CA1182409A - Plasmids constructed by gene manipulation, strains of escherichia coli carrying them, and process of tryptophan production using said strains - Google Patents
Plasmids constructed by gene manipulation, strains of escherichia coli carrying them, and process of tryptophan production using said strainsInfo
- Publication number
- CA1182409A CA1182409A CA000386858A CA386858A CA1182409A CA 1182409 A CA1182409 A CA 1182409A CA 000386858 A CA000386858 A CA 000386858A CA 386858 A CA386858 A CA 386858A CA 1182409 A CA1182409 A CA 1182409A
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- tryptophan
- strain
- escherichia coli
- strains
- trp
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/227—Tryptophan
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Abstract
Abstract of the Disclosure A recombinant plasmid having introduced thereinto a tryptophan operon which encodes an anthranilate synthase desensitized to feedback inhibition by tryptophan; a strain of Escherichia coli carrying said recombinant plasmid;
and a process for producing tryptophan using said strain of Escherichia coli.
and a process for producing tryptophan using said strain of Escherichia coli.
Description
~ his i~ve~tion relatec; t;o a .recombinax:lt pla~d having a tryptophan ope:ron which e~codes anthranilate s;~thase ~Sase3 dese~sitized -to f eedback ir~hibition b~
tr~ptvphan9 a~d to a strain o~ ch~ichia coli b~vir:~g high ~r~ptoph~producing abilit,y as a re~ult o:E intro-duct~on thereanto of a ~corlb:inant plasmid obtai~d by gene marlipulationO It also pertai~s to a p~oc~8s for produci~g tr~p~op~2~ ef~ic~tly Wi~ a short p0r~0d o`
time by cultivati~; the g~raill of ~ coli i~ a 10 ~lutrienl; m~dium and reco~eri~s t~h~n :Erom ~h~ culture bI~ot~,.
ha~ be~rl we~l ~ow~ that ~lu~ c acid, l~r~ine9 etc.. ar~ produ~ed by :EerDle~ta~io~ ~echni~ues u~ing micro-orga~i~ms, i~ particular b~ u~ing artificiall~ mutatcd 15 ~licroo:~;a~m st:r~ins o~ high prQduct~ it;y" Co~lrentional ~Eerme~tation methods, how~ver~ do ~ot g:ive rise to high productivi~y ~ID f ~ a~ t~ ophan, a }dnà o~ ~no acid i9 concarrled, ~o f ermen~tio~ m~3thod that satis~
eco~omic f oasibili~ ha~ yet been esta~lish~d o~ ael ~n-2Q du~trial basia. Orl the other harld9 the rec~a~ ~dvance~o~ genetic en~ eeri~g ha~e e~abled us~ ubsta~ces ~uch a~ i~suli~ or growth 3:Lox~aor~e to be ~od~ced b~
ïerme~tat 2~ chniqu~s usin~; s~rain~ o i~to ~Jhich ~enes of hi~;her ~rLLmals or pl8~ h~e be~r 25 i~troduc ed .
W~ e ~ow suce.eeded i~ produci~g tr,~ypto~?haz:
ly b~ ~a~s~ ; a ~t~ o~
with a recom~ pla~mid co~structed b~y gen~ ipula~io~
that imparts tryptophan-s~nthesizi~g activity to said strai~ and by cultivati~g the t;rans~ormed strain.
Thus, one characteristic of this inventioD is to provide a recombinant plasmLd having a tryptophan operon of ~ coli which encodes ASase desen~
sitized to tryptophan inhibition, said plasmid being able to replicate in a strain of Es~herichie coli~
Another feature of the in~ention pertains -to a strain of ~ co~i transformed with said plasmid, said ~ coli strain havi~g ASase desensitized to feedback inhibitlon by try~topha~ and being a de~icie~t m~tant of tr~ptophan repressor a~d tr~ptophanase.
S~ill a~o~her ~eature of the inve~tion is to provide a process for producing tr~ptophan, which comprises cultivating the above trans~ormed ~scherichia coli strai~
in a nutrient medi~ particularly in a ~utrient medium containing anthra~ilic acid supp~emented, if desired3 ~ith a tetracycline~type antibiotic~ and separating and recovering L-tI~tophan from the cultur~ broth in a man~er l~own ~ se~.
The present i~verltiorl is described in detail here-inb~l ow .
The recombinan t p`lasmid in this i~vention ca~ be obtained in t;he f ollowin~; manner.
~'he Iirst enzyme in the tr~-ptophan synthesizin~;
system in a microorganism is ~Sase. .A6ase i5 USllally subJect to :Eeedback inhibition by t~yptopha~. Some of mioroorganis~ strains wbich are r esistan-t to 5-~ethyl~
~ 3 --DLrtryptophan~ an analog of t~tophan, have ASase that is insensitive to feedback in~.bi-tlon by t~yptophan, Accordingly, if such strains ~ cultivated, tryptopha~
can be produced in lc~rge quanti.~ies.
~hus, Escheric ia co]i W~llO ~ (pSC101-tr~), i.e. an Escherichia coli strain carryin~ a plasmid, havi~g a tryptophan operon introduced thereinto~ is physically or chemica~ly mutated by a k~own muta-ting means such as W irradia~ion or treatment with N-methyl ~'-ni~ro-N-nitrosoguanidine, ethyl methanesulfonate, etc, ~rom the muta~ts obtained$ those which are resistant to 5-methyl-nL-tryptophan and hc1~e ASase insensiti.ve to tryptophan inhibition are selectedO Plasmids extracted from these selected strai~s (e.g~pS~l0~_-trp~I15) haviog a tryptophs~
~peron xesistan's to feedbac'~ inhibition by ~ryptophan~
Recombi~ant plasmids having a -tryptophan operon as used in this in~ention, such as p~C101-trp~ can be ob~
tained by recombining a ~no~m tryptophan operon with a vector such as a plasmid. for Escherichia coli.by the method 2t) described in Gene, VolO 1, pages 141 to 152 ~1977~ or by the modi~ied methodsO
It has been asce.rtained -that the :Eollowing strai~s can be used as hos-ts into which the recombinant plasmid pro~r-lded by -this inve~tion can be in-troduced by 25 ~transI ormationO
~scherichia coli W3110 ~ (AEl for short), _~__ __ ~scherichia coli W3110 ~ E~_m~Z ~Xam for ~____ _ short ~, ;Esche:r hi coli W3110 ~ ~ ~L (Tna f OI` short ) O
Escherlc'[li~ coli (E~ocOli) iS a stra~n which is non-ly~ogenic for lambda (';. ) and does ~ot have a 5 fert~lity factor F ~F ). It is ~ mut~nt ~ATCC 27325 derived :Fr~m a parent stxai~1 ~E. coli Kl29 which is lysoge~ic ~o~ lsmbds and has a fertility factor F~ rl:hus, accordin~ to this invention, all E. coli str~ins derived from E,. coli K12 are eligible as hosts of the plasmid of the m vent ion . When it is desired to produce tryptophan, the use of an Eo coli strain which has the followi~g three p.roperties, i.e.~ tryptophan operon deletion mutation ~trpA:E;l), ~r~ptophan repressor defici~eIIt mut~tioII (trpR) __ _ ar~d tr~ptophanase deficient mutation ~), thQt iS9 15 ~ coll W3110 ~ tnaA is most preferred.
~ he properties which the host E~ coli cells should ha~e are described below~
~~
'rhe tryptophan operon of ~O coli linl~s a tonB
gene on -the chromo~ome~ Since tonB is necessary for the adsorption o~ a phage Tl to Eo coli., m~lta5ion of tonB
results in a ~allure o~ phage '~1 adsorptionO Accordingly, the O coli becomes resistant to 5he phage Tl by the mutation o~ ton:B, ~en double mutants~ tryptophan requirement and re~istance to pha~e Tl ~Tr.p ~ Tlr~ are obtained by ultra violet irradiation~ it is seen that in mos-t of these mutants, DN~ segments extendlng from the tonB to the t ~
tryptophan operon are par-tly o:r to-tally de]eted from the chromosome. The mutagenic trea~men-t and the method o~
deter~ination in~olved iD the :foregoing are described in detail, for example~ in ~enletics, VolO 49~ p~ge~
tr~ptvphan9 a~d to a strain o~ ch~ichia coli b~vir:~g high ~r~ptoph~producing abilit,y as a re~ult o:E intro-duct~on thereanto of a ~corlb:inant plasmid obtai~d by gene marlipulationO It also pertai~s to a p~oc~8s for produci~g tr~p~op~2~ ef~ic~tly Wi~ a short p0r~0d o`
time by cultivati~; the g~raill of ~ coli i~ a 10 ~lutrienl; m~dium and reco~eri~s t~h~n :Erom ~h~ culture bI~ot~,.
ha~ be~rl we~l ~ow~ that ~lu~ c acid, l~r~ine9 etc.. ar~ produ~ed by :EerDle~ta~io~ ~echni~ues u~ing micro-orga~i~ms, i~ particular b~ u~ing artificiall~ mutatcd 15 ~licroo:~;a~m st:r~ins o~ high prQduct~ it;y" Co~lrentional ~Eerme~tation methods, how~ver~ do ~ot g:ive rise to high productivi~y ~ID f ~ a~ t~ ophan, a }dnà o~ ~no acid i9 concarrled, ~o f ermen~tio~ m~3thod that satis~
eco~omic f oasibili~ ha~ yet been esta~lish~d o~ ael ~n-2Q du~trial basia. Orl the other harld9 the rec~a~ ~dvance~o~ genetic en~ eeri~g ha~e e~abled us~ ubsta~ces ~uch a~ i~suli~ or growth 3:Lox~aor~e to be ~od~ced b~
ïerme~tat 2~ chniqu~s usin~; s~rain~ o i~to ~Jhich ~enes of hi~;her ~rLLmals or pl8~ h~e be~r 25 i~troduc ed .
W~ e ~ow suce.eeded i~ produci~g tr,~ypto~?haz:
ly b~ ~a~s~ ; a ~t~ o~
with a recom~ pla~mid co~structed b~y gen~ ipula~io~
that imparts tryptophan-s~nthesizi~g activity to said strai~ and by cultivati~g the t;rans~ormed strain.
Thus, one characteristic of this inventioD is to provide a recombinant plasmLd having a tryptophan operon of ~ coli which encodes ASase desen~
sitized to tryptophan inhibition, said plasmid being able to replicate in a strain of Es~herichie coli~
Another feature of the in~ention pertains -to a strain of ~ co~i transformed with said plasmid, said ~ coli strain havi~g ASase desensitized to feedback inhibitlon by try~topha~ and being a de~icie~t m~tant of tr~ptophan repressor a~d tr~ptophanase.
S~ill a~o~her ~eature of the inve~tion is to provide a process for producing tr~ptophan, which comprises cultivating the above trans~ormed ~scherichia coli strai~
in a nutrient medi~ particularly in a ~utrient medium containing anthra~ilic acid supp~emented, if desired3 ~ith a tetracycline~type antibiotic~ and separating and recovering L-tI~tophan from the cultur~ broth in a man~er l~own ~ se~.
The present i~verltiorl is described in detail here-inb~l ow .
The recombinan t p`lasmid in this i~vention ca~ be obtained in t;he f ollowin~; manner.
~'he Iirst enzyme in the tr~-ptophan synthesizin~;
system in a microorganism is ~Sase. .A6ase i5 USllally subJect to :Eeedback inhibition by t~yptopha~. Some of mioroorganis~ strains wbich are r esistan-t to 5-~ethyl~
~ 3 --DLrtryptophan~ an analog of t~tophan, have ASase that is insensitive to feedback in~.bi-tlon by t~yptophan, Accordingly, if such strains ~ cultivated, tryptopha~
can be produced in lc~rge quanti.~ies.
~hus, Escheric ia co]i W~llO ~ (pSC101-tr~), i.e. an Escherichia coli strain carryin~ a plasmid, havi~g a tryptophan operon introduced thereinto~ is physically or chemica~ly mutated by a k~own muta-ting means such as W irradia~ion or treatment with N-methyl ~'-ni~ro-N-nitrosoguanidine, ethyl methanesulfonate, etc, ~rom the muta~ts obtained$ those which are resistant to 5-methyl-nL-tryptophan and hc1~e ASase insensiti.ve to tryptophan inhibition are selectedO Plasmids extracted from these selected strai~s (e.g~pS~l0~_-trp~I15) haviog a tryptophs~
~peron xesistan's to feedbac'~ inhibition by ~ryptophan~
Recombi~ant plasmids having a -tryptophan operon as used in this in~ention, such as p~C101-trp~ can be ob~
tained by recombining a ~no~m tryptophan operon with a vector such as a plasmid. for Escherichia coli.by the method 2t) described in Gene, VolO 1, pages 141 to 152 ~1977~ or by the modi~ied methodsO
It has been asce.rtained -that the :Eollowing strai~s can be used as hos-ts into which the recombinant plasmid pro~r-lded by -this inve~tion can be in-troduced by 25 ~transI ormationO
~scherichia coli W3110 ~ (AEl for short), _~__ __ ~scherichia coli W3110 ~ E~_m~Z ~Xam for ~____ _ short ~, ;Esche:r hi coli W3110 ~ ~ ~L (Tna f OI` short ) O
Escherlc'[li~ coli (E~ocOli) iS a stra~n which is non-ly~ogenic for lambda (';. ) and does ~ot have a 5 fert~lity factor F ~F ). It is ~ mut~nt ~ATCC 27325 derived :Fr~m a parent stxai~1 ~E. coli Kl29 which is lysoge~ic ~o~ lsmbds and has a fertility factor F~ rl:hus, accordin~ to this invention, all E. coli str~ins derived from E,. coli K12 are eligible as hosts of the plasmid of the m vent ion . When it is desired to produce tryptophan, the use of an Eo coli strain which has the followi~g three p.roperties, i.e.~ tryptophan operon deletion mutation ~trpA:E;l), ~r~ptophan repressor defici~eIIt mut~tioII (trpR) __ _ ar~d tr~ptophanase deficient mutation ~), thQt iS9 15 ~ coll W3110 ~ tnaA is most preferred.
~ he properties which the host E~ coli cells should ha~e are described below~
~~
'rhe tryptophan operon of ~O coli linl~s a tonB
gene on -the chromo~ome~ Since tonB is necessary for the adsorption o~ a phage Tl to Eo coli., m~lta5ion of tonB
results in a ~allure o~ phage '~1 adsorptionO Accordingly, the O coli becomes resistant to 5he phage Tl by the mutation o~ ton:B, ~en double mutants~ tryptophan requirement and re~istance to pha~e Tl ~Tr.p ~ Tlr~ are obtained by ultra violet irradiation~ it is seen that in mos-t of these mutants, DN~ segments extendlng from the tonB to the t ~
tryptophan operon are par-tly o:r to-tally de]eted from the chromosome. The mutagenic trea~men-t and the method o~
deter~ination in~olved iD the :foregoing are described in detail, for example~ in ~enletics, VolO 49~ p~ge~
2~7-278 (1964)~
trpRam27~ trpR (tr~tophan repressor de~icient ~utation) Tryptophan repressor mu-t~nts are preliminarily selected for their resistance to -tryp-tophan analogs (such as 5-~ethyl-DL-tryptophan, or 6-methyl-DL-tryptophan).
q`he analog-resistant strains may possibly include (13 those which ~re derepressed mutants9 (2) those which are ~sensitive to feedback inhibition by tryptophan 7 and (3) those which cannot take the analogs into their cells because of changes in membrane structureO
Thus7 i~ tr~yptophan s~nthase is constitutively synthesiæed in the analog-resi~tant strains when culti-v~ted in a medium containing tryptophan (with wild strains tryptopha~ synthase is scarcely syrlthesized under these conditions because of repression), these str~ins are determined as derepressed mutantsO As -to whether the derepression is due to the repressor mut~tion or the operator mutation~ the analog-resistant strain is sub-jected to transduction by a ph~ge PIKC (a generalized transducing phage of ~ coli) to examine whether the genotype of this p~rticular analog-resistant mutation is linked to the thr(threonine-auxotrophic) gene or to ~(-tr~ptophsn-au~otrophic) geneO lf it is linked with thr gene, it can be ascertained that repres~or mutation has taken placeO Such a strai:n can be used in this in-ve~tion~
Incidentally~ ~27 is tryptophan repressor 5 nonsense mut~tion, and ~ is tryptophan repressor missense mutation. ~or details~ reference m~y be h~d9 ~or example, to J. Mol~ Biol., Vol 44, page~ 185-193 (1969), and Genetics, Vol. 52, pages 130~--1316 (1965)o A wild type of Eo coli strain c~n grow b~
uti~ izing tryptophan as ~ sole c~rbon source becaus~ of tryptophanase ~ctivity (tryptoph~n ~ indole+ pyruvic ~cid + NH3), whereas a tryptophan~e-deficient mut~nt of E.
coli c~nnot~ By utilizing this physiclo~ic~l tr~it, the ~5 tryptophanase~de~icient mutant c~n be separated. For ex~mple, a _~ coli 6trai~ is sub~ected to mu-ta~enic treat-m~ts eith~r ~y ultraviolet irr~diatio~ N-methyl-N'-nitro-N~nitrosoguanidine (NTG) or ethyl msth~esulfon~te (E~lS~ to form colo~ies on a minim~l ~gar mediwm cont~i~ing 20 gluoose as a carbon ~ource~ The colo~ies were tr~nsferred GlltQ 8 minimal ~gE~r medium contaill~ng tryptophan as a sole carbon ~ource ~ ~hus ,, those str~ which ~srow when th~
carbon source is glucose but c~nnot grow on tr~ptophaD
~re s~lected.. The tr~ptophan~se aotivity is determined 25 b;y ~dding ~r~ indol~2 reagent to the culture broth obtained by cultiv~ting the sforesaid strain in I.~brothO When red color specl;E ic to indole is not Iormed upon the ~3adition ~a color reaction on indole), the tryptoph~n~s~
7 _ is regarded as deficient.
In this regard~ reference may be had to, for example~ J. Bacteriologyt Vol~ &99 pages 680-686 (1965).
E~ coli W3110 ~ tnaA strain can be obtained by using the aforesaid methods i~ combination.
Specifically, this can be accomplished by the following procedureO
(1) Subject E. eoli W3110 ~ strain ~cystein-auxotroph) to -trpR mutation, (2) ~ransduce a ~ strairl with the PIKC
lysate prep~red on E. coli W3110 ~ strain to obtain W3110 ~ strain9 and
trpRam27~ trpR (tr~tophan repressor de~icient ~utation) Tryptophan repressor mu-t~nts are preliminarily selected for their resistance to -tryp-tophan analogs (such as 5-~ethyl-DL-tryptophan, or 6-methyl-DL-tryptophan).
q`he analog-resistant strains may possibly include (13 those which ~re derepressed mutants9 (2) those which are ~sensitive to feedback inhibition by tryptophan 7 and (3) those which cannot take the analogs into their cells because of changes in membrane structureO
Thus7 i~ tr~yptophan s~nthase is constitutively synthesiæed in the analog-resi~tant strains when culti-v~ted in a medium containing tryptophan (with wild strains tryptopha~ synthase is scarcely syrlthesized under these conditions because of repression), these str~ins are determined as derepressed mutantsO As -to whether the derepression is due to the repressor mut~tion or the operator mutation~ the analog-resistant strain is sub-jected to transduction by a ph~ge PIKC (a generalized transducing phage of ~ coli) to examine whether the genotype of this p~rticular analog-resistant mutation is linked to the thr(threonine-auxotrophic) gene or to ~(-tr~ptophsn-au~otrophic) geneO lf it is linked with thr gene, it can be ascertained that repres~or mutation has taken placeO Such a strai:n can be used in this in-ve~tion~
Incidentally~ ~27 is tryptophan repressor 5 nonsense mut~tion, and ~ is tryptophan repressor missense mutation. ~or details~ reference m~y be h~d9 ~or example, to J. Mol~ Biol., Vol 44, page~ 185-193 (1969), and Genetics, Vol. 52, pages 130~--1316 (1965)o A wild type of Eo coli strain c~n grow b~
uti~ izing tryptophan as ~ sole c~rbon source becaus~ of tryptophanase ~ctivity (tryptoph~n ~ indole+ pyruvic ~cid + NH3), whereas a tryptophan~e-deficient mut~nt of E.
coli c~nnot~ By utilizing this physiclo~ic~l tr~it, the ~5 tryptophanase~de~icient mutant c~n be separated. For ex~mple, a _~ coli 6trai~ is sub~ected to mu-ta~enic treat-m~ts eith~r ~y ultraviolet irr~diatio~ N-methyl-N'-nitro-N~nitrosoguanidine (NTG) or ethyl msth~esulfon~te (E~lS~ to form colo~ies on a minim~l ~gar mediwm cont~i~ing 20 gluoose as a carbon ~ource~ The colo~ies were tr~nsferred GlltQ 8 minimal ~gE~r medium contaill~ng tryptophan as a sole carbon ~ource ~ ~hus ,, those str~ which ~srow when th~
carbon source is glucose but c~nnot grow on tr~ptophaD
~re s~lected.. The tr~ptophan~se aotivity is determined 25 b;y ~dding ~r~ indol~2 reagent to the culture broth obtained by cultiv~ting the sforesaid strain in I.~brothO When red color specl;E ic to indole is not Iormed upon the ~3adition ~a color reaction on indole), the tryptoph~n~s~
7 _ is regarded as deficient.
In this regard~ reference may be had to, for example~ J. Bacteriologyt Vol~ &99 pages 680-686 (1965).
E~ coli W3110 ~ tnaA strain can be obtained by using the aforesaid methods i~ combination.
Specifically, this can be accomplished by the following procedureO
(1) Subject E. eoli W3110 ~ strain ~cystein-auxotroph) to -trpR mutation, (2) ~ransduce a ~ strairl with the PIKC
lysate prep~red on E. coli W3110 ~ strain to obtain W3110 ~ strain9 and
(3) Subjsct the resultin~ strain in (2) to t _ mutation.
The tryptophan synth~se (TSase) and anthranilate synthasc (A~ase) activities of a strain obt~ined by trans-~ormation with the plasmid pro-vided by this i~vention~
such as pSC101-trpdI5 or pSC101-trp-I15 are shown in ~able 1~ inhibition of ASa~e activity b~ tryptophan, in ~able 2; a~d the productivi-ty ot tryptcphan, in Table 30 The characteristics of -the Eo coli strains in this in~
vention will be urther descr1bed~
~ o o o o o o d~ .~
.~ ~3 ~
~~a ~
cq ~ ~ ~ ~ C ~D
a)E~ ,~ ~ ~ ~ ~JO
~ ~l ~ ~ ~
~ --r~3 r~ ~ tU ~ O
~; I r~ r~
----~
tn 0a~ ~C~co~ ~ ~
CQ ~ o ~ o ~ ~ c ~q 0 ~ ~ 0 ~ o ~
tQ r~ ~ O O
.~ ~l ~
~ --~
^
~1 U~ ~1 U~ r~
H H H H H H
E~
.~1~ ~ .
I I ~ rJ r~ I
O O O O ~ C) r-~l r~l r~ ~-~ r~
V C~ C~ ~ V
~ ~ ~ c3 u~ CQ
E~
.
a~
h Ul ~ .
____~
_ 9 _ The unit of the activities given in Table 1 is U~mg of pro-t~n~ The activities were measured usin~ 8 cell-disrupted solution (by sorlication; crude enzyme solution) obt~ined in a me~ium A to be described here-5 inbelow. ASase activity was measured in the absence (~Trp) and/or presence (+Trp)(().2mM) o:E -tryptop~an.
The TSase and ASase activitieæ are higher with Ram or Tna StrainB which are tryp-tophan repressor-, flsa . ~
de~icient than A:El straill. A~e is not inhibi-ted by 10 tryptophan in either ca~e.
_ 10 --able 2. Inhibition oî ASa~e acti~ity by tryptophan _ ~ . .___ Trp 50/0 in-Stra ill Trp ~mM ) ~ /0 ) hib it ion (mM ) ____~ ~
AEl (pSClOl_trp I5) 1~1 100 13.1 ___ __ In Ta~le 2, Trp 50% inhibition der~otes the conc~ntrat~o~ o~ trypt~?hal~ ~t which tryptopha~ ix~hibit~
5 by 5G~/o the ASass aetivity in the absence of tryptophar~c With regard to the inhibition oî A~3a~ actilrity by tryptopharl, no significant diî:~erence i~ see~ betwesn pSC101-trp - I5 ~nd pSC101- trp Il50 This suggests that the pl~smid pro~ d by the pre~;ent ir~veirlt iorl are not 10 alwa~s specif ic O
The 1~, col:~ cells trarlsform~d with the pla3mid i~ this i~vention, for exa~nple~ ~na(p~l(:l-trp-I15) str~in e~itiv~ to feed~ac:k inhibitiori by ~ryp~ophan t tryptophan repr~ssor is m~l~unction~g9 ar~d tr;srpt~ha~se 15 activitie~ are mi~sing~ Only tho~ :E. coli strains which ~eet the~e r~qu;~ments ac¢umulate t~;yptQphan oGnsider-a~bl;yO ~he ob jective OI high tryptophan production c~nnot be achieved, if ~ny one of these reguire~ents is lacking~
This is clearly seen from Table 3 below.
T~ble 3: Productivity of tryptophan (48_hour cultivation) ~ _ __ _ Repres- In- Trypto- Tryptophar ~traIn plasmid sio~ hibi-tion pha~ase (~g/m~) _ ___ ___ __ __ AEl pSC101-trp + + + 7 pSC101-trp I15 ~ _ + 11 Ram p5C101-trp ~ ~ 7 pSC101-trpoI15 _ _ ~ 7o Tha pSC101-trp _ + _ pSC101-trp,I15 _ _ _ 360 ~ __ __ __ ~
For accu~ulation of tryptophan i~ high con-ce~tratio~s~ both the removal of repression by tryptophan ~nd resistanc~ to feedback inhibition by tryptophan are es~enti~ nd the deficiency of tryptoph~nase is also neces~aryO The productivity of Tn~(pSClOl~trp~I15) is hi~her than that shown by a chro~o30~al dou~le mutant (repre~sio~ , inhi~ltion ) of _, coli [iOeO, 183 ~g/m~:
J. ~`er~ent. Technol., vol~ 43, page~ 302-306 (1965~].
T~is is believed to reflect ~he gene dosage effect of the plasmid on th~ amount of the ~in~l product~
E. coli W3110 ~ tnaA. ~pSC101-trp~
I15) mention~d above was deposited on October 28~ 19~0 i~
American ~yp~ Culture Collection (a d~posit number: ATCC
3~74~)~
~he medium ~or cultivating microorganisms i~
this invention may be ~ny natural or synthetic nutrient _ 12 -medium containing carbon sources, nitrogen sources, in-organic materials, etcO which may, as reguired, by supplemented with the drugs used in the preparation of the microorga~isms u~ed, and/or anthranilic acid. The carbon and nitrogen sources used in -the cultur~ mediu~
may ~e a~y kinds which the microorganisms can utilize.
~he carbon sources are, for examplel carbohydrate~ such as gluco~e, glycerol9 fructose, sucrose~ maltose, mannose, starch, starch hydrolyzate and molasses~ ~xamples of the nitrogen sources include ammonia, inorganic and organic ammo~ium salts such 85 ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate a~d a~o~ium acetate, and natural o~ganic nitrogen sources such as meat extract, yeast extract, corn steep liguor7 casein hydrolyzateS defatted soybaan residue or the digested product thereof. Man~ of natural organic nitrogen sources can serve both as the nitrogen and carbon sources~
Examples o~ the inorganic material~ include ~ 6r}~ potassium monohydrogen phospha-te (K ~P0~
---c potassium dih~dro~en phosphate (KH ~04~, magnesium ~ul~ate, sodium chloride, ferrous sulfate, calcium chloride, zinc ¢hloride, copper sulfate, manganese chloride, cobalt chloride, ammonium ~olybdate, and boric acid~
Whe~ the created microorganism has resista~ce to an antibiotic may be added to the culture m~dium to prevent antibiotic cvntamina-tion ~he addition of anthranilic acid, a precursor of tryptophan, is also useful for enhancement of tryptophan production.
~ - The cultivation is c~rri~d out;under aerobic conditions by shaking or submerg~d culture under aeration and stirxing. The culti~ation temperature is usu~lly 20 to 50C, preferably 35 to 45G. Desirably, the pH
of the culture medium is maint~ined nearly neutral, iOe~
about 6 to 7. 87 during the cultivation~ l'he cultivation period is usually ~rom 1 to 3 days~
'~ryptophan accumulated in the cul-ture mediu~
may be separated and recovered by conventional methods, ~or example, by using an ion exchange resin~
~ he following Examples illustrate the prese~t invention without any intention of limiting the invention thereby. Before proceeding to these Examples, the con-struction of pSClOl~trp is described.
~e~
pSC101 (tetracycli~e resisbant, ~cr a~d copy number per chrsmoso~e7 5-10) was used as a vector7 a~d a specialized ~ran~ducing phage ~ ~ wa~ used as do~or o~ tr~ptoph~n operon in E. coli~
(1) Treatment with res-triction endonuclease, EcoRI
-,~t~ Q=~ D~A (about 0.1 ~g) and pSC101 plas~id D~ (about lo9 ~g~ were digested ~or 1 hour at 37C ln the following re~ction soluti~n~ The~, the mixtur~
wa~ heat~d at 65~C for 5 minutes to inac-ti~te EcoRI.
~ coRI reaction solution ~50 ~ in to~al):-Tris-HCl (pH 7~4~ 90 mM
MgC12 10 m~-l DNA 2 ~g EcoRI 2 units EcoRI is commercially ~vail~ble f~m Miles ~abor~tories Inc.~ ~ethesda R~s~arch I,~boratories Inc.
etc~
~2) Treatment with T4-DN~ ligase The T4-D~A ligase trea-tment was carrie~ out at
The tryptophan synth~se (TSase) and anthranilate synthasc (A~ase) activities of a strain obt~ined by trans-~ormation with the plasmid pro-vided by this i~vention~
such as pSC101-trpdI5 or pSC101-trp-I15 are shown in ~able 1~ inhibition of ASa~e activity b~ tryptophan, in ~able 2; a~d the productivi-ty ot tryptcphan, in Table 30 The characteristics of -the Eo coli strains in this in~
vention will be urther descr1bed~
~ o o o o o o d~ .~
.~ ~3 ~
~~a ~
cq ~ ~ ~ ~ C ~D
a)E~ ,~ ~ ~ ~ ~JO
~ ~l ~ ~ ~
~ --r~3 r~ ~ tU ~ O
~; I r~ r~
----~
tn 0a~ ~C~co~ ~ ~
CQ ~ o ~ o ~ ~ c ~q 0 ~ ~ 0 ~ o ~
tQ r~ ~ O O
.~ ~l ~
~ --~
^
~1 U~ ~1 U~ r~
H H H H H H
E~
.~1~ ~ .
I I ~ rJ r~ I
O O O O ~ C) r-~l r~l r~ ~-~ r~
V C~ C~ ~ V
~ ~ ~ c3 u~ CQ
E~
.
a~
h Ul ~ .
____~
_ 9 _ The unit of the activities given in Table 1 is U~mg of pro-t~n~ The activities were measured usin~ 8 cell-disrupted solution (by sorlication; crude enzyme solution) obt~ined in a me~ium A to be described here-5 inbelow. ASase activity was measured in the absence (~Trp) and/or presence (+Trp)(().2mM) o:E -tryptop~an.
The TSase and ASase activitieæ are higher with Ram or Tna StrainB which are tryp-tophan repressor-, flsa . ~
de~icient than A:El straill. A~e is not inhibi-ted by 10 tryptophan in either ca~e.
_ 10 --able 2. Inhibition oî ASa~e acti~ity by tryptophan _ ~ . .___ Trp 50/0 in-Stra ill Trp ~mM ) ~ /0 ) hib it ion (mM ) ____~ ~
AEl (pSClOl_trp I5) 1~1 100 13.1 ___ __ In Ta~le 2, Trp 50% inhibition der~otes the conc~ntrat~o~ o~ trypt~?hal~ ~t which tryptopha~ ix~hibit~
5 by 5G~/o the ASass aetivity in the absence of tryptophar~c With regard to the inhibition oî A~3a~ actilrity by tryptopharl, no significant diî:~erence i~ see~ betwesn pSC101-trp - I5 ~nd pSC101- trp Il50 This suggests that the pl~smid pro~ d by the pre~;ent ir~veirlt iorl are not 10 alwa~s specif ic O
The 1~, col:~ cells trarlsform~d with the pla3mid i~ this i~vention, for exa~nple~ ~na(p~l(:l-trp-I15) str~in e~itiv~ to feed~ac:k inhibitiori by ~ryp~ophan t tryptophan repr~ssor is m~l~unction~g9 ar~d tr;srpt~ha~se 15 activitie~ are mi~sing~ Only tho~ :E. coli strains which ~eet the~e r~qu;~ments ac¢umulate t~;yptQphan oGnsider-a~bl;yO ~he ob jective OI high tryptophan production c~nnot be achieved, if ~ny one of these reguire~ents is lacking~
This is clearly seen from Table 3 below.
T~ble 3: Productivity of tryptophan (48_hour cultivation) ~ _ __ _ Repres- In- Trypto- Tryptophar ~traIn plasmid sio~ hibi-tion pha~ase (~g/m~) _ ___ ___ __ __ AEl pSC101-trp + + + 7 pSC101-trp I15 ~ _ + 11 Ram p5C101-trp ~ ~ 7 pSC101-trpoI15 _ _ ~ 7o Tha pSC101-trp _ + _ pSC101-trp,I15 _ _ _ 360 ~ __ __ __ ~
For accu~ulation of tryptophan i~ high con-ce~tratio~s~ both the removal of repression by tryptophan ~nd resistanc~ to feedback inhibition by tryptophan are es~enti~ nd the deficiency of tryptoph~nase is also neces~aryO The productivity of Tn~(pSClOl~trp~I15) is hi~her than that shown by a chro~o30~al dou~le mutant (repre~sio~ , inhi~ltion ) of _, coli [iOeO, 183 ~g/m~:
J. ~`er~ent. Technol., vol~ 43, page~ 302-306 (1965~].
T~is is believed to reflect ~he gene dosage effect of the plasmid on th~ amount of the ~in~l product~
E. coli W3110 ~ tnaA. ~pSC101-trp~
I15) mention~d above was deposited on October 28~ 19~0 i~
American ~yp~ Culture Collection (a d~posit number: ATCC
3~74~)~
~he medium ~or cultivating microorganisms i~
this invention may be ~ny natural or synthetic nutrient _ 12 -medium containing carbon sources, nitrogen sources, in-organic materials, etcO which may, as reguired, by supplemented with the drugs used in the preparation of the microorga~isms u~ed, and/or anthranilic acid. The carbon and nitrogen sources used in -the cultur~ mediu~
may ~e a~y kinds which the microorganisms can utilize.
~he carbon sources are, for examplel carbohydrate~ such as gluco~e, glycerol9 fructose, sucrose~ maltose, mannose, starch, starch hydrolyzate and molasses~ ~xamples of the nitrogen sources include ammonia, inorganic and organic ammo~ium salts such 85 ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate a~d a~o~ium acetate, and natural o~ganic nitrogen sources such as meat extract, yeast extract, corn steep liguor7 casein hydrolyzateS defatted soybaan residue or the digested product thereof. Man~ of natural organic nitrogen sources can serve both as the nitrogen and carbon sources~
Examples o~ the inorganic material~ include ~ 6r}~ potassium monohydrogen phospha-te (K ~P0~
---c potassium dih~dro~en phosphate (KH ~04~, magnesium ~ul~ate, sodium chloride, ferrous sulfate, calcium chloride, zinc ¢hloride, copper sulfate, manganese chloride, cobalt chloride, ammonium ~olybdate, and boric acid~
Whe~ the created microorganism has resista~ce to an antibiotic may be added to the culture m~dium to prevent antibiotic cvntamina-tion ~he addition of anthranilic acid, a precursor of tryptophan, is also useful for enhancement of tryptophan production.
~ - The cultivation is c~rri~d out;under aerobic conditions by shaking or submerg~d culture under aeration and stirxing. The culti~ation temperature is usu~lly 20 to 50C, preferably 35 to 45G. Desirably, the pH
of the culture medium is maint~ined nearly neutral, iOe~
about 6 to 7. 87 during the cultivation~ l'he cultivation period is usually ~rom 1 to 3 days~
'~ryptophan accumulated in the cul-ture mediu~
may be separated and recovered by conventional methods, ~or example, by using an ion exchange resin~
~ he following Examples illustrate the prese~t invention without any intention of limiting the invention thereby. Before proceeding to these Examples, the con-struction of pSClOl~trp is described.
~e~
pSC101 (tetracycli~e resisbant, ~cr a~d copy number per chrsmoso~e7 5-10) was used as a vector7 a~d a specialized ~ran~ducing phage ~ ~ wa~ used as do~or o~ tr~ptoph~n operon in E. coli~
(1) Treatment with res-triction endonuclease, EcoRI
-,~t~ Q=~ D~A (about 0.1 ~g) and pSC101 plas~id D~ (about lo9 ~g~ were digested ~or 1 hour at 37C ln the following re~ction soluti~n~ The~, the mixtur~
wa~ heat~d at 65~C for 5 minutes to inac-ti~te EcoRI.
~ coRI reaction solution ~50 ~ in to~al):-Tris-HCl (pH 7~4~ 90 mM
MgC12 10 m~-l DNA 2 ~g EcoRI 2 units EcoRI is commercially ~vail~ble f~m Miles ~abor~tories Inc.~ ~ethesda R~s~arch I,~boratories Inc.
etc~
~2) Treatment with T4-DN~ ligase The T4-D~A ligase trea-tment was carrie~ out at
4~C for 18 hours usi.ng a }igase solutio~ o~tained by adding the ~ollowing reagent~ to the a~or~said EcoRI
reaction solution, and adjusting the amount o~ the mixture to 100 ~ h~ ligase is also availa~le.
~E~
50 mM MgC12 10 100 mM d}thio~ry~hritol 10 11 0~5 ~M adeno$ine tri~
phosph~te (ATP) 10 Watcr ~ ~
T~ A ligase 0~2 u~it ~3) ~ran3~0rmatio~ of E~ coli ~- coli a60~ rk ~ mk 1 Trp strain waa culti-~ted at 37C in 20 ml of ~roth (~or co~positions.
per liter~pepto~e 10 g, yeast extr~c~ 5 g~ NaCl 5 ~
pE 7) ~o an op~ical de~slty ~OD660~ of 0047 The cells were collected, washed with 20 ml of 100 mM MgCl~, ~uspended in 10 ml of 100 mM C~C12, and allowed to stand i~ an ice bath for 20 minutes~ The cells were re-collected by centri~ugation, and again suspended in 1 ml of 100 mM CaC12. In an ice b~-th, 200 ~L~ of this cell suspension was mixed with 100 u~ of the a~ore~aid T4-D~A
ligas~ reaction solution~ and the mixture was allowed to stand ~or 30 minutes~ ~he mixture was then treated at 4~C for 2 minutes, and again allowed to s-tand in an ice bath for 20 minutes. 2.7 ml o:E the I.-broth was ~dded, and the bacteria were cultiva-ted a-t 37C for 2 hours~ The culture broth was spread on a cult~e medium A of the fol-~0 lowing composition containing 15 g~ agar ana 10 ~gJml o~tetracycl m e (~c), and a trans~ormed str~in ~er and ~rp was selected~
~ith regard to -the aforesaid E. coli C600, the symbol rk denotes the property of the bacterium to di~tîn~
guish its ow~ D~A molecules (the bases on DNA are specifi~
cally m~thyl~ted~ from foreign DNA molecules (which differ from its own DN~ molecules in the site of methylation), restricting the entr~ of foreign D~A. ~he symbol m~ de-notes the property of the bacterium -to modif~ the foreign DNA to innate DNA molecules in the site of methylation.
~husg rk and m~ show tha-t these properties are absent~
Gulture medium A (Vogel and Bonner minimal medium, pH 7~ contain~d per liter.
K~IP04 10 g NaNH~EP0~4~I20 3-5 g r1~S04 ~ 7~2 0 . 2 g Citric acid~H20 2 g Glucose 2,5 g Casamino acids 0~5 g (4) Extraction o.~ p~C101-trp plasmid ~NA
The TcrTrp trans~orman-t was cultivated at 37C
i~ 100 ~1 o~ the I~broth to an optical density (OD660) of 0.8~ The cells were collected, and suspended in 1 ml of Tris~sucrose [Tris-HCl pH 8 (50 ~M)~ sucrose (2~/o wt/vol)].
I~ an ice bath, Oa~ ml of lysozyme (5 mg/~l) was added, and the mixture was allowed to stand for 5 minutes~ Then, 0.4 ml of Na2EDTA (250 mM, pH 3) was added, a~d the mix-ture was allowed to st~nd for 5 minut~s. Further, 0.5 ml of 5M NaCl and 0.2 ml of l~o (w~v) sodium dodecylsulfate ~SD~) were successively added. The mixture was stirred and then allowed to stand over~ight in an ice bath. The mixture wa3 then cen~rifuged at 4C for 30 minutes (30,000x g~. The resulti~g supernatant liguid was ad-justed to a volume of 5 ml using 50 mM Na2EDTA (pH 8~, and 407 g of CsCl and 1~8 mg of ethidium bromide were added to adjust the de~sity of the mixture to 1.57 g~ml. The resulti~g sample was subjected to CsCl-ethidium bromide density gradient centrifugation us~ a~ RP65 rotor (made by Hitachi Works ~tdo) at 38,000 rpm ~or ~0 hours. hfter th~ centrifugation, fractions containing plasmid DNA wer~
collected, and ~thid;L~ bromide was e:~iFacted from them using butanol and dialyzed against T:E buffer ~Tris~HCl 10 mM 7 ~la 2EDqlA O ~ 1 m~l 9 p~ 8 ) .
The -theory o:E the above densi-ty gradient cen-tri~uga-tion is as follows~
Plasmid D~A (covalently closed circular DNA) a~d chxomosomal DNA (li~ear D~A; although they are _ l7 _ circular in the cells, Ihey change to linear in the course of extrac-tion because of their high molecular weight) differ from each other in the form of molecules. Ethidium bro~ide intercalates between bases of DNA to a greater exten-t in -the linear DNA molecules than in the cir-cular. Consequently, a difference in density arises be-tween the plasmid and the chromosomal DNAs. The plasmid DNA is separated by utilizing this density difference.
pSC101-trp extracted by the above method had a molecular weight oE about 16.5 x 106 daltons. It was digested with restriction endonucleaseJ EcoRI and subjected to electrophoresis on an agarose gel.
Two DNA fragments were noted, one corresponding to the fragment ~10.8 x 106 daltons) containing tryptophan operon and the other to the original vector plasmid, pSC101 (5.7 x 10 daltons).
Example 1 A plasmid having a tryptophan operon insensitive to feedback inhibition by tryptophan was separated in the following manner.
~1) Separation of strains resistant to 5-methyl-DL-tryptophan ~5MT) 5M~, an analog of tryptophan, as does tryptophan, represses transcription of a tryptophan operon to a messenger RNA (mRNA) and inhibits the activity of anthranilate synthase (ASase). A wild strain cannot grow in the presence of 5MT because of no production of o~
tryptophan. But if tryptophan operon is derepressed by tryptophan repressor ~utation~ etc. or the activity of ASase is not mhibited as a result of mutation, this str~in synthesizes a minimal amount of tryptophan and can grow (5MTr).
~ . coli W3110 ~ (whole tryptophan operon on chromosome was deleted, and therefore, Trp , design-ated as ~AEl] ) was tra~sformed with DNA of pSC101-trp to obtain a transformant ~1 (pSC101-trp~ whvse phenotype:
(~c~rp~). The AEl(pSC101-trp) s~rain was treated with NTG (200 ~g~m~), and the cells were spread on an agar medium A (medium A added by 15 g~ of ~gar) co~tai~ing 200 ~g/m~- of 5~, and cultivated overnight at 37C~ By this method, ab~ut seventy 5M~r strai~s were separated 15 independently.
(2) Separation of strains resistant to ~eedback inhibition Seventy 5M~r strains were cultivated each at 37C for 16 hours in 100 ~4 of the culture medium A. The cells were collected, washed with 0.~/0 NaCl, and suspended in 5 m~ of 100 ~M tris-I~Cl (pX 7.8)~ The cells were then so~icated by a~ ultrasonic generator (Model 42~0, Kaijo Denki Co., 10 KHz) to disrupt the cells7 ~nd centrifuged at 18~000 rpm for 30 minutes by using a rotor ~Sorval SS~34). '~he ~upern~t~nt liquid was obtained as a crude enzyme solution~
The .ASase ~ctivity of the crude enzyme solution was assayed and the ~ctivity of ~Sase in the presence of 0.2 ~M o~ tryptophan was checked. It was found that the activity of ASase in two strains (I5 and I15) was not inhibited by 0~2 mM of tryphophan and these strai~ had resistance to feedback i~hibi-t ion ~
(3) Praparation o~ a plasmid h~ving a trypto-phan operon desensitiæed to ~eedback inhibition Plasmids pSC101-trp I5 and pSC101-trp I15 wer~
extracted~ respectively by the same method as described iO here maboveO 'rhe pSC101-trp-I5 and pSC101-trp I15 were treated, respect ively with EcoRI 9 a~d ~ubjacted to agarose gel electrophoresisv Two bands corresponding to 10.8x 106 and 5.7x 106 daltons were noted.
It was confirmed ~rom the following fact t~at resistance to the ~eedback inhibition was ascribable to the plasmid mutation. Specifically, AEl strain, ~. coli W3110 ~ ~3~ (tryptophan operon deletion, tryptopha~ xepressor de~icient, ~esi~nated a~ [Ram]), ~ coli W3110 ~ tnaA (tryptophan operon deletion, 20 tryptopha~ repressor deficient~ tryptophansse deficient, desi~ated as ~'~na]) were tra~sformed with the pSC101-trp~I5 or pS&101--trpoI15s respecti~ely. U5ing the trans~ormants carrying pSC101--trp- I5 or pSC101-trp~I15, crude enæyme solutions were prepared a~ described here-inabove, and the activity of ASase was measured. In allcase~ the ~c tivity of ASase was insensitive to trypto-phan Accordi~ly, the resistance to ~eedback inhibition was attributable to the mutation of tryptoph~n opero~
20 ~
of pSC101-trp.I5 or pSC101-trp~Il50 A loop of the Tna(pSClOl-trp~l15) strain obtained by transforming Tna strain wit:h pSC101-trp I15 ~ s in-oculated into L-broth having the following composition (per liter~ and containi~g 20 ~g/m~ o~ tetracycline, and precultivated a-t 37C for 9 to 15 hours. Thenl 0.5 m~ (o~ 5%) of the pre-cul-ture was inoculated in 100 m~ of medium B having the followin~ composition (per liter) in a 500 m~ flask, and cultivated at 37C. The pH of the culture medium was adjusted to 7 with 2N NaOH every 3 to 5 hours. After the cultivation for 48 houxs~ trypto-phan (360 ~g/m~) was assayed by the Xanthydrol ~ethod.
~-b Bacto-tryptone 10 g Yeast ex~ract 5 ~
~aCl 5 g pH 7 Culture medium B
KH ~0~ 3 ~
K ~ 04 7 g NH4C1 3 g M~S0~7H~0 0.2 g pH 7 Glucose 30 g Casamino acids 1 g ~5 ~
The Tna strain (pSC101~-t~I15) was .inoculated in medium B supplemented with an-thra~ilic acid and Casamino acids as shown in the following table, and cultivated at 37 C ~or 48 hours. ~he pH of the culture mediu~ was adJusted to 7 with 2N NaOH. Tryptophan produced is shown in the following ta~le.
When the amount of anthranilic ~ci~ added ini-tially was 500 mg/e, it was consumed completely in about 24 hours. When the amount was 800 mg/~, anthranilic acid was consumed completely in about 48 hours. Whan the con-cen-t~ation of anthra~ilic acid was much higher, it was inhibitory to the bacterial g~owth~ ~he cultivatio~ time reguired was 63 hours when the concen-tration of' anthranilic acid was 1,000 mg~, a~d ~ore than 63 hours if the con-ce~tration was 1~500 mg~ he anthranilic acid consumed was converted almost completely to tryptophan.
_~
Amount o~ tryptophan accumulated (m~e) Anthranilic Cas~mino ___________ acid acids 48 hours 63 hours (~g/~) (%) later l~ter __~
500 0.2 1140 11~5 ~00 0~2 1626 1557 800 o L~ 1 L~07 1404 1000 0.4 1233 L575 1500 0.4 1233 1614 ____ ~
15 ~
Tna~pSC101-trp.I15) strain was precultivated at 37C for 9 to 15 hours in L~broth supplemented wi-th 20 y.gf~ of tetracycline" 100 m~ of the preculture broth was inoculated in a ~ar fermentor (2 ~;.Model MD-250;
- 2~ -arubishi Go., Tokyo) containing 1.5 ~ of a culture ~edium having the ~ollowing composition (per liter), and cultivated at ~7C, The cultu.re medium was stirrsd at 500 rpm, and air was supplied at a rate of 1 v.v~m. ~Le pH of the medium was adjusted to 7 with 1'~% N~-I40H.
A~thranilic acid as a substrate was added at a const~nt rate of 50 ~g/~/hr after 8 h~ur~ from the s-tarting of the cultivationO ~he cultivation was carried out for ~8 hours. ~he amount of tryptophan accumul~ted was 5~6 10 g/.eO
T ~re ~
KH ~O~ 3 ~
K2HPO4 7 g NH4C1 3 g MgSO407H20 0.2 g ~eso4 7H20 o. 01 g Anthranilic acid 005 g Glucose 50 g Casamino acids 10 ~
29 Tetracycline O~Ol g pH 7 One liter of -the resulti~g cul~ure broth was centrifuged, and the supernat~nt liquid obtained was pas~ed throu~h a chromatographic column packed with 900 ~ o~ activated carbon to cause adsorption of trypto-~5 phan. ~he column was washed with water and eluted with
reaction solution, and adjusting the amount o~ the mixture to 100 ~ h~ ligase is also availa~le.
~E~
50 mM MgC12 10 100 mM d}thio~ry~hritol 10 11 0~5 ~M adeno$ine tri~
phosph~te (ATP) 10 Watcr ~ ~
T~ A ligase 0~2 u~it ~3) ~ran3~0rmatio~ of E~ coli ~- coli a60~ rk ~ mk 1 Trp strain waa culti-~ted at 37C in 20 ml of ~roth (~or co~positions.
per liter~pepto~e 10 g, yeast extr~c~ 5 g~ NaCl 5 ~
pE 7) ~o an op~ical de~slty ~OD660~ of 0047 The cells were collected, washed with 20 ml of 100 mM MgCl~, ~uspended in 10 ml of 100 mM C~C12, and allowed to stand i~ an ice bath for 20 minutes~ The cells were re-collected by centri~ugation, and again suspended in 1 ml of 100 mM CaC12. In an ice b~-th, 200 ~L~ of this cell suspension was mixed with 100 u~ of the a~ore~aid T4-D~A
ligas~ reaction solution~ and the mixture was allowed to stand ~or 30 minutes~ ~he mixture was then treated at 4~C for 2 minutes, and again allowed to s-tand in an ice bath for 20 minutes. 2.7 ml o:E the I.-broth was ~dded, and the bacteria were cultiva-ted a-t 37C for 2 hours~ The culture broth was spread on a cult~e medium A of the fol-~0 lowing composition containing 15 g~ agar ana 10 ~gJml o~tetracycl m e (~c), and a trans~ormed str~in ~er and ~rp was selected~
~ith regard to -the aforesaid E. coli C600, the symbol rk denotes the property of the bacterium to di~tîn~
guish its ow~ D~A molecules (the bases on DNA are specifi~
cally m~thyl~ted~ from foreign DNA molecules (which differ from its own DN~ molecules in the site of methylation), restricting the entr~ of foreign D~A. ~he symbol m~ de-notes the property of the bacterium -to modif~ the foreign DNA to innate DNA molecules in the site of methylation.
~husg rk and m~ show tha-t these properties are absent~
Gulture medium A (Vogel and Bonner minimal medium, pH 7~ contain~d per liter.
K~IP04 10 g NaNH~EP0~4~I20 3-5 g r1~S04 ~ 7~2 0 . 2 g Citric acid~H20 2 g Glucose 2,5 g Casamino acids 0~5 g (4) Extraction o.~ p~C101-trp plasmid ~NA
The TcrTrp trans~orman-t was cultivated at 37C
i~ 100 ~1 o~ the I~broth to an optical density (OD660) of 0.8~ The cells were collected, and suspended in 1 ml of Tris~sucrose [Tris-HCl pH 8 (50 ~M)~ sucrose (2~/o wt/vol)].
I~ an ice bath, Oa~ ml of lysozyme (5 mg/~l) was added, and the mixture was allowed to stand for 5 minutes~ Then, 0.4 ml of Na2EDTA (250 mM, pH 3) was added, a~d the mix-ture was allowed to st~nd for 5 minut~s. Further, 0.5 ml of 5M NaCl and 0.2 ml of l~o (w~v) sodium dodecylsulfate ~SD~) were successively added. The mixture was stirred and then allowed to stand over~ight in an ice bath. The mixture wa3 then cen~rifuged at 4C for 30 minutes (30,000x g~. The resulti~g supernatant liguid was ad-justed to a volume of 5 ml using 50 mM Na2EDTA (pH 8~, and 407 g of CsCl and 1~8 mg of ethidium bromide were added to adjust the de~sity of the mixture to 1.57 g~ml. The resulti~g sample was subjected to CsCl-ethidium bromide density gradient centrifugation us~ a~ RP65 rotor (made by Hitachi Works ~tdo) at 38,000 rpm ~or ~0 hours. hfter th~ centrifugation, fractions containing plasmid DNA wer~
collected, and ~thid;L~ bromide was e:~iFacted from them using butanol and dialyzed against T:E buffer ~Tris~HCl 10 mM 7 ~la 2EDqlA O ~ 1 m~l 9 p~ 8 ) .
The -theory o:E the above densi-ty gradient cen-tri~uga-tion is as follows~
Plasmid D~A (covalently closed circular DNA) a~d chxomosomal DNA (li~ear D~A; although they are _ l7 _ circular in the cells, Ihey change to linear in the course of extrac-tion because of their high molecular weight) differ from each other in the form of molecules. Ethidium bro~ide intercalates between bases of DNA to a greater exten-t in -the linear DNA molecules than in the cir-cular. Consequently, a difference in density arises be-tween the plasmid and the chromosomal DNAs. The plasmid DNA is separated by utilizing this density difference.
pSC101-trp extracted by the above method had a molecular weight oE about 16.5 x 106 daltons. It was digested with restriction endonucleaseJ EcoRI and subjected to electrophoresis on an agarose gel.
Two DNA fragments were noted, one corresponding to the fragment ~10.8 x 106 daltons) containing tryptophan operon and the other to the original vector plasmid, pSC101 (5.7 x 10 daltons).
Example 1 A plasmid having a tryptophan operon insensitive to feedback inhibition by tryptophan was separated in the following manner.
~1) Separation of strains resistant to 5-methyl-DL-tryptophan ~5MT) 5M~, an analog of tryptophan, as does tryptophan, represses transcription of a tryptophan operon to a messenger RNA (mRNA) and inhibits the activity of anthranilate synthase (ASase). A wild strain cannot grow in the presence of 5MT because of no production of o~
tryptophan. But if tryptophan operon is derepressed by tryptophan repressor ~utation~ etc. or the activity of ASase is not mhibited as a result of mutation, this str~in synthesizes a minimal amount of tryptophan and can grow (5MTr).
~ . coli W3110 ~ (whole tryptophan operon on chromosome was deleted, and therefore, Trp , design-ated as ~AEl] ) was tra~sformed with DNA of pSC101-trp to obtain a transformant ~1 (pSC101-trp~ whvse phenotype:
(~c~rp~). The AEl(pSC101-trp) s~rain was treated with NTG (200 ~g~m~), and the cells were spread on an agar medium A (medium A added by 15 g~ of ~gar) co~tai~ing 200 ~g/m~- of 5~, and cultivated overnight at 37C~ By this method, ab~ut seventy 5M~r strai~s were separated 15 independently.
(2) Separation of strains resistant to ~eedback inhibition Seventy 5M~r strains were cultivated each at 37C for 16 hours in 100 ~4 of the culture medium A. The cells were collected, washed with 0.~/0 NaCl, and suspended in 5 m~ of 100 ~M tris-I~Cl (pX 7.8)~ The cells were then so~icated by a~ ultrasonic generator (Model 42~0, Kaijo Denki Co., 10 KHz) to disrupt the cells7 ~nd centrifuged at 18~000 rpm for 30 minutes by using a rotor ~Sorval SS~34). '~he ~upern~t~nt liquid was obtained as a crude enzyme solution~
The .ASase ~ctivity of the crude enzyme solution was assayed and the ~ctivity of ~Sase in the presence of 0.2 ~M o~ tryptophan was checked. It was found that the activity of ASase in two strains (I5 and I15) was not inhibited by 0~2 mM of tryphophan and these strai~ had resistance to feedback i~hibi-t ion ~
(3) Praparation o~ a plasmid h~ving a trypto-phan operon desensitiæed to ~eedback inhibition Plasmids pSC101-trp I5 and pSC101-trp I15 wer~
extracted~ respectively by the same method as described iO here maboveO 'rhe pSC101-trp-I5 and pSC101-trp I15 were treated, respect ively with EcoRI 9 a~d ~ubjacted to agarose gel electrophoresisv Two bands corresponding to 10.8x 106 and 5.7x 106 daltons were noted.
It was confirmed ~rom the following fact t~at resistance to the ~eedback inhibition was ascribable to the plasmid mutation. Specifically, AEl strain, ~. coli W3110 ~ ~3~ (tryptophan operon deletion, tryptopha~ xepressor de~icient, ~esi~nated a~ [Ram]), ~ coli W3110 ~ tnaA (tryptophan operon deletion, 20 tryptopha~ repressor deficient~ tryptophansse deficient, desi~ated as ~'~na]) were tra~sformed with the pSC101-trp~I5 or pS&101--trpoI15s respecti~ely. U5ing the trans~ormants carrying pSC101--trp- I5 or pSC101-trp~I15, crude enæyme solutions were prepared a~ described here-inabove, and the activity of ASase was measured. In allcase~ the ~c tivity of ASase was insensitive to trypto-phan Accordi~ly, the resistance to ~eedback inhibition was attributable to the mutation of tryptoph~n opero~
20 ~
of pSC101-trp.I5 or pSC101-trp~Il50 A loop of the Tna(pSClOl-trp~l15) strain obtained by transforming Tna strain wit:h pSC101-trp I15 ~ s in-oculated into L-broth having the following composition (per liter~ and containi~g 20 ~g/m~ o~ tetracycline, and precultivated a-t 37C for 9 to 15 hours. Thenl 0.5 m~ (o~ 5%) of the pre-cul-ture was inoculated in 100 m~ of medium B having the followin~ composition (per liter) in a 500 m~ flask, and cultivated at 37C. The pH of the culture medium was adjusted to 7 with 2N NaOH every 3 to 5 hours. After the cultivation for 48 houxs~ trypto-phan (360 ~g/m~) was assayed by the Xanthydrol ~ethod.
~-b Bacto-tryptone 10 g Yeast ex~ract 5 ~
~aCl 5 g pH 7 Culture medium B
KH ~0~ 3 ~
K ~ 04 7 g NH4C1 3 g M~S0~7H~0 0.2 g pH 7 Glucose 30 g Casamino acids 1 g ~5 ~
The Tna strain (pSC101~-t~I15) was .inoculated in medium B supplemented with an-thra~ilic acid and Casamino acids as shown in the following table, and cultivated at 37 C ~or 48 hours. ~he pH of the culture mediu~ was adJusted to 7 with 2N NaOH. Tryptophan produced is shown in the following ta~le.
When the amount of anthranilic ~ci~ added ini-tially was 500 mg/e, it was consumed completely in about 24 hours. When the amount was 800 mg/~, anthranilic acid was consumed completely in about 48 hours. Whan the con-cen-t~ation of anthra~ilic acid was much higher, it was inhibitory to the bacterial g~owth~ ~he cultivatio~ time reguired was 63 hours when the concen-tration of' anthranilic acid was 1,000 mg~, a~d ~ore than 63 hours if the con-ce~tration was 1~500 mg~ he anthranilic acid consumed was converted almost completely to tryptophan.
_~
Amount o~ tryptophan accumulated (m~e) Anthranilic Cas~mino ___________ acid acids 48 hours 63 hours (~g/~) (%) later l~ter __~
500 0.2 1140 11~5 ~00 0~2 1626 1557 800 o L~ 1 L~07 1404 1000 0.4 1233 L575 1500 0.4 1233 1614 ____ ~
15 ~
Tna~pSC101-trp.I15) strain was precultivated at 37C for 9 to 15 hours in L~broth supplemented wi-th 20 y.gf~ of tetracycline" 100 m~ of the preculture broth was inoculated in a ~ar fermentor (2 ~;.Model MD-250;
- 2~ -arubishi Go., Tokyo) containing 1.5 ~ of a culture ~edium having the ~ollowing composition (per liter), and cultivated at ~7C, The cultu.re medium was stirrsd at 500 rpm, and air was supplied at a rate of 1 v.v~m. ~Le pH of the medium was adjusted to 7 with 1'~% N~-I40H.
A~thranilic acid as a substrate was added at a const~nt rate of 50 ~g/~/hr after 8 h~ur~ from the s-tarting of the cultivationO ~he cultivation was carried out for ~8 hours. ~he amount of tryptophan accumul~ted was 5~6 10 g/.eO
T ~re ~
KH ~O~ 3 ~
K2HPO4 7 g NH4C1 3 g MgSO407H20 0.2 g ~eso4 7H20 o. 01 g Anthranilic acid 005 g Glucose 50 g Casamino acids 10 ~
29 Tetracycline O~Ol g pH 7 One liter of -the resulti~g cul~ure broth was centrifuged, and the supernat~nt liquid obtained was pas~ed throu~h a chromatographic column packed with 900 ~ o~ activated carbon to cause adsorption of trypto-~5 phan. ~he column was washed with water and eluted with
5~/O etha~ol c~olution conta}~ing o.70/o of ammonia to sepa-rate trypt~phan~ ~he eluate was concantrated under reduced pressure to xemove ~mmonia and eth~nol~
The resulting agueous solutio~ Qf -tryptop:han was adjusteà to ~ p:~l of 4~5, and passed through a column (10 x 40 cm) of Dowex 50x8 (Na form) (a tradename for a product of Dow Chemical Co. ~ buîI ered with a t)O lM citrate 5 ~olu~ion ~pH 3.4)~
~ hen, 4.5 ~ of a O~lM citrate bufîer tpH 5.0) was passed through the column, and the colum~ was elut~d with a 50% a9ueous solut io~ of ethanol conta in ing 0O 2~/o ~mmonia to sep~rate tryptophan. ~h~ eluate was concen-~0 trated to dryness under reduced pres~ure to ~ive crudetryptoph~n as a crystalline powder. ~ne crude tryptophan powder was dissolved in a small amount Of 5C% hot ethaDol, decolorized with a small amo~t of activated carbon, and cooled to gi~T~ 3O5 g of tryptophan as white scale-like 15 crystals~
t ~h~ result~ shown i~ thase l~amples demonstrate that tryptophan ca~ be obtained efîiciently within shorter periods o~ time by using Eo coli strains tr~nsIormed with recombina~t plasmids constructed by the gene manuipu~:a~or 2:) techni~ues i~ vitro than by using conve2ltionRl mut~nts o~ ;E. coli. Moreovar, by transformillg E. coli strains with recombinan t plasmids of larg~r copy numbers or by increasirlg their resis-tance to feedb~ck inhibition9 ths praduction of brylptopha~ will be rnors eff icient.
The resulting agueous solutio~ Qf -tryptop:han was adjusteà to ~ p:~l of 4~5, and passed through a column (10 x 40 cm) of Dowex 50x8 (Na form) (a tradename for a product of Dow Chemical Co. ~ buîI ered with a t)O lM citrate 5 ~olu~ion ~pH 3.4)~
~ hen, 4.5 ~ of a O~lM citrate bufîer tpH 5.0) was passed through the column, and the colum~ was elut~d with a 50% a9ueous solut io~ of ethanol conta in ing 0O 2~/o ~mmonia to sep~rate tryptophan. ~h~ eluate was concen-~0 trated to dryness under reduced pres~ure to ~ive crudetryptoph~n as a crystalline powder. ~ne crude tryptophan powder was dissolved in a small amount Of 5C% hot ethaDol, decolorized with a small amo~t of activated carbon, and cooled to gi~T~ 3O5 g of tryptophan as white scale-like 15 crystals~
t ~h~ result~ shown i~ thase l~amples demonstrate that tryptophan ca~ be obtained efîiciently within shorter periods o~ time by using Eo coli strains tr~nsIormed with recombina~t plasmids constructed by the gene manuipu~:a~or 2:) techni~ues i~ vitro than by using conve2ltionRl mut~nts o~ ;E. coli. Moreovar, by transformillg E. coli strains with recombinan t plasmids of larg~r copy numbers or by increasirlg their resis-tance to feedb~ck inhibition9 ths praduction of brylptopha~ will be rnors eff icient.
Claims (6)
1. A recombinant plasmid having introduced there-into a tryptophan operon which encodes an anthranilate syn-thase desensitized to feedback inhibition by tryptophan, said plasmid being able to replicate in a strain of Escherichia coli.
2. A strain of Escherichia coli carrying a re-combinant plasmid and being a deficient mutant of tryptophan repressor and tryptophanase, said recombinant plasmid having a tryptophan operon which encodes an an-thranilate synthase desensitized to feedback inhibition by tryptophan.
3. The strain of Escherichia coli of claim 2 which is Escherichia coli W3110 trpAE1 trpR tnaA (pSC101-trp.
I15) (ATCC 31743).
I15) (ATCC 31743).
4. A process for producing tryptophan, which com-prises: cultivation in a nutrient medium of a strain of Escherichia coli carrying a recombinant plasmid and be-ing a deficient mutant of tryptophan repressor and tryptophanase, said recombinant plasmid having a trypto-phan operon which encodes an anthranilate synthase de-sensitized to feedback inhibition by tryptophan, and separation and recovery of tryptophan from the culture broth.
5. The process of claim 4 wherein the nutrient medium contains anthranilic acid.
6. The process of claim 4 wherein the nutrient medium contains tetracycline.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP154,706/80 | 1980-11-05 | ||
| JP55154706A JPS5780398A (en) | 1980-11-05 | 1980-11-05 | Plasmid produced by genetic manipulation, coliform bacillus having the same and preparation of tryptophan with said bacillus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1182409A true CA1182409A (en) | 1985-02-12 |
Family
ID=15590169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000386858A Expired CA1182409A (en) | 1980-11-05 | 1981-09-29 | Plasmids constructed by gene manipulation, strains of escherichia coli carrying them, and process of tryptophan production using said strains |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPS5780398A (en) |
| AU (1) | AU549686B2 (en) |
| CA (1) | CA1182409A (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6180373B1 (en) | 1992-09-28 | 2001-01-30 | Consortium f{umlaut over (u)}r elektrochemische Industrie GmbH | Microorganisms for the production of tryptophan and process for the preparation thereof |
| EP1715056A1 (en) | 2005-04-23 | 2006-10-25 | Degussa AG | Process for the production of L-amino acids using improved Enterobacteriaceae strains |
| DE102007051024A1 (en) | 2007-03-05 | 2008-09-11 | Evonik Degussa Gmbh | Process for the preparation of L-amino acids using strains of the family Enterobacteriaceae |
| EP1975241A1 (en) | 2007-03-29 | 2008-10-01 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| EP2036979A1 (en) | 2007-09-15 | 2009-03-18 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| EP2055785A1 (en) | 2007-11-02 | 2009-05-06 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| EP2060636A1 (en) | 2007-11-14 | 2009-05-20 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| DE102008040352A1 (en) | 2008-07-11 | 2010-01-14 | Evonik Degussa Gmbh | Process for the preparation of L-tryptophan using improved strains of the family Enterobacteriaceae |
| DE102008044768A1 (en) | 2008-08-28 | 2010-03-04 | Evonik Degussa Gmbh | Process for the preparation of organochemical compounds using improved strains of the family Enterobacteriaceae |
| EP2628792A1 (en) | 2012-02-17 | 2013-08-21 | Evonik Industries AG | Cell with reduced ppGppase activity |
| WO2014117992A1 (en) | 2013-01-30 | 2014-08-07 | Evonik Industries Ag | Microorganism and method for production of amino acids by fermentation |
| EP3385275A1 (en) | 2017-04-07 | 2018-10-10 | Evonik Degussa GmbH | Method for producing aromatic l-amino acids using improved strains of the enterobacteriaceae family |
| US11053526B2 (en) | 2018-08-09 | 2021-07-06 | Evonik Operations Gmbh | Process for preparing L amino acids using improved strains of the enterobacteriaceae family |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA811368B (en) * | 1980-03-24 | 1982-04-28 | Genentech Inc | Bacterial polypedtide expression employing tryptophan promoter-operator |
| JPH074257B2 (en) * | 1983-06-17 | 1995-01-25 | 三菱油化株式会社 | Novel plasmid |
| JPS6043392A (en) * | 1983-08-20 | 1985-03-07 | Showa Denko Kk | Preparation of l-tryptophan |
| EP0190921A3 (en) * | 1985-02-04 | 1988-01-13 | Engenics, Inc. | Method for the overproduction of amino acids |
| EP0293207A3 (en) * | 1987-05-29 | 1989-11-02 | The Standard Oil Company | Eschericia coli carrying recombinant plasmid for the production of tryptophan |
| JPH0722510B2 (en) * | 1988-05-09 | 1995-03-15 | 三井東圧化学株式会社 | Gene encoding thermostable tryptophan synthase and its use |
| US6118047A (en) | 1993-08-25 | 2000-09-12 | Dekalb Genetic Corporation | Anthranilate synthase gene and method of use thereof for conferring tryptophan overproduction |
| BR112015026210B1 (en) * | 2013-04-16 | 2022-11-29 | Cj Cheiljedang Corporation | RECOMBINANT MICROORGANISM HAVING IMPROVED L-TRYPTOPHAN PRODUCTIVITY AND METHOD FOR PRODUCING L-TRYPTOPHAN |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4371614A (en) * | 1980-08-22 | 1983-02-01 | Ajinomoto Co., Inc. | E.Coli bacteria carrying recombinant plasmids and their use in the fermentative production of L-tryptophan |
-
1980
- 1980-11-05 JP JP55154706A patent/JPS5780398A/en active Granted
-
1981
- 1981-08-26 AU AU74644/81A patent/AU549686B2/en not_active Withdrawn - After Issue
- 1981-09-29 CA CA000386858A patent/CA1182409A/en not_active Expired
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6180373B1 (en) | 1992-09-28 | 2001-01-30 | Consortium f{umlaut over (u)}r elektrochemische Industrie GmbH | Microorganisms for the production of tryptophan and process for the preparation thereof |
| EP1715056A1 (en) | 2005-04-23 | 2006-10-25 | Degussa AG | Process for the production of L-amino acids using improved Enterobacteriaceae strains |
| DE102007051024A1 (en) | 2007-03-05 | 2008-09-11 | Evonik Degussa Gmbh | Process for the preparation of L-amino acids using strains of the family Enterobacteriaceae |
| EP1975241A1 (en) | 2007-03-29 | 2008-10-01 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| EP2036979A1 (en) | 2007-09-15 | 2009-03-18 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| DE102007044134A1 (en) | 2007-09-15 | 2009-03-19 | Evonik Degussa Gmbh | Process for the preparation of L-amino acids using improved strains of the family Enterobacteriaceae |
| EP2055785A1 (en) | 2007-11-02 | 2009-05-06 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| EP2060636A1 (en) | 2007-11-14 | 2009-05-20 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| DE102008040352A1 (en) | 2008-07-11 | 2010-01-14 | Evonik Degussa Gmbh | Process for the preparation of L-tryptophan using improved strains of the family Enterobacteriaceae |
| EP2147972A1 (en) | 2008-07-11 | 2010-01-27 | Evonik Degussa GmbH | Method for manufacturing L-tryptophane using improved strains of the enterobacteriaceae family |
| DE102008044768A1 (en) | 2008-08-28 | 2010-03-04 | Evonik Degussa Gmbh | Process for the preparation of organochemical compounds using improved strains of the family Enterobacteriaceae |
| EP2163613A2 (en) | 2008-08-28 | 2010-03-17 | Evonik Degussa GmbH | Method for manufacturing organic chemical compounds using improved strains of the enterobacteriaceae family |
| EP2628792A1 (en) | 2012-02-17 | 2013-08-21 | Evonik Industries AG | Cell with reduced ppGppase activity |
| WO2014117992A1 (en) | 2013-01-30 | 2014-08-07 | Evonik Industries Ag | Microorganism and method for production of amino acids by fermentation |
| EP3385275A1 (en) | 2017-04-07 | 2018-10-10 | Evonik Degussa GmbH | Method for producing aromatic l-amino acids using improved strains of the enterobacteriaceae family |
| US11053526B2 (en) | 2018-08-09 | 2021-07-06 | Evonik Operations Gmbh | Process for preparing L amino acids using improved strains of the enterobacteriaceae family |
Also Published As
| Publication number | Publication date |
|---|---|
| AU549686B2 (en) | 1986-02-06 |
| JPH0226955B2 (en) | 1990-06-13 |
| JPS5780398A (en) | 1982-05-19 |
| AU7464481A (en) | 1982-05-13 |
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