United States Patent f!9J
`Anderson et al.
`
`[II]
`
`[45]
`
`4,371,614
`Feb. 1, 1983
`
`[54] E. COLJ BACTERIA CARRYING
`RECOMBII'<ANT PLASMIDS AND THEIR
`USE IN THE FERMENTATIVE
`PRODUCTION OF L-TRYPTOPHAN
`Inventors: David M. Anderson, Rockville, Md.;
`Klaus M. Herrmann; Ronald L.
`Somerville, both of West Lafayette,
`Ind.
`
`[75]
`
`[73] Assignee: Ajinomoto Co., Inc., Tokyo, Japan
`[21] Appl. No.: 180,296
`Aug. 22, 1980
`[22] Filed:
`[51]
`Int. CJ.3 .......................... C121'< 1/00; Cl2R 1/19;
`C12N 9/00; C12P 13/22; CI2P 21/00; CI2N
`15/00; C12N 1/20
`[52] U.S. 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 PUBLICA TJONS
`Tribe and Pittard, Applied and Environmental Microbi(cid:173)
`ology, v. 38, pp. 181-190 (Aug. 1979).
`Nagahari et al., Gene 1: 141-152 (1977).
`Fredericq and Cornelis, Journal of General Microbiol(cid:173)
`ogy 105: 357-349 (1978).
`Oxender et al. Proceedings of the National Academy of
`Sciences USA V. 76 5524-5528 (Nov. 1979).
`
`ReznikofT 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).
`Hershfield 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) Chern. Abstr. 90:200102e (1979).
`Hallewell et al. Gene 9, 27-47 (1980).
`Somerville et al. Abstract of J. Mol. Bioi. II, 747 (1965)
`Chern. Abstr. 63:2152d.
`Watson, Molecular Biology of the Gene, W. A. Benjamin,
`Inc. (I 977), pp. 398-400.
`Bertrand et al. Abstract of J. Mol. Bioi. 117, 227-247
`(1977) in Chern. Abstr. 88:85816k (1978).
`
`Primary Examiner-Alvin E. Tanenholtz:
`Assistant Examiner-James Martinell
`Auorney, Agent, or Firm-Obion, Fisher, Spivak,
`McClelland & Maier
`[57]
`ABSTRACT
`A bacterium which comprises a host of the genus Esch(cid:173)
`erichia deficient in the enzyme tryptophanase carrying a
`plasmid with genetic information to control L-trypto(cid:173)
`phan production is useful for the fermentative produc(cid:173)
`tion of L-tryptophan in high yields.
`
`34 Claims, 4 Drawing Figures
`
`Sanofi/Regeneron Ex. 1 034, pg 952
`
`Merck Ex. 1034, pg 978
`
`

`
`F/6. 1
`
`INSERT CDITAINING
`SERINE B GENE
`
`Bgll
`
`Bum HI
`
`CLONING PLASMID
`p Ser 859·1
`
`Bam HI
`
`ENlYM£S
`WITHOJT
`SITES
`Xba I
`Sac!
`Sst!
`Bell
`Xhol
`Kpnl
`
`(f)
`Q)
`::::J
`
`0 -::::::.:
`
`:::0
`CD
`(()
`CD
`::::J
`
`CD a
`::::J
`m
`;><
`.....>.
`0
`0)
`.t:>.
`"'0
`(()
`<D
`01
`0)
`
`-(cid:173)
`
`_..-
`.--- _.
`
`AMPICILLIN RESIST/WCE
`GENE
`.... ---
`~---,r------' c:::3
`EcoRI
`
`..... -----f/IJII
`
`1--------1----~'-----
`1 KILO BAS£
`
`_ ........
`
`.... -
`--
`
`PROBABLE
`LOCATION
`SER 8 GENE
`r------,.
`..... ____ _.
`BgiJI
`. Bgll
`Bg/1
`
`----------
`
`Hpol
`
`pBR 322 VECTOR
`
`ORIGIN OF
`REftiCATION
`
`------
`
`._._SPLIT TETRACYCUNE
`RESISTANCE GENE
`--
`So/1~118gll -----. .....
`
`Hpol
`
`Pvuii PvuH
`l--
`SEGMENTS RETAINED IN pGx6
`
`BomHI
`
`Pvuii
`
`; '
`
`~
`en .
`;?
`('D g
`
`~
`?'
`.....
`.....
`
`\C)
`00
`w
`
`Vl
`::r
`0
`0 .....
`
`0 .....,
`~
`
`_.:::..
`...
`w
`-.....]
`...
`""""'"
`0\
`""""'" ~
`
`Merck Ex. 1034, pg 979
`
`

`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 2 of 4
`
`4,371,614
`
`CLJJNING STRATEGY FOR MTR'#2 trp GENES FROM CELL DNA
`
`Amp o-BomHI
`
`VECTOR
`pBR 322
`~Bam HI
`
`T4LIGAS£ CELL AND VECTOR
`FRAGMENTS AT £(}.)/MOLAR
`AND HIGH CONCENTRATION
`
`CELL DNA
`Sou3o!
`
`I Hosr
`
`Bam HI
`Sou 3o
`
`EcoRI
`
`I' V I
`
`Bam HI Bam HI
`Sou .3o Sou 3a
`
`Eco Rl
`
`~
`
`pBR .322
`
`v
`
`£co RI
`
`f ~ V I·
`J!gmjJ1. Bam H 1
`Sou .3o "SaiiJO
`
`1~"1 £co RI
`~
`
`Trp OPERON
`
`pBR 322
`
`l T4L1GAS£
`
`Eco RI
`
`FRAGMENTS AT
`WW CONCENTRATION
`
`FIG.2
`
`Trp OPERON
`
`V = pBR 322 VECTOR
`
`Sanofi/Regeneron Ex. 1 034, pg 954
`
`Merck Ex. 1034, pg 980
`
`

`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 3 of 4
`
`4,371,614
`
`CONSTFWCTION OF PLASM/OS
`
`pSer 8 59·1
`
`MTR#2 DNA
`
`p8R 322 0~
`
`lsodo
`~ T4 Ligase
`pGx/-pGII /3
`
`!Analysis
`
`pGx6 I
`
`( Ser B J
`
`pCC3
`
`T4 Ligase
`
`t Pst I
`
`( ser 8- wild type .!!.R J
`
`T4 Ligase
`~ Pst I
`
`FIG. ~
`
`Sanofi!Regeneron Ex. 1 034, pg 955
`
`Merck Ex. 1034, pg 981
`
`

`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 4 of 4
`
`4,371,614
`
`CONSmUCTION OF HOST STRAINS
`
`E. Coli 37-1
`
`E. Coli SP 338
`
`E. Coli 61-1
`
`P1
`
`Plkc
`
`Seleot
`thr+ frp-ser-
`
`I AG)AG'51
`
`AGxl
`
`I AGx6-AGx13J
`
`AGX6
`
`£Coli
`5161
`
`pGX35 DNA
`
`Select
`tetR lyr-
`I \
`Spontaneous
`\
`I
`tet5 tg-file-8
`~
`
`Select
`te,S tyr-
`
`Select
`
`[AGx6 (pGx35)}
`
`KB3/00~ I
`PJ l
`
`Spontaneoos
`
`I \
`\
`I
`I AG X/9 I
`~8
`FIG. 'I
`
`Select
`tets lyr A
`
`Select
`tets t~r-phe-
`
`Select
`{3-2-thienyi(cid:173)
`D, L -alanine
`resistance
`
`Sanofi/Regeneron Ex. 1 034, pg 956
`
`Merck Ex. 1034, pg 982
`
`

`
`l
`
`4,371,614
`
`2
`authors however, did not further attempt to maximize
`tryptophan production, as their aim was solely to dem(cid:173)
`onstrate the utility of the Col El plasmid as a molecular
`vehicle for cloning and amplification of DNA.
`The biosynthetic pathways for the synthesis of aro-
`matic amino acids (tryptophan, tyrosine, phenylalanine)
`in bacteria, are shown in Chart I:
`
`sJ .. at least
`··,:=.:-t-/'""""" ~
`
`15 steps
`
`E. COLI BACTERIA CARRYING RECOMBINANT
`PLASMIDS AND THEIR USE IN THE
`FERMENTATIVE PRODUCTION OF
`L-TRYPTOPHAN
`
`5
`
`BACKGROUND OF THE INVENTION
`I. Field of the Invention
`The present invention relates to E. coli microorgan(cid:173)
`isms carrying recombinant plasmids constructed in 10
`vitro and their use for producing L-tryptophan by fer(cid:173)
`mentation.
`2. Brief Description of the Prior Art
`The production of L-tryptophan from carbohydrates
`in wild type microorganism strains, has been obtained in 15
`the prior art through artificial mutants therefrom.
`Among the known examples of such artificial mutants
`are those of the genera Brevibacterium resistant to 5-
`methyl-tryptophan (U.S. Pat. No. 3,700,539), Bacillus
`resistant to 5-fluorotryptophan (Japanese Published 20
`Unexamined Patent Application Number 20391/1974),
`and Enterobacter resistant to 5-methyl-tryptophan (Jap(cid:173)
`anese Published Unexamined Patent Application Num(cid:173)
`ber 57888/1976).
`The most efficient known microorganism to produce 25
`tryptophan
`is Corynebacterium glutamicum A TCC
`21851, which requires phenyl-alanine and tyrosine, and
`is resistant to 4-methyl-tryptophan, 6-fluoro-trypto(cid:173)
`phan, 4-amino-phenylalanine, 4-fluoro-phenylalanine,
`tyrosine-hydroxamate,
`and phenylalanine-hydroxa- 30
`mate. This strain produced 16.8 mglml 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 fulfill commercial
`requirements.
`The possibility of utilizing recently developed ge(cid:173)
`netic recombination techniques, to engineer a microor(cid:173)
`ganism capable of producing high levels of tryptophan
`is appealing. The general techniques for the introduc(cid:173)
`tion of genes, and the amplification in bacteria capable 40
`of expressing them have recently been described by
`Gilbert and Villa-Komaroffin Scientific 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 45
`circular DNA) in vitro, by means of a splicing/ligation
`sequence using endonuclease and ligase enzymes re(cid:173)
`spectively. The hybrid plasmid containing the gene or
`genes is then mixed with cells of a host organism, usu(cid:173)
`ally a prokaryotic bacterial microorganism. A dilute 50
`solution of calcium chloride renders the bacteria perme(cid:173)
`able and the cells will take up plasmids from solution.
`Reproduction of the plasmid-carrying host microorgan(cid:173)
`isms then produces millions of identical copies of the
`recombinant DNA. If the appropriate genetic control 55
`sequences are present, the amplified gene or genes will
`produce corresponding enzymes using the available
`protein-synthesizing apparatus of the host.
`Hershfield et a!, for example (Proceedings of the
`National Academy of Sciences, USA, 71: 3455-3459 60
`(1974)), have reported the insertion of a DNA fragment
`of E. coli possessing genetic information related to tryp(cid:173)
`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 65
`hybrid plasmid (Col El trp), the tryptophan auxotroph
`became a tryptophan prototroph. Elevated levels of
`tryptophan biosynthetic enzymes were reported. These
`
`35
`
`Anthrarc acid
`
`/Prephenic\
`
`N-(5' -phosphoribosyl-)·
`anthranili~acid
`
`p-Hydroxyphenyl
`
`pyruvJ acid
`
`Phenylpyruvic
`
`r
`
`Enol-1-(0-carboxyphenyl(cid:173)
`amino-)-
`1-deoxyribulr•-5-phosphate
`
`Tyrosine
`
`Phenylalanine
`
`lndole-3-glyJrolphosphate
`
`L·tryptophan
`~ V tryptophanase
`
`Breakdown producls
`(Indole)
`
`Tyrosine, phenylalanine and tryptophan are produced
`from a common biosynthetic intermediate, chorismic
`acid. Tyrosine and phenylalanine are furthermore de(cid:173)
`rived from another common intermediate, prephenic
`acid. The first 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 I.
`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(cid:173)
`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 deficient 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
`
`Sanofi/Regeneron Ex. 1 034, pg 957
`
`Merck Ex. 1034, pg 983
`
`

`
`3
`recovering the L-tryptophan produced from the culture
`medium.
`
`4,371,614
`
`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(cid:173)
`ings, wherein:
`FIG. 1 shows the plasmid pSerB59-l which is de(cid:173)
`rived from the pBR322 vector and the E. coli serB gene.
`The abbreviations denote restriction endonuclease en(cid:173)
`zymes from the following sources:
`
`10
`
`BB
`
`1111
`
` .. :
`
`Hpal:
`Sail·
`acba
`.:
`
`4
`mants and result in loss of the plasmids (see, e.g.,
`Herschfield et al, supra; Hallewell, 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(cid:173)
`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(cid:173)
`ferred embodiment, the plasmids additionally contain a
`15 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
`gaml I:Hl·.
`Bacillus globiggi
`Bacillus amyloliquefacie"s H
`plasmids or vectors available. These plasmids may be E.
`E. coli R YlJ
`EcoR I:
`coli plasmids or plasmids capable of replicating in E.
`Pro•idencia stuortii t 64
`Pst 1:
`20 coiL Among useful plasmids, the following can be listed:
`HBaint dEI
`Haemophi/us influenzae Rd
`Bacillus stearoJermop/Jilus ET
`ColE!, pSCIOI, pSF2124, pmB8, pMB9, ACYC184,
`Haemophilusparoinfluenzae
`pAcYCI77, pCK1, R6K, pBR312, pBR313, pBR317,
`StreptomycesalbisG
`pBR318, pBR320, pBR321, pBR322, pBR333, pBR341,
`Xanrhomonas badni
`Streptomyces adrromogenes
`pBR345, pBR350, pBR351, pML2, pML21, ColEIAp,
`Streptomyces stanfOTd
`25 RSFIOIO, pVH51, pVHI51, pVH!S3 (Recombinant
`Bacilluscoldolyticus
`Molecules: Impact on Science and Society: Beers, R. F.,
`Xanthomonas ho/icola
`E
`Klebsiella pneumoniae
`and Bassett,
`. G. eds., Raven Press, New York (1977)).
`Proteus •ulgaris
`Other plasmids are pBR327, pBR32S and pBRJlS (So(cid:173)
`heron, et al, Gene 9: 287-305 (1980)); still others are
`FIG. 2 shows the cloning strategy for MTR #2 trp 30 described in "DNA Insertion Elements, Plasmids and
`genes from cell DNA using the vector pBR322 as don-
`Episomes", Bukhari et a! (eds), Cold Spring Harbor
`ing vehicle. Abbreviations used:
`Sau Ja: 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: HfrH(gal-bio-attX)17
`deo=serB-trpR)17thi-
`strain of E. coli.
`SP338: trpED, tna2, thr- strain of E. coli.
`61-1: (deoB-serB)Vthi- (gal-bio-attX)17 strain of E.
`coli.
`5161: tyrA: tn!O insertion strain
`PI: bacteriophage Pike (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(cid:173)
`tive procedures. The preparation ofplasmids containing
`DNA from a donor, of the recipient strains, and of the
`transformants among recipients and donor DNA are as
`follows:
`
`Laboratory (1977). The preferred plasmids are the mul-
`ticopy plasmids of the type of pBR and its derivatives,
`35 and ColE! 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
`40 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
`45 loss of the trp gene-containing plasmid. At the time of
`Joss 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
`50 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(cid:173)
`ics, 176:361-368 (1979)). This plasmid called pSerB59-l
`60 is described in FIG. 1, including the specific endonucle(cid:173)
`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
`65 plasmid copies per cell in the final strain, it is preferred
`to prepare deletions of pSerB59-I. pSerB59-l DNA can
`be partially digested to various extents with endonucle(cid:173)
`ase Sau 3a, which cuts at the sequence
`
`1I
`
`X8
`
`Sst 1:
`Bell:
`Xho 1:
`Kpn I·
`Pvu ,j,
`
`55
`
`A. Plasmid Construction
`The plasmids used in this invention are either strin(cid:173)
`gent (single copy plasmids) or relaxed (multicopy plas(cid:173)
`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-
`
`Sanofi/Regeneron Ex. 1 034, pg 958
`
`Merck Ex. 1034, pg 984
`
`

`
`4,371,614
`
`5
`
`i ~· ... GATC . . 3'
`. CTAG ... 5' t
`
`J'
`
`and leaves the tetranucleotide GATC single stranded
`on the 5' end of each fragment. Since Sau 3a has a tetra(cid:173)
`meric recognition sequence, it cuts very frequently
`within pSerB59-I DNA. Partial digestion produces a
`somewhat random distribution of fragments representa(cid:173)
`tive of each segment of the plasmid. The digest can then
`be ligated with phage T 4ligase at low concentration to
`produce circles, or at higher concentrations with the
`endonuclease Bam HI digested pBR 322 DNA. Bam HI
`cuts at the hexanucleotide
`
`5' . . . GGATCC.
`
`~
`3' . . . CCTAGG t
`
`3'
`5'
`
`20
`
`25
`
`6
`mids, such as those described above: (a) wild type trp
`operon and (b) feedback resistant anthranilate synthe(cid:173)
`tase trp operon. The latter mutant trp gene is one
`wherein anthranilic acid synthetase is resistant to feed(cid:173)
`back inhibition by tryptophan. It can be found in high
`frequency in mutants resistant to tryptophan-antago(cid:173)
`nists. These antagonists inhibit the growth of Escher(cid:173)
`ichia strains, but the inhibition is suppressed partially or
`completely when tryptophan is in the medium. Exam-
`10 pies of tryptophan antagonists are: 4-fluoro-tryptophan,
`7-tluoro(cid:173)
`5-fluoro-tryptophan, 6-Huoro-tryptophan,
`tryptophan, 4-methyl-tryptophan,
`5-methyl-trypto(cid:173)
`phan,
`6-methyl-tryptophan,
`7-methyl-tryptophan,
`naphthyl-alanine, indole-acrylic acid, naphthyl-acrylic
`15 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(cid:173)
`uator region of the trp operon. (Oxender et a!, 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
`tRNA1'P, 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(cid:173)
`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(cid:173)
`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 exemplifies such a tech(cid:173)
`nique using MTR #2 (Somerville, R. and Yanofsky, C.,
`J. Mol. Bioi., II: 747 (1965)) as donor for cell DNA and
`pBR322 as the plasmid. A first ligation can preferably
`be carried out at high DNA concentration so that for(cid:173)
`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(cid:173)
`tant trp operon-containing plasmids can be done by
`transforming a (trpA-E)V strain (Zalkin, H., et al, J.
`Bioi. Chern., 249:465-475 (1974)) therewith and allow(cid:173)
`ing the cells to grow on minimal media. Resistance to
`5-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(cid:173)
`ously. The MTR#2 trp+ operon is only exemplary and
`any feedback resistant anthranilate synthetase express(cid:173)
`ing gene can be used.
`Recombination of mutant trp operon (e.g., MTR#2)
`containing plasmids with serE-containing plasmid
`yields trp+-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 serB+-containing plasmid DNA.
`The high molecular weight ligated DNA can be di(cid:173)
`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 serB+ /mutaot trp-containing plasmids will
`
`leaving the same tetranucleotide single stranded at the 30
`5' ends of fragments as Sau 3a. Therefore, Sau 3a di(cid:173)
`gested DNA can be readily ligated to Bam HI 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 35
`colonies which grow in the absence of serine. For exam(cid:173)
`ple, strain 37-1 E. coli W3110, HfrH, (gal-bio-att!.)"
`(deo-serB-trpR)9 , thi-, (Roeder and Somerville, Mo(cid:173)
`lecular and General Genetics 176: 361-368 (1979)) can
`be transformed for this purpose. Plasmids carrying the 40
`serB gene can then be isolated by screening the colonies
`(Eckhardt, T., Plasmid, 1: 584-588 (1978)). By such
`procedures, several plasmids can be iSCJ!ated which are
`smaller than pSerBS9-l having observable deletions in
`the range of 85 to 51% the size of pSerB59-I. An analy- 45
`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 50
`isolated. Both of these have no Eco Rl sites, 2 Bgl I
`sites, I Bst Ell site, I Bglll site, no Sail sites and I Pst
`I site. The restriction map of a plasmid derived from
`pBR322 carrying a serB+ gene is also shown in FIG. 1
`including the probable location of the serB+ gene in the 55
`plasmid. It can be ascertained that the enzymes Pst I and
`Bgl II c~n 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 60
`not located within the serB gene. This can be showo by
`cloning Sau 3a digested cell DNA into the Bgl II site of
`the plasmid 6 and demonstrating that the serB + gene
`function is retained. This plasmid is exemplary only. In
`a preferred embodiment however, this plasmid is uti- 65
`lized as the cloning vehicle.
`Two types of trp operon genes can be used alone in
`plasmids, or inserted into serB gene-containing plas-
`
`Sanofi/Regeneron Ex. 1 034, pg 959
`
`Merck Ex. 1034, pg 985
`
`

`
`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 trpR1' variant can be obtained from a trp-lac
`fusion E. coli strain, (Reznikoff et a! J. of Bact., 109:
`526-532 (I 972)), by transducing a PI 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 identified.
`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
`10 into trpR + 61-1 E. coli by PI transduction from E. coli
`KB 3100 (Brown, K. D., J. Bacterial. 106:71-81 (1971))
`and selection for resistance to /:l-2-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 ,6-2-thienyi-DL-alanine is suffi(cid:173)
`cient to allow selection of an aroP mutant. A phage PI
`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 /:l-2-thienyl(cid:173)
`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(cid:173)
`tion.
`The tryA and tryA-pheA mutations can be intro(cid:173)
`duced into the strains using a strain of E. coli (E. coli
`5161), which has a Tn!O
`transposable element
`(Kleckner, N. et al. Proc. Nat. Acad. Sci. USA 73:
`3838-3842 (1976)) inserted in the tyrA locus. Phage PI
`lysates of such E. coli 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 tel' colonies to grow. In this way, genes with tn I 0
`insertions are converted to non-revertable mutations.
`By screening the tet' 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(cid:173)
`ological Reviews: 44: 1-56 (1980)).
`
`C. Transformation of Host Strains
`Hosts are transformed with plasmid DNA (contain(cid:173)
`ing wild type trp-, wild type trp-ser, mutant trp, mutant
`trp-serB as well as plasmids with the attenuator dele(cid:173)
`tion) by calcium shock. This procedure makes the hosts
`50 competent for DNA uptake (see, e.g., Morrison, D. A.,
`J. Bact. 132: 349-35 I (1977)). Anthranilate synthetase
`specific activity determinations are made with cultures
`of plasmid-containing cells. Alternatively, tryptophan
`levels can be detected enzymatically or by microbioas(cid:173)
`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(cid:173)
`copy plasmids containing only trp genes have consider(cid:173)
`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 W3110.
`Tryptophan excretion experiments show that those
`strains which contain either the aroP mutations or the
`tyrA, tyrA pbeA mutations do excrete tryptophan.
`Without these mutations, very little tryptophan is made,
`
`B. Host Strain Constructions
`As the recipient microorganism for the hybrid DNA
`plasmids, tryptophanase-<leficient mutants of E. coli are
`used in this invention. Preferably trp operon-lacking 15
`strains (trp operon") and/or nonrevertable tryptopha(cid:173)
`nase-mutated (tna) strains are used. The lack of trypto(cid:173)
`phanase insures that tryptophan is not broken down and
`yields of excreted L-tryptophan remain high. A trypto(cid:173)
`phan auxotroph may also be used as the recipient, since 20
`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(cid:173)
`mid. Desirably, the host also has one or more of the
`following genetic deficiencies: (i) aroP gene: this gene 25
`controls the permeability characteristics of the cells to
`aromatic amino acids. In aroP mutant strains trypto(cid:173)
`phan excretion is proportional to anthranilate synthe(cid:173)
`tase activity; (ii) trpR gene (tryptophan repressor gene):
`this gene controls the repression of the tryptophan bio- 30
`synthetic pathway; in trpR mutant strains the pathway
`proceeds derepressed regardless of the levels of trypto(cid:173)
`phan produced; (iii) The trpR gene can also, in a pre(cid:173)
`i.e.,
`ferred embodiment, be temperature sensitive,
`trpRts. In such case, when the cells are maintained at 35
`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- 40
`tiona! trpR product synthesis: (iv) tyr A: this gene con(cid:173)
`trols the biosynthetic pathway for the formation of
`tyrosine; in tryA mutant strains the flow of aromatic
`precursor ( chorismic acid) can be diverted into the trp
`and the phenylalanine paths; (v) pheA: this gene con- 45
`trois the biosynthetic pathway for the formation of
`phenylalanine; in pheA mutant strains the flow of aro(cid:173)
`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 operon 17 strains, it is possible
`to use a trpED I 02 deletion strain. A threonine auxo- 55
`troph with the trpED deletion, E. Coli SP338 (trpEDI7
`I 02, tna2, thr-) (Roeder and Somerville, Molecular and
`general Genetics, 176: 36 I -368 (1979)), is transduced
`with phage PI lysates (Rosner, J. L., Virology 49:
`679-689 (I 972)) prepared on either E. Coli 61- I (Roeder 60
`and Somerville, Molecular and General Genetics, I 76:
`361-368 (1979)) [deob-serB), thi-, (gal-bio-att:>..)VJ or on
`E. Coli 37-1 (Roeder and Somerville, supra.) [HfrH,
`(gal-bio-attA.)" (deo-serB-trpR)I7, thi- ). Tryptophan
`auxotrophs are selected which no longer require threo- 65
`nine and have become serine auxotrophs. They can be
`prepared with the trpR- 37-1 cell line PI lysates and
`with trpR + 6 I- I cell line lysates.
`
`Sanofi/Regeneron Ex. 1 034, pg 960
`
`Merck Ex. 1034, pg 986
`
`

`
`9
`less than 10 1-'glml. High yields of tryptophan may be
`obtained by combining the aroP mutated hosts with a
`mutant trp-serB plasmid or a wild type trp-serB plas(cid:173)
`mid.
`The methods of culturing the L-tryptophan produc(cid:173)
`ing strains thus obtained are conventional, and are simi-
`lar to the methods for the cultivation of known L-tryp(cid:173)
`tophan producing microorganisms. Thus, the culture
`medium employed is one containing carbon sources,
`nitrogen sources, inorganic ions and, when required, 10
`minor organic nutrients such as vitamins or amino acids.
`Examples of suitable carbon sources include glucose,
`lactose, starch hydroysate and molasses. Gaseous am(cid:173)
`monia, aqueous ammonia and ammonium salts and
`other nitrogen containing materials can be used as the IS
`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- 20
`tryptophan ceases.
`The L-tryptophan accumulated in the culture me(cid:173)
`dium can be recovered by conventional procedures
`(Japanese Published Unexamined Patent Application
`No. 74293/1979).
`By the method of the present invention, L-trypto(cid:173)
`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
`specific examples which are provided herein for pur(cid:173)
`poses of illustration only and are not intended to be
`limiting unless otherwise specified.
`
`4,371,614
`
`10
`-continued
`E. coli trp·lac fusion
`
`NRRL B-4575
`
`MEDIA FORMULAS
`
`FUSZIN PLATES
`per liter
`agar 15g