`Anderson et al.
`
`[11]
`[45]
`
`4,371,614
`Feb. 1, 1983
`
`[54] E. COLI BACTERIA CARRYING
`RECOMBINANT PLASMIDS AND THEIR
`USE IN THE FERMENTATIVE
`PRODUCTION OF L-TRYPTOPHAN
`[75] Inventors: David M. Anderson, Rockville, Md.;
`Klaus M. I-Ierrmann; Ronald L.
`Somerville, both of West Lafayette,
`Ind.
`[73] Assignee: Ajinomoto Co., Inc., Tokyo, Japan
`[21] Appl.No.: 180,296
`[22] Filed:
`Aug. 22, 1980
`
`[51]
`
`Int. Cl.3 ........................ .. C12N l/00; C12R 1/19;
`C12N 9/00; Cl2P 13/22; C12P 21/00; C12N
`15/00; C12N 1/20
`[52] US. Cl. .................................. .. 435/108; 435/317;
`435/849; 435/183; 435/68; 435/172; 435/253
`[58] Field of Search .............. .. 435/108, 172, 317, 253
`[56]
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,237,224 12/1980 Cohen et al.
`
`435/68
`
`OTHER PUBLICATIONS
`Tribe and Pittard, Applied and Environmental Microbi
`ology, v. 38, pp. 181-190 (Aug. 1979).
`Nagahari et al., Gene 1: 141-152 (1977).
`Fredericq and Cornelis, Journal of General Microbiol
`ogy 105: 357-349 (1978).
`Oxender et a1. Proceedings of the National Academy of
`Sciences USA V. 76 5524-5528 (Nov. 1979).
`
`Reznikoff and Thornton, Journal of Bacteriology V.
`109: 526-532 (9172).
`Collins et al., Proceedings of the National Academy of
`Sciences USA vol. 73: 3838-3842 (1976).
`Enger-Valk et al. Gene 9: 69-85 (1980).
`Hersh?eld et al., Proceedings of the National Academy
`of Sciences USA V. 71: 3455-3459 (1974).
`Wagner Abstract of German Patent DT 2841642 Mar.
`1980.
`Nagahari et al., Abstract of Mol. Gen. Genet. 171, 115
`(1979) Chem. Abstr. 90:200102e (1979).
`Hallewell et al. Gene 9, 27-47 (1980).
`Somerville et al. Abstract of J. Mol. Biol. 1 l, 747 (1965)
`Chem. Abstr. 63:2152d.
`Watson, Molecular Biology of the Gene. W. A. Benjamin,
`Inc. (1977), pp. 398-400.
`Bertrand et 211. Abstract of J. Mol. Biol. 117, 227-247
`(1977) in Chem. Abstr. 88:858l6k (1978).
`Primary Examiner—A1vin E. Tanenholtz
`Assistant Examiner-James Martinell
`Attorney, Agent, or Firm-Oblon, Fisher, Spivak,
`McClelland & Maier
`[57]
`ABSTRACT
`A bacterium which comprises a host of the genus Esch
`erichia de?cient in the enzyme tryptophanase carrying a
`plasmid with genetic information to control L-trypto~
`phan production is useful for the fermentative produc
`tion of L-tryptophan in high yields.
`
`34 Claims, 4 Drawing Figures
`
`Sanofi/Regeneron Ex. 1034, pg 952
`
`
`
`U.S. Patent
`
`.D.eF
`
`3oo91
`
`11.,
`
`4,371,614
`
`2o:§.$E
`
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`
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`
`Sanofi/Regeneron Ex. 1034, pg 953
`
`
`
`U.S. Patent Feb. 1, 1983
`
`Sheet 2 of4
`
`4,371,614
`
`CLONING STRATEGY FOR MTR#2 frp GENES FROM CELL DNA
`
`Am
`
`GPK Barn HI
`
`VECTOR
`PER 5??
`
`lBam HI
`l
`
`T4 LIGASE CELL AND VECTOR
`FRAGMENTS AT EOUIMOLAR
`AND HIGH CONCENTRATION
`
`130'" "1
`
`TrpOPERN
`
`_ BamHI
`
`CELL DNA
`
`coRI
`
`Sau 301
`
`Eco RI
`
`Hw
`
`Eco RI
`
`l V l
`
`I g
`'1
`I
`
`Eco RI
`
`Eco RI
`
`7
`J. - 'r
`pBR 322
`rm opmou
`pBR 522
`
`T4LIGASE
`FRAGMENTS AT
`LOW CONCENTRATION
`
`Eco [-71
`
`SauJa/Bom HI\
`
`V
`
`Bar" HT/S?u 30
`
`V: pBR 322 VECTOR
`
`2
`
`Trp OPEHON
`
`Sanofi/Regeneron Ex. 1034, pg 954
`
`
`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 3 of4
`
`4,371,614
`
`CONSTRUCTION OF PUISMIDS
`
`pSer 359-:
`
`MrR#2 DNA
`
`[Sou 3a
`1 T4 Ligase
`
`pGX I —pG x (3
`
`ltlnalysis
`
`l Eco RI
`x T4Ligase
`
`pGx/4-pGxI9
`
`{MTR#21rg_)
`
`Analysis and
`Selection
`
`T4 Ligase
`
`l Ps!I
`
`T4 Ligose
`
`pGx3!-pGI45
`
`(ggg-wud rype Lrp)
`
`(ser 3-MrR#2 i_rg)
`
`(sera-mR#2 rm;
`
`FIG. 3
`
`Sanofi/Regeneron Ex. 1034, pg 955
`
`
`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 4 of4
`
`4,371,614
`
`CONSTRUCTION OF HOST STRAINS
`
`E.C0li SP338
`
`E.Coli SI-l
`
`Select
`tlrr* trp’ser ‘
`
`Select
`lhr‘ trp‘se"
`l
`
`AGXI
`
`E. Cali
`5I6l
`
`P1
`
`P1
`
`AG)!
`
`6
`
`AGX6
`
`pGX35 DNA
`
`Select
`tel’? tyr'
`/ \
`Spontaneous
`
`Select
`tetRtyr'
`/ \
`Spontaneous
`
`[Aex6lpex55l]
`
`KB 3l00P!
`
`/
`Select
`‘
`H5:
`9
`yr
`
`\
`Select
`rt‘
`'
`tyr phe
`e
`
`‘
`
`/
`Select
`/3
`ll‘!
`yr
`e
`
`\
`Select
`'
`tetstyr phe
`
`-
`
`Select
`K4 ‘3' W90)!’ '
`DL-alanine
`résismnoe
`
`FI6.'l
`
`Sanofi/Regeneron Ex. 1034, pg 956
`
`
`
`1
`
`ECOLI BACTERIA CARRYING RECOMBINANT
`PLASMIDS AND THEIR USE IN THE
`FERMENTATIVE PRODUCTION OF
`L-TRYPTOPI-IAN
`
`4,371,614
`2
`authors however, did not further attempt to maximize
`tryptophan production, as their aim was solely to dem
`onstrate the utility of the Col El plasmid as a molecular
`vehicle for cloning and ampli?cation of DNA.
`The biosynthetic pathways for the synthesis of aro
`matic amino acids (tryptophan, tyrosine, phenylalanine)
`in bacteria, are shown in Chart 1:
`
`Q2111
`Su gars
`at least
`15 steps
`V
`¢h°ri§mi° acid
`
`Anthranilic acid
`synthetase
`1/
`
`Anthranilic acid
`
`Prephenic Acid
`
`N'-(5'-ph0sphoribosyl-)_
`
`anthranilic acid
`
`p-Hydroxyphenyl
`
`pyruvli/acid
`
`Phenylpyruvic
`
`,j/ld
`
`Tyrosine
`
`Phenylalanine
`
`Enol'l '(o'carboxyphenyl-
`amino-)4
`l-deoxyribulose-5-phosphate
`
`lndole-3~glycerolphosphate
`
`L-tryptophan
`V tryptophanase
`
`Breakdown products
`(indole)
`
`30
`
`35
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The present invention relates to E. coli microorgan
`isms carrying recombinant plasmids constructed in
`vitro and their use for producing L-tryptophan by fer
`mentation.
`2. Brief Description of the Prior Art
`The production of L-tryptophan from carbohydrates
`in wild type microorganism strains, has been obtained in
`the prior art through arti?cial mutants therefrom.
`Among the known examples of such arti?cial mutants
`are those of the genera Brevibacterium resistant to 5
`methyl-tryptophan (US Pat. No. 3,700,539), Bacillus
`resistant to S-?uorotryptophan (Japanese Published
`Unexamined Patent Application Number 20391/1974),
`and Enterobacter resistant to S-methyl-tryptophan (Jap
`anese Published Unexamined Patent Application Num
`ber 57888/1976).
`25
`The most ef?cient known microorganism to produce
`tryptophan is Corynebacterium glutamicum ATCC
`21851, which requires phenyl-alanine and tyrosine, and
`is resistant to 4-methyl-tryptophan, ?-?uoro-trypto
`phan, 4-amino-phenylalanine, 4-?uoro-phenylalanine,
`tyrosine-hydroxamate, and phenylalanine-hydroxa
`mate. This strain produced 16.8 mg/ml tryptophan
`from 15 g/dl of sugar derived from cane blackstrap
`molasses. However, the yield of tryptophan in this best
`known method, is still insufficient to ful?ll commercial
`requirements.
`The possibility of utilizing recently developed ge
`netic recombination techniques, to engineer a microor
`ganism capable of producing high levels of tryptophan
`is appealing. The general techniques for the introduc
`tion of genes, and the ampli?cation in bacteria capable
`of expressing them have recently been described by
`Gilbert and Villa-Komaroff in Scienti?c American, 242:
`74-94 (1980). Briefly, one or more genes from a donor
`organism, such as a prokaryotic or eukaryotic cell are
`introduced into a vector or plasmid (extrachromosomal
`circular DNA) in vitro, by means of a splicing/ligation
`sequence using endonuclease and ligase enzymes re
`spectively. The hybrid plasmid containing the gene or
`genes is then mixed with cells of a host organism, usu
`ally a prokaryotic bacterial microorganism. A dilute
`solution of calcium chloride renders the bacteria perme
`able and the cells will take up plasmids from solution.
`Reproduction of the plasmid-carrying host microorgan
`isms then produces millions of identical copies of the
`recombinant DNA. If the appropriate genetic control
`sequences are present, the ampli?ed gene or genes will
`produce corresponding enzymes using the available
`protein-synthesizing apparatus of the host.
`Hersh?eld et al, for example (Proceedings of the
`National Academy of Sciences, USA, 71: 3455-3459
`(1974)), have reported the insertion of a DNA fragment
`of E. coli possessing genetic information related to tryp
`tophan (trp) production (trpA-E gene), into the Col El
`(colicinogenic factor El) plasmid. When a tryptophan
`auxotroph of E. coli was transformed with the resulting
`hybrid plasmid (Col El trp), the tryptophan auxotroph
`became a tryptophan prototroph. Elevated levels of
`tryptophan biosynthetic enzymes were reported. These
`
`Tyrosine, phenylalanine and tryptophan are produced
`from a common biosynthetic intermediate, chorismic
`acid. Tyrosine and phenylalanine are furthermore de
`rived from another common intermediate, prephenic
`acid. The ?rst major enzyme of the tryptophan pathway
`is anthranilate synthetase which, in E. coli is subject to
`feedback inhibition by L-tryptophan. The degradation
`of tryptophan into indole by the enzyme tryptophanase
`is also shown in Chart 1.
`A need continues to exist for microorganisms capable
`of producing high levels of tryptophan. A need also
`continues to exist for a method for the production of
`tryptophan in high yields, using a microorganism dis
`tinct from those obtained by the mutation techniques of
`the prior art.
`SUMMARY OF THE INVENTION
`These and other objects of the invention which will
`hereinafter become more readily apparent have been
`attained by providing:
`A bacterium which comprises a host of the genus
`Escherichia de?cient in the enzyme tryptophanase,
`carrying a plasmid with genetic information to control
`tryptophan production.
`Another object of the invention has been attained by
`providing a method for producing L-tryptophan by
`fermentation which comprises culturing in a culture
`medium a bacterium as described hereinbefore, and
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Sanofi/Regeneron Ex. 1034, pg 957
`
`
`
`4,371,614
`3
`recovering the L-tryptophan produced from the culture
`medium.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`A more complete appreciation of the invention and
`many of the attendant advantages thereof will be
`readily obtained as the same becomes better understood
`by reference to the following detailed description when
`considered in connection with the accompanying draw
`ings, wherein:
`FIG. 1 shows the plasmid pSerB59-l which is de
`rived from the pBR322 vector and the E. coli serB gene.
`The abbreviations denote restriction endonuclease en
`zymes from the following sources:
`
`10
`
`20
`
`25
`
`35
`
`45
`
`4
`mants and result in loss of the plasmids (see, e.g.,
`Hersch?eld et a1, supra; I-Iallewell, R. A., et al, Gene 9:
`27—47 (1980)). However, to prevent loss, it is possible in
`a preferred embodiment of this invention, to add a tem
`perature sensitive trpR gene (trpR"), or a trpR gene,
`which causes gene product synthesis to be regulated by
`temperature, see infra. With this gene, the stability of
`the multicopy plasmids carrying strains can be even
`further increased.
`Plasmids used in this invention contain a wild type trp
`operon or a mutated trp operon gene which expresses
`feedback resistant anthranilate synthetase. In a pre
`ferred embodiment, the plasmids additionally contain a
`serB+ gene. Plasmids containing the trp operon or plas
`mids containing both a trp operon and a serB+ gene,
`can be constructed by using any of the well known
`plasmids or vectors available. These plasmids may be E.
`coli plasmids or plasmids capable of replicating in E.
`coli. Among useful plasmids, the following can be listed:
`ColEl, pSClOl, pSF2l24, pmB8, pMB9, ACYC184,
`pAcYCl77, pCKl, R6K, pBR3l2, pBR3l3, pBR3l7,
`pBR3l8, pBR320, pBR32l, pBR322, pBR333, pBR341,
`pBR345, pBR350, pBR35l, pML2, pML2l, ColElAp,
`RSFlOlO, pVHSl, pVH151, pVHl53 (Recombinant
`Molecules: Impact on Science and Society: Beers, R. F.,
`and Bassett, E. G. eds, Raven Press, New York (1977)).
`Other plasmids are pBR327, pBR325 and pBR328 (So
`beron, et al, Gene 9: 287-305 (1980)); still others are
`described in “DNA Insertion Elements, Plasmids and
`Episomes”, Bukhari et al (eds), Cold Spring Harbor
`Laboratory (1977). The preferred plasmids are the mul
`ticopy plasmids of the type of pBR and its derivatives,
`and ColEl and its derivatives.
`The presence of both serB+ and trp genes in the same
`plasmid assures that if, during fermentation, this plasmid
`is spontaneously lost, the resulting ser auxotroph bacte
`rium will stop growing. Since some of the recipient
`microorganisms may be trp auxotrophs, these would
`naturally stop growing unless the medium contains a
`source of external tryptophan. However, because of the
`nature of the present invention, the bacteria have been
`producing high levels of tryptophan until the time of
`loss of the trp gene-containing plasmid. At the time of
`loss then, the medium does contain tryptophan and the
`plasmidless host would be able to continue growing.
`This would entail the undesired consumption of trypto
`phan from the medium and decrease overall yields of
`the amino acid. By constructing composite plasmids
`harboring both the trp+ and serB+ gene, it is assured
`that if a trp and ser auxotroph is used as host and if loss
`of the composite plasmid occurs, growth will stop and
`tryptophan yields will not decrease.
`A plasmid containing the serB gene may be prepared,
`for example, from pBR322 according to Somerville
`(Roeder and Somerville, Molecular and General Genet
`ics, 176: 361-368 (1979)). This plasmid called pSerBS9-l
`is described in FIG. 1, including the speci?c endonucle
`ase sites that have been determined. The plasmid is
`approximately 9.4 kilo bases long and contains more
`genetic information than necessary. In order to decrease
`the size and therefore potentially increase the number of
`plasmid copies per cell in the ?nal strain, it is preferred
`to prepare deletions of pSerB59-1. pSerB59-1 DNA can
`be partially digested to various extents with endonucle
`ase Sau 3a, which cuts at the sequence
`
`Bgl l:
`Barn HI:
`EcoR I:
`PS! I:
`Hind III:
`Bat E II:
`Hpa I:
`Sal l:
`Xba I:
`Sac I:
`Sst l:
`Bel I:
`Xho I:
`Kpn I:
`Pvu II:
`
`Bacillus globllai
`Bacillus amyloliquqfaciens H
`E. coli RY13
`Pravidencia stuarrii [64
`Haemophilus in?uenzue Rd
`Bacillus stearotermophilus ET
`Haemophilus pamin?uenzae
`Streptomyces albis G
`Xanthomonas badrii
`Streplomyces achromogenes
`Strepmmyces stanford
`Bacillus caldolvticus
`Xanrhomonas halicala
`Klebsiella pneumoniae
`Proteus vulgaris
`
`FIG. 2 shows the cloning strategy for MTR #2 trp
`genes from cell DNA using the vector pBR322 as clon
`ing vehicle. Abbreviations used:
`Sau 3a: endonuclease derived from Staphylococcus
`aureus 3a
`Amp: ampicillin resistance gene
`FIG. 3 is a flow chart showing the construction of
`composite plasmids carrying both a serB gene and a
`MTR #2 trpE gene. Abbreviations used:
`MTR #2 trp: Methyl tryptophan resistance E. coli
`feedback resistant anthranilate synthetase.
`pGx: plasmid numbering system
`FIG. 4 is a flow chart showing the construction of
`host strains. Abbreviations and numbering used:
`37-1:
`I-IfrH(gal-bio-attk)V deo=serB-trpR)Vthi—
`strain of E. coli.
`SP338: trpED, tna2, thr- strain of E. coli.
`61-1: (deoB—serB)vthi— (gal-bio-att7t)V strain of E.
`
`5161: tyrA: tnlO insertion strain
`Pl: bacteriophage Plkc (transducing phage)
`tetR: tetracycline resistance
`AGx: strain numbering system.
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`The present invention provides E. coli strains capable
`of producing high levels of L-tryptophan by fermenta
`tive procedures. The preparation of plasmids containing
`DNA from a donor, of the recipient strains, and of the
`transformants among recipients and donor DNA are as
`follows:
`
`55
`
`60
`
`A. Plasmid Construction
`The plasmids used in this invention are either strin
`gent (single copy plasmids) or relaxed (multicopy plas
`mids). The multicopy plasmids are preferred since the
`yields of tryptophan are higher. The use of multicopy
`plasmids may cause instability of the resulting transfor
`
`65
`
`Sanofi/Regeneron Ex. 1034, pg 958
`
`
`
`and leaves the tetranucleotide GATC single stranded
`on the 5' end of each fragment. Since Sau 3a has a tetra
`meric recognition sequence, it cuts very frequently
`within pSerB59-l DNA. Partial digestion produces a
`somewhat random distribution of fragments representa
`tive of each segment of the plasmid. The digest can then
`be ligated with phage T4ligase at low concentration to
`produce circles, or at higher concentrations with the
`endonuclease Bam H] digested pBR 322 DNA. Barn HI
`cuts at the hexanucleotide
`
`25
`
`45
`
`20
`
`4,371,614
`6
`mids, such as those described above: (a) wild type trp
`operon and (b) feedback resistant anthranilate synthe
`tase trp operon. The latter mutant trp gene is one
`wherein anthranilic acid synthetase is resistant to feed
`back inhibition by tryptophan. It can be found in high
`frequency in mutants resistant to tryptophan-antago
`nists. These antagonists inhibit the growth of Escher
`ichia strains, but the inhibition is suppressed partially or
`completely when tryptophan is in the medium. Exam~
`ples of tryptophan antagonists are: 4-?uoro-tryptophan,
`S-fluoro-tryptophan, ?-fluoro-tryptophan, 7-?uoro
`tryptophan, 4-methyl-tryptophan, S-methyl-trypto
`phan,
`6-methyl-tryptophan,
`7-methyl-tryptophan,
`naphthyl-alanine, indole-acrylic acid, naphthyl-acrylic
`acid, B-(2-benzo-thienyl)-alanine, styryl-acetic acid,
`indole and tryptazan. In addition, the plasmids may be
`constructed so that they contain a deletion of the atten
`uator region of the trp operon. (Oxender et al, Proc.
`Nat. Acad. Sci. USA., 76: 5524 (1979)). This region is
`present between the operator region and the beginning
`of the trpE gene. With the attenuator region in place
`and in the presence of excess tryptophan and functional
`tRNAl'l’, only one transcript in eight proceeds past the
`termination region in the tryptophan leader. Removing
`this control greatly increases the levels of trp operon
`enzymes. The absence of prematurely terminated tran
`scripts saves metabolic energy which can be used in
`tryptophan production.
`The feedback resistant trp genes can be cloned by
`partially or totally digesting the E. coli mutant trp op
`eron with any endonuclease that does not cut within the
`trp operon and then ligating (e.g., using T4 DNA ligase)
`with plasmid at a concentration so that the cell frag
`ments and plasmid fragments are at approximately equal
`molar concentration. FIG. 2 exempli?es such a tech
`nique using MTR #2 (Somerville, R. and Yanofsky, C.,
`J. Mol. Biol, ll: 747 (1965)) as donor for cell DNA and
`pBR322 as the plasmid. A ?rst ligation can preferably
`be carried out at high DNA concentration so that for
`mation of long concatenated molecules predominates.
`The resulting DNA is digested with an enzyme that cuts
`in the vector but not in the trp operon DNA, diluted to
`low concentration and religated; the low concentration
`favors circle formation. Detection and selection of mu
`tant trp operon-containing plasmids can be done by
`transforming a (trpA-E)V strain (Zalkin, H., et al, J.
`Biol. Chem., 249: 465-475 (1974)) therewith and allow
`ing the cells to grow on minimal media. Resistance to
`S-methyltryptophan is a property of the trp operon of
`the MTR#2 strain. The resulting plasmids can then be
`used for recombining the feedback resistant trp operon
`into the serB+ containing plasmids, described previ—
`ously. The MTR#2 trp1L operon is only exemplary and
`any feedback resistant anthranilate synthetase express
`ing gene can be used.
`Recombination of mutant trp operon (e.g., MTR#2)
`containing plasmids with serB-containing plasmid
`yields trp’r-ser+ composite plasmids. For example,
`partial digests of MTR#2 trp-containing DNA can be
`ligated (T4 DNA ligase) at high DNA concentrations
`with excess digested serBit-containing plasmid DNA.
`The high molecular weight ligated DNA can be di
`gested, with a second enzyme which does not cut in the
`trp operon, diluted to low concentration and ligated
`again to form circles. The resulting plasmids are se
`lected by transforming E. coli cells having trp and ser
`deletions, so serBt/ mutant trp-containing plasmids will
`
`leaving the same tetranucleotide single stranded at the
`5' ends of fragments as San 3a. Therefore, Sau 3a di
`gested DNA can be readily ligated to Barn H] digested
`DNA. In order to select plasmids which carry the serB
`gene, the resulting mixture of deleted ligated plasmids
`can be introduced into a serine auxotroph and tested for
`colonies which grow in the absence of serine. For exam
`ple, strain 37-1 E. coli W31 10, I-lfrH, (gal-bio-attk)‘7
`(deo-serB-trpR)V, thi—, (Roeder and Somerville, Mo
`lecular and General Genetics 176: 361—368 (1979)) can
`be transformed for this purpose. Plasmids carrying the
`serB gene can then be isolated by screening the colonies
`(Eckhardt, T., Plasmid, 1: 584-588 (1978)). By such
`procedures, several plasmids can be isolated which are
`smaller than pSerB59-l having observable deletions in
`the range of 85 to 51% the size of pSerB59-l. An analy
`sis of these smaller plasmids for expression of ampicillin
`resistance, size and restriction sites allows the search
`and selection of the most efficient cloning vehicle. For
`example, two plasmids, having 45% the size of
`pSerB59-l, and a size of about 4.2 kilo bases can be
`isolated. Both of these have no Eco RI sites, 2 Bgl 1
`sites, 1 Est EII site, 1 Bgl I] site, no Sal I sites and l Pst
`I site. The restriction map of a plasmid derived from
`pBR322 carrying a serB+ gene is also shown in H0. 1
`including the probable location of the serB+ gene in the
`plasmid. It can be ascertained that the enzymes Pst I and
`Bgl II can be used to insert genes into this plasmid. The
`Pst I site is located in what remains of the ampicillin
`resistance gene. Although the Bgl II site seems to have
`been preferentially retained in the deleted plasmids, it is
`not located within the serB gene. This can be shown by
`cloning Sau 3a digested cell DNA into the Bgl II site of
`the plasmid 6 and demonstrating that the serB’r gene
`function is retained. This plasmid is exemplary only. In
`a preferred embodiment however, this plasmid is uti
`lized as the cloning vehicle.
`Two types of trp operon genes can be used alone in
`plasmids, or inserted into serB gene-containing plas
`
`60
`
`65
`
`Sanofi/Regeneron Ex. 1034, pg 959
`
`
`
`4,371,614
`7
`confer on these cells the ability to grow in the absence
`of external tryptophan and serine. Analogous plasmids
`prepared from serB+ gene-containing plasmids, and
`wild type trp operon-containing plasmids such as pCC3
`(Collins, C. J., Proc. Nat. Acad. of Sci., USA 73: 3838
`(1976)) can also be prepared and tested in the same
`manner. The wild type or trp+ operon genes can also be
`obtained directly from a chromosone. The composite
`plasmids contain both the serB+ and wild or mutant trp
`gene integrated therein.
`
`8
`The trpR“ variant can be obtained from a trp-lac
`fusion E. coli strain, (Reznikoff et al J. of Bact., 109:
`526-532 (1972)), by transducing a P1 lysate thereof into
`any of the wild E. coli or mutant E. coli strains of this
`invention. In trp-lac fusion strains, trpR" mutations can
`be readily identi?ed.
`The aroP mutation (Kuhn, J. C. and Sommerville, R.
`L., Biochem. Biophys. Acta 332: 298-312 (1974);
`Brown K. D., J. Bact. 106: 70-81 (1971)) can be inserted
`into trpR+ 61-1 E. coli by P1 transduction from E. coli
`KB 3100 (Brown, K. D., J. Bacteriol. 106: 71-81 (1971))
`and selection for resistance to B-Z-thienyl-DL-alanine
`(Brown, K. D., J. Bact. 104: 177-188 (1970)).
`When trp-ser deleted cells contain one of the trp-ser
`plasmids, and are not supplemented with tryptophan,
`inhibition of growth by B-Z-thienyl-DL-alanine is suffi
`cient to allow selection of an aroP mutant. A phage Pl
`lysate of E. coli KB 3100 (arop-) can be used to infect
`the plasmid-containing trp ser deleted strain and the
`resulting colonies which grow rapidly on B-2-thienyl
`DL-alanine plates are selected. One of the rapidly
`growing strains having an enhanced ability to excrete
`tryptophan can be ascertained to contain the aroP muta
`tion.
`The tryA and tryA-pheA mutations can be intro
`duced into the strains using a strain of E. coli (E. coli
`5161), which has a Tnl0 transposable element
`(Kleckner, N. et al. Proc. Nat. Acad. Sci. USA 73:
`3838-3842 (1976)) inserted in the tyrA locus. Phage Pl
`lysates of such E. 0011' are used to transduce trpR+ and
`trpR— strains. Tetracycline resistant tyrosine auxo
`trophs (tetRtyr*) are selected. Tetracycline sensitive
`derivatives are selected by ampicillin enrichment
`(Miller, J. H., Experiments in Molecular Genetics
`(1972), supra) or by plating under conditions that only
`allow tets colonies to grow. In this way, genes with tn l0
`insertions are converted to non-revertable mutations.
`By screening the tets colonies, cells can be found with
`deletions or rearrangements that affect either the tyrA
`locus alone, or both the tyrA and pheA loci. The double
`mutants occur because the pheA map location is very
`close to tyrA (Bachmann, B. J. and Low, K. B. Microbi
`ological Reviews: 44: l-56 (1980)).
`
`- 5
`
`25
`
`35
`
`40
`
`B. Host Strain Constructions
`As the recipient microorganism for the hybrid DNA
`plasmids, tryptophanase-deiicient mutants of E. coli are
`used in this invention. Preferably trp operon-lacking
`strains (trp operonv) and/or nonrevertable tryptopha
`nase-mutated (tna) strains are used. The lack of trypto
`phanase insures that tryptophan is not broken down and
`yields of excreted L-tryptophan remain high. A trypto
`phan auxotroph may also be used as the recipient, since
`a transformant which produces tryptophan can be eas
`ily selected from the recipients. The host may also be a
`serine auxotroph when the serB+ gene is on the plas
`mid. Desirably, the host also has one or more of the
`following genetic deficiencies: (i) aroP gene: this gene
`controls the permeability characteristics of the cells to
`aromatic amino acids. In aroP mutant strains trypto
`phan excretion is proportional to anthranilate synthe
`tase activity; (ii) trpR gene (tryptophan repressor gene):
`this gene controls the repression of the tryptophan bio
`synthetic pathway; in trpR mutant strains the pathway
`proceeds derepressed regardless of the levels of trypto
`phan produced; (iii) The trpR gene can also, in a pre
`ferred embodiment, be temperature sensitive, i.e.,
`trpR“. In such case, when the cells are maintained at
`low temperatures trp repressor will be produced to
`allow normal cell growth. During production, when
`the cell mass has proceeded to a sufficient level, the
`fermentation cooling is turned off, the temperature rises
`and inactivates the repressor, preventing further func
`tional trpR product synthesis: (iv) tyrA: this gene con
`trols the biosynthetic pathway for the formation of
`tyrosine; in tryA mutant strains the ?ow of aromatic
`precursor (chorismic acid) can be diverted into the trp
`and the phenylalanine paths; (v) pheA: this gene con
`45
`trols the biosynthetic pathway for the formation of
`phenylalanine; in pheA mutant strains the flow of aro
`matic precursor (chorismic acid) can be diverted into
`the trp and tyr pathways; (vi) tyrA pheA: controls both
`the phenylalanine and tyrosine pathways.
`Preferably the host strain may contain more than one
`of the aforementioned mutations in order to maximize
`the biosynthetic flow and excretion of tryptophan.
`In order to prepare trp operonV strains, it is possible
`to use a trpED 102 deletion strain. A threonine auxo
`troph with the trpED deletion, E. Coli SP338 (trpED‘7
`102, tna2, thr—) (Roeder and Somerville, Molecular and
`general Genetics, 176: 361-368 (1979)), is transduced
`with phage Pl lysates (Rosner, J. L., Virology 49:
`679-689 (1972)) prepared on either E. Coli 61-1 (Roeder
`and Somerville, Molecular and General Genetics, 176:
`361-368 ( l979)) [deob-serB), thi“, (gal-bio-attMV] or on
`E. Coli 37-1 (Roeder and Somerville, supra.) [I-IfrH,
`(gal-bio-atOt)V (deo-serB-trpRW, thi—]. Tryptophan
`auxotrophs are selected which no longer require threo
`nine and have become serine auxotrophs. They can be
`prepared with the trpR- 37-1 cell line Pl lysates and
`with trpR+ 61-] cell line lysates.
`
`60
`
`C. Transformation of Host Strains
`Hosts are transformed with plasmid DNA (contain
`ing wild type trp-, wild type trp-ser, mutant trp, mutant
`trp-serB as well as plasmids with the attenuator dele
`tion) by calcium shock. This procedure makes the hosts
`competent for DNA uptake (see, e.g., Morrison, D. A.,
`J. Bact. 132: 349-351 (1977)). Anthranilate synthetase
`specific activity determinations are made with cultures
`of plasmid-containing cells. Alternatively, tryptophan
`levels can be detected enzymatically or by microbioas
`say. The cells are grown in a medium containing
`casamino acids which is tryptophan-free but contains
`serine, or a synthetic medium which contains all amino
`acids except serine and tryptophan. Strains with multi
`copy plasmids containing only trp genes have consider
`able elevations in anthranilate synthetase. In trpR+
`strains, feedback resistant mutant trp-containing plas
`mids display a considerable increase in anthranilate
`synthetase specific activity over the level observed in
`wild-type E. coli W3ll0.
`Tryptophan excretion experiments show that those
`strains which contain either the aroP mutations or the
`tyrA, tyrA pheA mutations do excrete tryptophan.
`Without these mutations, very little tryptophan is made.
`
`55
`
`65
`
`Sanofi/Regeneron Ex. 1034, pg 960
`
`
`
`10
`~continued
`NRRL 8-4575
`E. coli trp-lac fusion
`MEDIA FORMULAS
`
`LB
`ELLCL
`
`tryptone 10g
`yeast extract 5g
`sodium chloride 10g
`
`5
`
`FUSZIN PLATES
`Er liter
`agar 15g
`Dii'co @ tryptone 10g
`Difco ® yeast extract 5g
`sodium chloride 10g
`glucose 2g
`chlorotetracycline 50mg
`NaH1PO4J-IZO
`
`L8 plates = LB + 1% agar
`LB Mg = LB + 0.0IM MgClz
`LB Ca = LB + 0005M CaClz
`L8 Topagar = L8 + 0.75% agar
`Saline = 0.85% NaCl
`
`Supplements to Minimal Plates
`amino acids 20 tag/ml
`tetracycline 25 ug/ml
`B-Z-thienyl-DL-alanine 22 ug/ml
`Difco @ casamino acids 0.4%
`
`biotin 1 pg/ml
`
`thiamim l ‘Lg/ml
`
`autoclave, cool, then add
`6m] Zmg/ml fusaric acid
`Sml Zinc chloride ZOmM
`Minimal plates
`per liter
`K;HPO4 10.5g
`KH2PO4 4.5g
`(P11102804 1-08
`Sodium citrateZHZO 0.5g
`glucose 4g
`(autoclave separately)
`agar 1.5g
`(autoclave separately)
`after cooling adjust to
`MgClz lrnM
`FezSOa 10 PM
`Solutions for Transformations
`Wash:
`lwmM NaCl
`Solution:
`SmM MgClZ
`SmM Tris HCl pH 7.6
`
`4,371,614
`9
`less than 10 pg/ml. High yields of tryptophan may be
`obtained by combining the am? mutated hosts with a
`mutant trp-serB plasmid or a wild type trp-serB plas
`mid.
`The methods of culturing the L-tryptophan produc
`ing strains thus obtained are conventional, and are simi
`lar to the methods for the cultivation of known L-tryp
`tophan producing microorganisms. Thus, the culture
`medium employed is one containing carbon sources,
`nitrogen sources, inorganic ions and, when required,
`minor organic nutrients such as vitamins or amino acids.
`Examples of suitable carbon sources include glucose,
`lactose, starch hydroysate and molasses. Gaseous am
`monia, aqueous ammonia and ammonium salts and
`other nitrogen containing materials can be used as the
`nitrogen source.
`Cultivation of the recombinant microorganisms is
`conducted under aerobic conditions, in which the pH
`and the temperature of the medium are adjusted to a
`suitable level, and continued until the formation of L
`tryptophan ceases.
`The L-tryptophan accumulated in the culture me
`dium can be recovered by conventional procedures
`(Japanese Published Unexamined Patent Application
`No. 74293/ 1979).
`By the method of the present invention, L-trypto
`phan can be produced in higher yields than has been
`achieved in previously known methods using mutants
`of Escherichia.
`Having generally described this invention, a further
`understanding can be obtained by reference to certain
`speci?c examples which are provided herein for pur
`poses of illustration only and are not intended to be
`limiting unless otherwise speci?ed.
`A. Experimental Methods
`Table 1 shows strains and plasmids used for the fol
`lowing experiments:
`
`25
`
`30
`
`35
`
`Calcium
`Solution:
`
`l00rnM CaClz
`250mM KC]
`SmM MgCl;
`SmM Tris TCl pH 7.6
`M9 casamino acids medium
`Er liter
`4g
`casamino acids
`6g
`Na1HPO4
`3g
`Kl-lzPOa
`0. 5g
`NaCl
`1.0g
`NH4CL
`Make 0.1mM in CaCl and 0.4% glucose after autoclaving
`Solutions for DNA Preparation
`Wash:
`0.2M Tris/HCl pH 8.0
`A ( 0.8% NaCl
`0.01M EDTA
`8
`SOmM Tris pH 8.0, 20% sucrose
`C
`0.25M EDTA pH 8.0
`5 ug/ml lysozyme in 0.25M Tris/
`HCl pH 8.0
`10 tag/ml lysozyme in 0.25M Tris/
`HCl pH 11.0
`60mM EDTA
`SmM Tris
`0.1% Triton X-l00
`
`l22-l(pSer859-l)
`
`pCC3
`
`MTR #2
`
`TABLE I
`Mam
`Description
`Reference
`E. coli Barn H1 DNA
`(a)
`fragment cloned in
`8am H1 site of pBR322
`trp operon derived from
`d>80ptA~E cloned in C01 El
`along with gal operon genes
`Mutant with feedback resis-
`