc gaa cct tgg 732 pro his asp leu lys leu arg leu gly ile phe gly ala glu pro trp acc gat gaa cta cgt cgc tcg atc gag cag cgc ctg ggc atc aat gcc 780 thr asp glu leu arg arg ser ile glu gln arg leu gly ile asn ala ctc gac atc tat ggt ttg tcg gaa atc atg ggc ccc ggg gtg gcc atg 828 leu asp ile tyr gly leu ser glu ile met gly pro gly val ala met gaa tgc atc gaa acc aag gac ggc ccg acc ata tgg gaa gac cac ttc 876 glu cys ile glu thr lys asp gly pro thr ile trp glu asp his phe tac ccc gaa atc atc gac ccg gtc acc ggc gaa gta ttg cca gac ggt 924 cag ctg ggc gaa ctg gtg ttc acc tcg cta agc aaa gag gcg ctt ccg 972 gln leu gly glu leu val phe thr ser leu ser lys glu ala leu pro atg gtg cgc tac cgc acc cgt gac ctc acc cgc ctg ctg ccc ggc acc 1020 gcc agg ccg atg cgg cgg atc ggc aag att acc ggg cgc agt gac gac 1068 atg ctg atc att cgc ggc gtc aac gtg ttc ccg acc cag atc gag gaa 1116 met leu ile ile arg gly val asn val phe pro thr gln ile glu glu cag gta tta aaa ata aaa cag ctt tcc gag atg tat gag att cat ttg 1164 tat cgc aat ggc aac ctg gac agc gta gag gtg cat gta gag ttg cgt 1212 gcg gag tgc cag cac ctc gat gaa ggc cag cgc aag ctg gtt atc ggg 1260 ala glu cys gln his leu asp glu gly gln arg lys leu val ile gly gag ctg agc aaa cag atc aag acc tac atc ggc atc agc acc cag gtg 1308 cac ctg cag gct tgc ggc acg ctc aag cgt tcc gag ggc aag gcg tgc 1356 his leu gln ala cys gly thr leu lys arg ser glu gly lys ala cys cac gtg tac gac aaa cgg ttg gcc agc tga ttcattcggc tgcct 1401 trp ser leu lys his ala tyr asp asn val pro leu tyr arg gln arg leu ala lys phe pro phe thr gly lys asn asp leu arg asp asn tyr pro tyr gly met phe ala val pro gln glu glu val val arg leu his cys thr val ile pro met ser gly gly gln thr glu lys gln val gln leu ile arg asp phe gln pro asp ile ile met val thr pro ser tyr met leu asn leu ala asp glu ile glu arg gln gly ile asp pro his asp leu lys leu arg leu gly ile phe gly ala glu pro trp thr asp ile tyr gly leu ser glu ile met gly pro gly val ala met glu cys ile glu thr lys asp gly pro thr ile trp glu asp his phe tyr pro gly glu leu val phe thr ser leu ser lys glu ala leu pro met val cys gln his leu asp glu gly gln arg lys leu val ile gly glu leu gln ala cys gly thr leu lys arg ser glu gly lys ala cys his val sequence is the peptide encoded by the dna of seq id no 11 .