`Anderson et a1.
`
`[19]
`
`[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. Herrmann; Ronald L.
`Somerville, both of West Lafayette,
`incl.
`
`[73] Assignee: Ajinomoto Co., Inc., Tokyo, Japan
`[21] Appl. No.: 180,296
`[22] Filed:
`Aug. 22. 1980
`[51]
`Int. Cl.’ ........................ .. C12N 1/00; C12R 1/I9;
`CIZN 9/00;C12P13/22;C12P 21/00; Cl2N
`15/00; C12N 1/20
`[52] U.S. C1. .................................. .. 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 12/1930 Cohen et al,
`OTHER PUBLICATIONS
`
`435/68
`
`Tribe and Pittard, Applied and Environmental Microbi-
`ology. v. 38, pp. 181-190 (Aug. 1979).
`Nagahari et 211., Gene 1: 141-152 (1977).
`Fredericq and Cornelis, Journal of General Microbiol-
`ogy 105: 357-349 (1978).
`Oxender et al. 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 211., Proceedings of the National Academy of
`Sciences USA vol. 73: 3838-3842 (1976).
`Enger-Valk et al. Gene 9: 69-85 (I980).
`Hershficld et a1., 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 (I979).
`Hallewell et al. Gene 9. 27-47 (1980).
`Somerville et al. Abstract of J. M01. Biol.
`Chem. Abstr. 63:2l52d.
`Watson, Molecular Biology ofthe Gene, W. A. Benjamin,
`Inc. (1977), pp. 398-400.
`Bertrand et a1. Abstract of J. Mol. Biol. lI7, 227-247
`(1977) in Chem. Abstr. 88:858l6k (1978).
`Primary Examiner—A1vin E. Tanenholtz
`Assistant Exam1'ner—-James Martinell
`Attorney, Agent, or Firm-Oblon, Fisher, Spivak,
`McClelland & Maier
`
`1 1, 747 (1965)
`
`ABSTRACT
`[57]
`A bacterium which comprises a host of the genus Esch-
`erichia deficient 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
`
`Mylan Ex. 1034, pg 952
`
`
`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 1 of4
`
`4,371,614
`
`
`
`2oE<S.I.uE
`
`.6EGEQ
`
`///1:353:a.Hwas823$:
`
`m22.9G<m.E..3%///
`
`mamammzsmm
`
`3%K38Emma.
`
`
`
`92%.:$239
`
`..%mam..
`
`Hmam
`
`::.am
`
`HGI
`
`.5012:
`
`8286
`
`E5
`
`:8Ham23Hax
`
`HE:HEx
`
`EsqHim
`
`
`
`
`
`1\l*:|I:\||»96¢2Sm2§EmfizmzummII\:lIlIIA1Tlullnl.
`
`
`
`Sam5%Esamansz:&
`
`mmqmSE.
`
`Sanofi/Regeneron Ex. 1034, pg 953
`
`Mylan 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 I’ Bum HI
`0
`
`vecron
`pan 322
`
`lBam HI
`
`5'” 31
`
`Bam HI
`
`
`4
`
`Trp opsnon
`BarnHI
`
`CELL DNA
`
`
`Eco nz
`
`Sau.301
`
`
`
`T4 LIGASE CELL AND VECTOR
`FRAGMEN73 AT EWIMOLAR
`AND HIGH CONCENTRATION
`
`
`
`Eco RI
`
`1
`
`Eco RI
`
`Eco RI
`
`l
`
`pm? 322
`
`Trp OPERON
`
`pm .322
`
`T4LIGASE
`FRAGMENTS AT
`LOW CONCENTRATION
`
`gm 3;
`
`Saula/Bam HI& V
`
`B“”'HI/5°” 3”
`
`F 2
`
`Trp OPERON
`
`V=p8R 322 VECTOR
`
`Sanofi/Regeneron Ex. 1034, pg 954
`
`Mylan Ex. 1034, pg 954
`
`
`
`
`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 3 of4
`
`4,371,614
`
`comsmucnom or Pmsmlos
`
`p.S‘er 359-:
`
`Mrf?#2 om
`
`pBFl' 322 am
`
`[Sou 3a
`1 T4Ligase
`pGxI—pGx!.3
`l/Jnalysis
`
`(Ser 3)
`
`
`
`ism R1
`4 T4Ligase
`
`{MTR#2Lr2)
`
`p00 3
`
`Analysis and
`Selection
`
`LPSII
`
`
`
`
`T4 Ligase
`
`
`
`{gfi-wildfypejfgl
`
`
`
`T L‘ ase
`T4 Ligase
`4 '9
`
`
`l Ps!I
`T4 Lfgase
`
`(sera-MrR#2:rp)
`
`(;eL§-MTR#Z1;g)
`
`FIG. 3
`
`Sanofi/Regeneron Ex. 1034, pg 955
`
`Mylan Ex. 1034, pg 955
`
`
`
`U.S. Patent
`
`Feb. 1, 1983
`
`Sheet 4 of4
`
`4,371,614
`
`CONSYRUCTION OF HOST STRAINS
`
`E. Cofi 37-!
`
`E. Coli SP338
`
`E.Coh' 61-!
`
`P1 kc
`
`Select
`fhr*frp‘ser '
`
`AGXI
`
`E. Cali
`5I6I
`
`P1
`
`P1
`
`Selec!
`Ihr‘ rrp'se*'
`¢
`
`AGK
`
`6
`
`AGX6
`
`pGX35 DNA
`
`Select
`fetRIyr'
`/ \
`Spontaneous
`
`Select
`ter"ryr‘
`/ \
`Spontaneous
`
`[AG"5{”G"55’]
`KB 3100p!
`
`,
`
`//
`SeIec1_
`fer‘ Iyr
`
`\\
`.S‘eIec_f
`retsryr phe
`
`_
`
`/
`Select _
`fefstyr A
`
`\\
`Select
`fef5!yr"phe'
`
`Seleqr
`fl '3' ""9"?! '
`lags-,.;‘f’ggg"9
`
`F I 5. ‘I
`
`Sanofi/Regeneron Ex. 1034, pg 956
`
`Mylan Ex. 1034, pg 956
`
`
`
`1
`
`4,371,614
`
`E. COLI BACTERIA CARRYING RECOMBINANT
`PLASMIDS AND THEIR USE IN THE
`FERMENTATIVE PRODUCTION OF
`L-TRYPTOPI-IAN
`
`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 amplification of DNA.
`The biosynthetic pathways for the synthesis of aro-
`matic amino acids (tryptophan, tyrosine, phenylalanine)
`in bacteria, are shown in Chart 1:
`
`Cl-[ART 1:
`Su
`
`rs
`at least
`15 steps
`
`Amhmnmc mid
`synthetase
`
`chorismic acid
`
`P
`
`Anthranilic acid
`
`l
`
`Prephenic Acid
`
`N..(5',phospho,.ibmy1_)_
`
`p-Hydroxyphenyl
`
`Phenylpyruvic
`
`anthranilic acid
`
`Enol-1-(0-carboxyphenyh
`amino-)-
`1-deoxyribulose~5-phosphate
`
`Pvflwic “id
`
`ii/id
`
`Tyrone
`
`Phenylalanine
`
`10
`
`I5
`
`20
`
`25
`
`BACKGROUND OF‘ THE INVENTION
`1. Field of the Invention
`The present invention relates to E. calf microorgan-
`isms carrying recombinant plasmids constructed in
`vitro and their use for producing L-tryptophan by fer-
`mentalion.
`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 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—tluorotryptophan (Japanese Published
`Unexamined Patent Application Number 2039l/ 1974),
`and Enterobacter resistant to 5—methyl-tryptophan (Jap-
`anese Published Unexamined Patent Application Num-
`ber 57888/1976).
`The most efficient known microorganism to produce
`tryptophan is Corynebacterium glutamicum ATCC
`21851, which requires phenyl-alanine and tyrosine, and
`is
`resistant
`to 4-methyl-tryptophan, 6-fluoro-trypto-
`phan, 4-amino-phenylalanine, 4-fluoro-phenylalanine,
`tyrosine-hydroxamate,
`and
`phenylalanine-hydroxm
`mate. This strain produced 168 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 fulfill 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 amplification in bacteria capable
`of expressing them have recently been described by
`Gilbert and Villa-Komaroff in Scientific American, 242:
`74-94 (1980). Briefly, one or more genes from a donor
`organism, such as a proltaryotic 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 amplified gene or genes will
`produce corresponding enzymes using the available
`protein-synthesizing apparatus of the host.
`Hershfield 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
`
`30
`
`indole-Lglycerolphosphate
`
`35
`
`L-tryptophan
`|/ tryptophanas-c
`
`Breakdown products
`(indole)
`
`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 first major enzyme of the tryptophan pathway
`is anthranilate synthetase which, in E. cali 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 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
`
`45
`
`50
`
`55
`
`65
`
`Sanofi/Regeneron Ex. 1034, pg 957
`
`Mylan Ex. 1034, pg 957
`
`
`
`3
`recovering the L-tryptophan produced from the culture
`medium.
`
`4,371,614
`
`4
`
`in loss of the plasmids (see, e.g.,
`mants and result
`Herschfield et al, 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‘3'), 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.
`
`5
`
`10
`
`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 pSerB59-1 which is de-
`rived from the pBR322 vector and the E. cali serB gene.
`The abbreviations denote restriction endonuclease en-
`zymes from the following sources:
` —_:
`Bgl I:
`Bacillus globiggi
`Barn HI:
`Bacillus amyloliquefuciens H
`Ecol! 1:
`E. coli RY13
`P5! 1:
`Pruvideneiu stuarlir‘ [64
`Hind III:
`Haemophilus influenzae Rd
`Bat E II:
`Bacillus stearotermaphilus ET
`lips I:
`Haemopllilus parainfluenzae
`Sa] 1:
`Slrepromyces albis G
`Xba I:
`Xtzm‘/iomona: badrii
`Sac I:
`Streptamyces achromogenes
`5st I:
`Streplamycas stanfard
`Bcl I:
`Bacillus caldalyticu:
`Xho I:
`Xaniho.-none: halicala
`Kpn I:
`Klebsiella pneumoniae
`Pvu II:
`Proteus vulgari:
`
`15
`
`20
`
`25
`
`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, pSCl0l, pSF2l24, pmB8, pMB9, ACYCIS4,
`pAcYC177, pCKl, R6K, pBR3l2, pBR3l3, pBR3l7,
`pBR3l8, pBR320, pBR32l, pBR322, pBR333, pBR34l,
`pBR345, pBR350, pBR35l, pML2, pML2l, ColElAp,
`RSFIOIO, pVH51, pVHl5l, p\/H153 (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 pSerB59-1
`is described in FIG. 1, including the specific 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 final 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
`
`FIG. 2 shows the cloning strategy for MTR #2 trp 30
`genes from cell DNA using the vector pBR322 as clon-
`ing vehicle. Abbreviations used:
`Sau 3a: endonuclease derived from Staphylococcus
`aureus 321
`
`Amp: ampicillin resistance gene
`FIG. 3 is a flow chart showing the construction of
`composite plasmids carrying both a serB gene and a
`MT” #2 trpE gene. Abbreviations used:
`MTR #2 trp: Methyl tryptophan resistance E coli
`feedback resistant anthranilate synlhetase.
`pGx: plasmid numbering system
`FIG. 4 is a flow chart showing the construction of
`host strains. Abbreviations and numbering used:
`37-1: HfrH(gaI—bio-attA)V deo=serB-trpR)Vthi—
`strain of E. coli.
`SP338: trpED, tna2, thrr strain of E. cali.
`61-1: (deoB——serB)Vthi— (gal-bio-att)t)V strain of E.
`coli.
`5161: tyrA: tnl0 insertion strain
`Pl: bacteriophage Plkc (transducing phage)
`tetR: tetracycline resistance
`AGx: strain numbering system.
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`35
`
`40
`
`45
`
`50
`
`55
`
`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
`transforrnants among recipients and donor DNA are as 60
`follows:
`
`A. Plasmid Construction
`
`The plasmids used in this invention are either strin-
`gent (single copy plasmids) or relaxed (multicopy p1as- 65
`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. 1034, pg 958
`
`Mylan Ex. 1034, pg 958
`
`
`
`5'...GA'l"C...3'
`3’...CTAG...5'
`
`T
`
`and leaves the tetranucleotide GATC single stranded
`on the 5‘ end of each fragment. Since Sau 3a has a tet.ra-
`meric recognition sequence,
`it cuts very frequently
`within pSerB59-1 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 Taligase 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
`
`15'...GGATCC...3’
`
`3’ .
`
`.
`
`. CCTAGG .
`
`5’
`
`T
`
`leaving the same tetranucleotide single stranded at the
`5' ends of fragments as Sau 3a. Therefore, Sau 3a di-
`gested DNA can be readily ligated to Barn 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
`colonies which grow in the absence of serine. For exam-
`ple, strain 37-1 E. calf W3ll0, HfrH, (gal-bio-attA)V
`(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 pSerB59-1 having observable deletions in
`the range of 85 to 51% the size of pSerB59-1. 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
`pSerB59-1. 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 Bst EII site,
`1 Bgl 11 site, no Sal 1 sites and 1 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
`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 11 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 11 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-
`lized as the cloning vehicle.
`Two types of trp operon genes can be used alone in
`plasmids, or inserted into serB gene-containing plas-
`
`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-fluoro-tryptophan,
`5-fluoro-tryptophan,
`6-fluoro-tryptophan,
`7-t'luoro-
`tryptophan,
`4-methyl-tryptophan,
`5-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
`tRNA"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-
`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 exemplifies such a tech-
`nique using MTR #2 (Somerville, R. and Yanofsky, C.,
`J. M01. Biol., 11: 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-
`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 trp+ 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 +-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-
`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+/mutant trp-containing plasmids will
`
`I0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Sanofi/Regeneron Ex. 1034, pg 959
`
`Mylan 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.
`B. Host Strain Constructions
`
`8
`The trpR" variant can be obtained from a trp-lac
`fusion E. coil‘ strain, (Reznikofi et al J. of Bact., 109:
`526-532 (1972)), by transducing a P1 lysate thereof into
`any of the wild E. call’ or mutant E coli strains of this
`invention. In trp-lac fusion strains, trpR“ mutations can
`be readily identified.
`The a.roP 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 Pl transduction from E. coli
`K13 3100 (Brown, K. D., J. Bacteriol. 106: 71-81 (1971))
`and selection for resistance to B-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 B-2-thienyl—DL-alanine is suffi-
`cient to allow selection of an aroP mutant. A phage Pl
`lysate of E. colt‘ 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.
`
`10
`
`15
`
`20
`
`As the recipient microorganism for the hybrid DNA
`plasmids, tryptophanase-deficient mutants of E. colt’ 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 two? 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 flow of aromatic
`precursor (chorismic acid) can be diverted into the trp
`and the phenylalanine paths; (V) pheA: this gene con-
`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 SP33B (trpEDV
`102, tnal, 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 (1979)) [deob-serB), thi‘-, (gal-bio-attA)V] or on
`E. Coli 37-1 (Roeder and Somerville, supra.) [HfrH,
`(gal-bio-att7i)V (deo-serB-trpR)V,
`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+ 61-1 cell line lysates.
`
`25
`
`30
`
`35
`
`4-0
`
`45
`
`50
`
`55
`
`65
`
`The tryA and tryA-pheA mutations can be intro-
`duced into the strains using a strain of E. Cali (E. call‘
`5161), which has
`a Tn10 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. 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 tets colonies to grow. In this way, genes with tnl0
`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: 1-56 (1980)).
`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 arol’ mutations or the
`tyrA,
`tyrA pheA mutations do excrete tryptophan.
`Without these mutations, very little tryptophan is made,
`
`Sanofi/Regeneron Ex. 1034, pg 960
`
`Mylan Ex. 1034, pg 960
`
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`4,371,614
`
`10
`~continued
`E coli trp-lac fusion
`NRRL B-4575
`MEDIA FORMULAS
`LB
`Er liter
`
`
`
`9
`less than l0 pg/ml. High yields of tryptophan may be
`obtained by combining the arol’ 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
`specific examples which are provided herein for pur-
`poses of illustration only and are not intended to be
`limiting unless otherwise specified.
`
`PCC3
`
`A. Experimental Methods
`Table 1 shows strains and plasmids used for the fol-
`lowing experiments:
`TABLE I
`
`Origin of Strains and Plasmids
`Reference
`
`Description
`E. colt‘ Barn HI DNA
`(3)
`122-l(pSerBS9-I)
`fragment cloned in
`Barn HI site of pBR322
`trp operon derived from
`da80ptA-E cloned in Col El
`along with gal operon genes
`Mutant with feedback resis-
`(c)
`MT“ #2
`tant anthranilate synthetase
`37-1
`(I)
`(serB-trpR)V strain
`61-1
`(a)
`serBV strain
`5161
`(d)
`tyrA: tnl0 insertion strain
`(a)
`SP338
`trpEDV, tna2, thr'
`arol’ strain
`
`K3310!)
`(e)
`(a) Roeder, W. and Somervxlle. R. Molecular & General Genetics, 176: 361-368
`(I979)
`(b) Collins et :1. Proc. Nat. Acnd. Sci. 73: 383E—J84Z (I976)
`(C) Pabst el al. J. Biol. Chem., 248- 901-914 (I973)
`dd) Kleckner. N.. et al, J. Mol. Biol. l27: 89-113 (1976); also see reference (b)
`((3) Brown. K.D., J. Bact. I12: l77—lIiil (1970)
`
`(bl
`
`The following strains were deposited at the NRRL,
`Peoria, Ill., USA on Aug. 14, 1980:
`
`E coli 37-1
`NRRL B4568
`E. cull’ SP 338
`NRRL B-4569
`E. cali fil-1
`NRRL B-4570
`E. 5l6l
`NRRL B-4-57l
`E, coli MT® #2
`NRRL B-4572
`E cali AEl
`NRRL B-4573
`E. cali KBJIOU
`NRRL B-4574
`
`l0
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`45
`
`S0
`
`55
`
`60
`
`65
`
`tryptone mg
`yeast extract 5g
`sodium chloride log
`
`LB plates = LB + 1% agar
`LB Mg = LB + l].0lM MgCl1
`LB Ca = LB + 0.005M CaClz
`LB Topagar = LB + 0.75% agar
`Saline = 0.85% NaCl
`
`Supplements to Minimal Plates
`amino acids 20 pg/ml
`tetracycline 25 pg/ml
`B-2—thienyl-DL-alanine 22 pg/ml
`Difco ® casamino acids 0.6%
`
`biotin l pg/ml
`
`thiamirn 1 pg/ml
`
`FUSZIN PLATES
`Er liter
`agar l5g
`Difco ® tryptone 10g
`Difeo ® yeast extract Sg
`sodium chloride lOg
`glucose 2g
`chlorotetracycline 50mg
`NaH2POq.H2O
`autoclave, cool. then add
`6ml 2mg/ml fusaric acid
`Sml Zinc chloride 20mM
`Minimal plates
`per liter
`KzHP04 l0..'>g
`KI-I2PO4 4.5g
`(NH4)2S04 1-03
`Sodium cixrate.2H2