EP0232262A1 - Tryptophan producing microorganism - Google Patents

Abstract

Escherichia coli microorganisms carrying genetic information carried by the plasmids and relating to the production of L-tryptophan, and methods for producing or increasing the production of L-tryptophan by fermentation.

Description

TRYPTOPHAN PRODUCING MICROORGANISM

Cross reference to Related Applications This application is a continuation-in-part of application Serial No. 642,303, filed August 20, 1984.

Background of the Invention The present invention relates to Escherichia coli (E. coli) microorganisns carrying plasmid borne genetic information for the prcduction of L-tryptophan and methods for prcducing and increasing the fermentative production of L-tryptophan.

Brief Description of Relevant Art Tryptophan is an amino acid that is an essential component in animal nutrition. Its fermentative production by microorganisns from inexpensive carbohydrate substrates is highly desirable.

Fermentative production of tryptophan, using microorganism strains artificially mutated and selected for increased tryptophan production is known. Microorganisms used to this end include Brevibacterium (U.S. Pat. 3,700,539), Enterobacter (U.S. Pat. 4,439,627), Bacillus, particularly Bacillus subtilis [using anthranilate and carbon, nitrogen and mineral sources (U.S. Pat. 4,363,875; Japanese Patent Application J58017190, 1,3-5A, and European Patent Application EP 81-107)] and Corynebacterium glutamicum ATCC 21851.

The application of recombinant DNA methods for the construction of E. coli strains for tryptophan production, has been described in a limited way in U.S. Pat. 4,371,614. The E. coli strains described therein carry a specific plasmid having thereon genes of the wild type tryptophan operon ( trpA-E ) with a wild type or temperature sensitive tryptophan repressor gene (trpR or trpRts, respectively) for control of tryptophan biosynthesis. Such plasmid or multicopy plasmid according to U.S. Patent 4,371,614 may also carry a Serine B gene (SerB). This is particularly desirable when the host strain is a tryptophan or L-serine auxotroph (i.e., Trp-, Ser-). Maximum yields disclosed are about 230 ppm of tryptophan. This same patent also decribes a number of general methods known to those skilled in the art for carrying out alterations to chromosomal DNA and plaanid DNA, the transformation of microorganism with plasmid DNA, and the selection of microorganian so altered or transformed. U.S. Patent 4,371,614 is hereby incorporated by reference for the purpose of illustrating the general state of the known methods in the field to which the present invention pertains.

A need for inexpensively produced tryptophan and methods for the prcduction of tryptophan as well as methods for increasing yields of tryptophan produced by microorganisms persists.

Objects of the Invention The present invention is intended to meet the persistent need for inexpensively produced tryptophan by providing a bacterial host microorganism, of the genus Escherichia, deficient in the enzyme tryptophanase carrying plaanid borne information for the production of tryptophan wherein said information is divided between at least two plaanids.

Another object of the invention is to provide a method for producing tryptophan by culturing the above-mentioned bacterial host and plaanids therein in a culture medium and recovering L-tryptophan from the fermented culture medium.

Still another object of the invention is to provide inexpensively produced tryptophan by providing a bacterial host microorganian of the genus Escherichia deficient in the enzyme tryptophanase carrying plaanid borne genetic information for the prcduction of tryptophan wherein a portion of said information for the production of tryptophan is under lac operator/promoter control.

Yet another object of the invention is to provide a method for producing tryptophan by culturing the above-mentioned bacterial host with plaanid borne information for the prcduction of tryptophan, a portion of said information being under lac operator/promoter control in a culture medium and recovering tryptophan from the fermented culture median. A still further object of the invention is to increase the production of tryptophan by a bacterial host of the genus Escherichia deficient in the enzyme tryptophanase by transforming said host with plasmid borne information to control tryptophan production divided between at least two plaanids; or by transforming the bacterial host as described with plaanid borne information to control tryptophan production a portion of said information being under lac promoter/operator control, or by transforming said host as described with plasmid borne information to control tryptophan prcduction divided between at least two plaanids, a portion of said information to control tryptophan prcduction being under lac operator/promoter control.

Summary of the Invention The plaanids used in this invention may be E. coli plaanids or plaanids capable of replicating in E. coli. If the information to control tryptophan prcduction is distributed on more than one plasmid, it is desirable to use plaanids that are replication compatible. Thus, for example, when the information to control tryptophan prcduction is distributed on two plaanids, plaanids having a ColE1 origin of replication may be used to carry part of the information and plaanids having a p15A origin of replication may be used to carry another part of the information.

Useful in the plasmid constructions of the type described in the invention are the following: ColEl, pSC101, pSF2124, pMB3, pMB9, pACYC184, pACYC177, pCK1, R6X, pBR312, pBR313, pBR317, OBR318, pBR320, PSR321, pBR322, pBR333, pBR341, pBR345, pBR350, pBR351, pML2, pML21, ColElaD, RSF1010, pVH51, pVh151, pVH153 (Recombinant Molecules: Impact on Science and Society: Beers, R.F., and Bassett, E.G., eds. Raven Press, New York (1977)). Other plaanids are pBR327, pBR325 and pBR328 (Soberon, et al., Gene, 9:287-305 (1980). Plasmid p15A is described in Cha ng A.C.Y and Cohen, S., J. Bacteriol., 135: 1148-1156 (1978). pHH509 is described in Barth et al., J. Bacteriol., 135:760-765 (1978), and pLG339 is described in Stoker et al., Gene 18:335-341 (1982). Still others are described in "DMA Insertion Elenents, Plaanids and Episomes," Bukhari et al. (eds.) Cold Spring Harbor Laboratory (1977). The preferred plasmids are the multicopy plaanids of the type of pBR and its derivatives, ColEl and its derivatives, p15A and its derivatives, pHH509 and its derivatives and pSC101 and its derivatives.

To ensure equal distribution of the plaanids in daughter cells during growth of the microorganism, it may furthermore be desirable for at least one of the plaanids to contain a par locus (Meacock, P.A. and Cohen, S.N., Cell, 20:529-542 (1980)). If the information to control tryptophan production is distributed anong more than two plaanids it is preferred that all plaanids be replication compatible with each other, by constructing them such that they each carry different origins of replication.

The plaanids used in the invention are single copy (stringent) or multicopy (relaxed) plasmids. Because tryptophan yields can be increased by increasing the copy nunber of plaanids carrying information multicopy plaanids are preferred. Although multicopy plaanids can be frequently lost from the host cell, i.e., are unstable, plasmid copy nunber may be maintained by constructing the plasmids such that determinants of antibiotic resistance are carried thereon. By adding antibiotics to the culture medium, microorganisms that lose plasmids are eliminated or fall to insignificant nunbers, depending on the mode of antibiotic action.

Plasmids used in this invention bear genetic information to control tryptophan production. The term "information to control tryptophan production" according to the invention includes:

1. information controlling the biosynthesis of the common precursors of the aromatic anino acids: tryptophan, tyrosine and phenylalanine; 2. information for the biosynthesis of tryptophan from chorianic acid via several intermediate steps;

3. information controlling the oiosynthesis of serine which is required in the final step of the tryptophan biosynthetic pathway; and 4. controlling elements for the information of 1 and 2.

The controlling elements used in the invention are nucleotide sequences derived from the lactose operon and include the lac operon promoter/ operator and lac repressor sequences. According to the invention, various portions of 1 and 2 are under lac control and produce significant amounts of functional gene products only in the presence of lactose. According to the invention it is preferred that tandem lac operator/promoters are used to achieve greater levels of gene expression than are possible with a single lac promoter.

The genetic information to control tryptophan prcduction according to the invention comprises a large nαnber of DNA sequences ceding for genes and regulatory sequences thereof, which are plaanid borne. The terms "gene" or "genes" as used herein are meant to encompass a DNA sequence ceding for an active product of the gene. Thus, DNA sequences ceding for an active gene product are genes within this definition even though the particular DNA sequence may not include the complete structural gene for the active product of the gene.

In placing this genetic information to control tryptophan production on plaanids it is convenient to divide the genetic information between at least two plasmids. By dividing the genetic information to control tryptophan prcduction between at least two plasmids, the elenents can be positioned relative to information they control and their gene products can be produced in sufficient concentration to insure that the information controlling biosynthesis of the common aromatic amino acid precursors, information controlling biosynthesis of tryptophan from chorismic acid and/or information controlling biosynthesis of serine are expressed under optimum conditions and at the desired time in a fermentation cycle. Thus, in a preferred embodiment the plaanid-borne information to control tryptophan prcduction is divided between at least two plasmids.

Brief Description of the Drawing The invention and its advantages over the known prior art can be best appreciated by those skilled in the art from the following detailed description of the invention considered along with the accompanying drawings.

Figure 1 and Figure 2 are schematic drawings of the preferred embediments of the plaanids carrying information to control tryptophan production according to the invention. Fragment Numbers 1-11 of Table 1 read in conjunction with Figure 1 identifying each restriction endonuclease segment of each plaanid. Similarly, Fragment Numbers 12-14 identify the restriction endonuclease segments of the plaanid within Figure 2. Table 1 further identifies each segment by approximate size in kilobases (kb), restriction nuclease ends of each fragment, genes/loci on the fragment, sequence coordinates if known, and journal reference for each known sequence coordinate. The depicted segment sizes in Figure 1 are only approximately proportional to the size of the complete plaanid as shown.

In the drawings, table and in the detailed description of the invention which follows the abbreviations indicated below are used and denote restriction endonuclease enzymes from the following organisms:

Restriction Endonculease Source organism

EcoRI Escherichia coli RY13

Eglll Bacillus globigii

SaIl Streptcmyces albis G

PstI Providencia stuartii 164

Sau3Al Staphylococcus aureus 3A(1)

SphI Streptcmyces phaechrcmogenes

Narl Nocardia argentinensis

Clal Caryophanon 1aturn

Aval Anabaena variabilis

BamHI Bacillus amyloliguifaciens H

Xhol Xanthcmonas holicola

Ddel Desulfovibrio desulfuricans

Nrul Nocardia rubra

Xmnl Xanthomonas manihotis

MstI Microcoleus sp.

Ncol Nocardia coralina

Hindlll Haemophilus influenzae Rd

Ahalll Apanatheca holophytica

SstI Streptomyces Stanford

Experimental Methods In the construction of the host strains and plasmids according to the invention, a number of methods known to those skilled in the art are employed. These methods are substantially described in numerous journals and those cited herein are well known examples of such method descriptions.

Transduction using P1 phage lysates is described in Rosner, J.L., Virology, 49:679-689 (1972). The use of Tn10 transposable elements for insertion into and deletion of chromosomal regions is described in Kleckner, N. et al., Proc. Nat. Acad. Sci., USA 73:3838-3842 (1976). Transformation of host strains with the plaanids according to the invention is carried out by calcium shock of the host strain rendering the host capable of incorporating DNA as described, for example, in Morrison, D.A. et al., J. Bacteriol., 132:342-351 (1977). Alternatively, E. coli host strains may be transformed by growing them to an optical density of about 0.6 at 600 nanometers in liquid median, chilling the cell suspension on ice for approximately 5 minutes, harvesting the cells by centrifugation, washing the cells in chilled 10 mM MgCl2, harvesting the cells, resuspending in chilled 50 mM CaCl₂ for about 30 minutes and adding plaanid DNA in suspension and maintaining the mixture at 0°C for about 45 minutes; the cells are resuspended in growth median for about one hour at 37 °C. Transformed host cells are selected or screened on appropriate medium.

The λ 1059 Sau3A E. coli genomic DNA clone bank was constructed by ligating Sau3A partial digests of E. coli genomic DNA to BamHI-cleaved λ1059 arms, thus generating a bank of recombinant phage containing randan pieces of the entire E. coli genome. (Karn et al., Proc. Nat'l. Acad. Sci., USA vol. 77 9:5172-5176 (1980).) The general methods for generating such a λ 1059 Sau3A E. coli genomic DNA clone bank are well known to those skilled in the art. Clone banks using other vectors and restriction endonuclease digests are also known.

Plaanids are generally isolated from host cells by the Birnboim cesium chloride gradient method. (Birnboim, H.C., Doly, J., Nucleic Acids Research, 7:1513-1523 (1979).) After restriction endonuclease digestion, plaanid fractions are generally fractionated by electrophoresis on an agarose or acrylamide gel, with appropriate molecular weight standards, using a buffer of 4 mM Tris, 1 mM EDTA adjusted to pH 8.2. Fragments are subsequently washed in ethanol and resuspended in Tris EDTA buffer at pH 8.0.

Ligation of restriction endonuclease-cut DNA fragments is generally carried out with T₄ ligase in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl₂, 20 mM dithiothreitol, 1 mM ATP and 50 ug/ml bovine serum albunin. T₄ ligase may be used to catalyze the formation of phosphcdiester bonds between juxtaposed 5' phosphate and 3'-hydroxyl termini in double-stranded DNA. Both blunt and cohesive end restriction fragments may be ligated using T₄ ligase.

Unpaired terminal bases of restriction endonuclease cut DNA fragments are made blunt-ended with DNA polymerase I (Poll) in the presence of deoxyribonucleotides (dNTP's). In general, approximately 1 micro¬gram (ug) of the purified DNA fragment having unpaired terminal bases is incubated with 2 units of Klenow DNA Polymerase I (Jacobsen et al., Eur. J. Biochem., 45:623-627 (1974)), 10 mM β-mercaptoethanol, 6 mM Tris-HCl (pH 7.5), 8 mM magnesiαn chloride and 0.2 mM each of the following dNTP's: deoxyadenosine triphosphate (dA-CP), deoxycytidine triphosphate (dCTP), thymidine triphosphate (TTP) and deoxyguanosine triphosphate (dGTP). The reaction is run at 16ºC for 60 minutes and is terminated by heat inactivation at 65ºC for 10 minutes followed by rapid cooling on ice.

A number of strains used in making the host strain according to the invention were obtained from the E .coli Genetic Stock Center (CGSC) Department of Human Genetics, Yale University School of Medicine, P.O. Box 3333, Cedar Street, New Haven, Connecticut 06510, USA. Strains obtained from the CGSC are designated herein by a researcher's name "via B. Bachnann", curator for CGSC.

Media for antibiotic selection or screening of plasmid containing microorganians is formulated to sustain growth of the microorganian of interest theredn, but contains, unless otherwise indicated, 20 mg/l of the antibiotic of interest. Media used in the construction of the host strains and plasmid described hereinbelow are as follows: LB per liter tryptone 10 grans (g) yeast extract 5 g sodium chloride 5 g glucose 2 g

LB plates = LB + 1.5% agar LB Mg = LB + 0.01M MgCI₂ LB Ca = LB + 0.005M CaCl₂ LB Top Agar = LB + 0.75% agar Saline = 0.85% NaCl

Fusaric Acid Plates agar 15 g

Difcc^® tryptone 10 g

Difcc^® yeast extract 5 g scdiun chloride 10 g glucose 2 g chlortetracycline 50 milligrams (mg)

NaH₂PO₄H₂O 10 g

autoclave, cool, then add 6 ml 2 mg/ml fusaric acid 5 ml zinc chloride 20 mM

Minimal Media per liter citric acid 2 g 80% H₃PO₄4.9 g KC14 g

30% NH₃ solution 5.86 g FeSO₄.7H₂O 1 mg MnSO₄ 1 mg MgSO₄.7H₂O 1 g thiamine-HCl 3.5 mg Supplements to Minimal Plates amino acids 20 micrcmoles/milliliter (ug/ml) tetracycline 10 ug/ml

-2-thienyl-DL-alanine 20 ug/ml Difcc^® casamino acids 0.4% 3-fluoro tryptophan (20 ug/ml) 7-methyl tryptophan (20 ug/ml) biotin 1 ug/ml thiamine 1 ug/ml triphenyltetrazolium chloride (20 ug/ml)

Vogel-Bonner Minimal Medium (per liter)

MgSO₄.7H₂O 0.2 g citric acid 2 g K₂H PO₄ 10 g NaNH₄PO₄.4H₂O 3.5 g

Make 0.4% glucose after autoclaving. Plates ( solid medium) are made by adding agar ( 1 .5%) .

CONSTRUCTION OF SPECIFIC PLASMIDS Construction of Plaanid pD2643 Plasmid pD2643 comprising trpA-E genes of which trpA-D are under lac promoter control, lacking a trp attenuator region, carrying a tetracycline resistance gene ( tet^r) and having a ColEl-type origin of replication was constructed as follows.

Tandem lac promoters were removed from pKB252 ( lac PUV5) (K.

Backman et al. , Proc. Nat' 1. Acad. Sci. USA, 73: 4124-4178 (1976 ) ) by partial digestion with EcoRI and complete digestion with SaIl restriction endonucleases under buffer conditions suggested by the manufacturer, and were ligated using T₄ ligase into plasmid pBR327 which had been previously digested to completion by SaIl and EcoRI. After trans forming E. coli , tetracycline resistant (Tet^r) ampicillin resistant (Amp^r) colonies were isolated. The resident plaanid in one of these was designated pEMl 26.

pCM126 was partially digested with Bglll and completely digested with SaIl restriction endonucleases. A 5.8 kilobase ( kb) Bglll-Sall fragment derived from pCB107 (Mascarenhas et al. , Virology, 24: 100-108 (1 983 ) ) carrying the trpD, C, B, A genes and lacking most of trpE gene as well as the attenuator and promoter regions of the trp operon (corresponding to nucleotides 1190-7015 identified by Yanofsky et al., Nucl. Acids Fes., 9:6647-6668 (1981)) was ligated using T₄ ligase into the Bglll, Salldigested pCM126 forming a mixture of ligated DNA. Plaanid pEMl36, having trpA-D under lac control was identified by transforming strain AE2

(Eenedik et al., Virology, 126:653-668 (1983)), which lacks trpA-E, with the ligated DNA and selecting transformed microorganisms on minimal medium containing ampicillin and anthranilate.

A Sall-EcoRI fragment containing a gene for anthranilate synthetase resistant to feed-back inhibition by tryptophan was obtained as follows. An EcoRI digest of Serratia marcescens gencmic DNA was prepared, and frαn an agarose gel a 4-9 kb fraction thereof was isolated. Said 4-9 kb fraction was ligated into EcoRI-digested pACYC184 under conditions favoring formation of hybrid molecules, and the resultant plaanid was used to transform E. coli strain M717 (D. Helsinki) deleted for trpE. Colonies were selected that grow on minimal medium. Plaanid pC501 carrying trpC-E was isolated from one of the selected colonies.

Plasmid-linked 7-methyltryptophan (7 MT) resistance was selected by treating a strain carrying pC501 with ethyl methane sulfonate and selecting for resistant mutants on minimal medium containing 7MT and tetracycline. One such mutant had a plasmid gene (trpE) ceding for an anthranilate synthetase resistant to inhibition by tryptophan.

A 1.6 kb Sall-EcoRI fragment of the mutant derivative of pC501, having an altered trpE gene encoding an anthranilate synthetase resistant to inhibition by tryptophan was ligated into Sall-PstI digested pCM136. An EcoRI-PstI fragment of pBR327 containing a tet^r gene was added to this ligation mixture. Plasmid pD2623 ceding for ^"the activities of the trpA-D genes under lac control and for trpE (feedback resistant anthranilate synthetase) and which specifies Tet^r and Amp^r was identified by transforming strain MV17 which is a tryptophan auxotroph, selecting for Tet^r-Amp^r and screening these colonies for growth in the absence of tryptophan (i.e., tryptophan independence) on minimal mediun. Plaanid pD2643 was constructed by deleting Amp^r activity from pD2623 by digestion with Ahalll restriction endonuclease, ligation with the T₄ ligase, transformation of an E. coli host microorganism to Tet^r and screening for ampicillin sensitivity.

Construction of Plasmid pD2625 Plaanid pD2625 coding for aroG activity which is feedback resistant to inhibition by aromatic amino acids (under lac control), serA and lacl activity, a par locus and a p15A origin of replication was constructed in the manner described immediately below.

A. Construction of serA-Containing Donor Plaanids

A nucleotide sequence ( λ1059 serA) derived from an E. coli K12 genomic clone bank (Karn et al., Proc. Nat'l. Acad. Sci., 77, 9:5172-5176 (1980); clone bank obtained from M. Benedik, Stanford University) was isolated, digested with Ncol restriction endonuclease and ligated into Ncoldigested pACYC184 under conditions favoring formation of hybrid molecules. E. coli serine auxotroph JC158 (A.J. Clark via B. Bachmann) was transformed to Tet^r and serine independence using aliquots of the hybrid molecules. pD2528 was isolated from a serine independent Tet^r transformant of JC158. pD2528 DNA was digested with EcoRI and SaIl and was ligated using T₄ ligase into EcoRI-Sall digested pBR327 under conditions favorable for plaanid formation. pD2537 was isolated from transformants of E. coli (JC158) that were Amp^r, Tet^s and Ser⁺.

B. Construction of lad Activitv Donor Plasmids

A bacteriophage carrying the genes of the lac operon was obtained from the λ1059-E. coli genomic bank indicated above. Lac activity was identified by complenentation of a Lac- E. coli strain (B1361) carrying plaanid pDE4081 (Mascarenhas et al. Virology, 26:658-668 (1983)) on lactose minimal plates. The λ 1059 lac phage thus selected was digested with Bglll. A 10.5 kb Bglll fragment obtained from this digest was ligated to BamHI-digested pACYC184 and the hybrid was used to transform B1361 to Cam^r Lac⁺. A 4.2 kb Eco Rl-SphI sub-fragment of this plaanid was then ligated to a 2.7 kb Eco Rl-SphI fragment previously obtained frcm pBR327, to form pD2329. Finally, digestion of ρD2329 with Narl restriction endonuclease yielded a 1.85 kb Narl fragment with lacI sequences frcm about 100 base pairs upstrean of the lacl promoter to base pair nunber 1020 in the lacI DNA sequence reported by Farabaugh in Nature, 274: 765-769 ( 1978) . This 1 .35 kb Narl fragment was ligated into pACYC184 digested with Clal restriction endonuclease-forming plaanid pD2333 having lacI activity, Cam^r and a P15A origin from plasmid pACYC184. A SaIl restriction site located between an Aval and BamHI site on plasmid pD2333 was eliminated by digestion with Aval followed by addition of Klenow Pol I, dNTP ' s, BamHI linkers and T₄ ligase. This was followed by digestion with BanHI and re-circularization of the resulting large fragment with T₄ ligase, to form pD2348.

Plasmid pD2348 was digested with restriction endonucleases Nrul and SaIl. The digest was combined with a 1 . 1 kb EcoRI- SaIl fragment of pEM31 containing a partition (par) locus (Meacock and Cohen, Cell , 20: 529542 (1980 ) ) , and T₄ ligase, Klenow Poll and dNTP ' s. The resulting plasmid pD2349 which is Cam^r and has a P15A origin of replication, par locus and lacl activity was isolated by screening for Cam^r transformants.

C. Construction of aroG^fbr Donor Plasmid A bacteriophage carrying the arcG gene of E. coli was selected from the λ 1059-E. coli genomic bank indicated above by complementation of an Aro- (arcG) strain, C531 , described below, carrying plaanid pDE4081. A 5.8 kb BamHI- SaIl fragment of DNA from this bacteriophage was subcloned into BamHI-Sall digested pACYC184 to form pC520.

Construction of pD2625 From Donor Plasmids Plasmid pC520 was digested with Bglll and BanHI restriction endonucleases, and a 1.6 kb Bglll-BamHI fragment from this digest was ligated into Bglll- BamHI digested plasmid pCM126 using T4 ligase to form pD2149, a plaanid in which aroG activity is under tandem lac promoter transcriptional control.

pD2149 was digested with PstI to form PstI fragments. These fragments were ligated to a BamHI adaptor sequence which converts the PstI fragments ends to BamHI ends. From the converted BamHI fragments, a 2.8 kb BamHI fragment carrying the aroG gene under tandem lac promoter control was isolated by gel electrophoresis and was ligated using T₄ ligase into BanHI digested pD2349 to form pD2351 which is characterized as follows: Cam^r, p15A origin, par locus, lacl activity, tandem lac promoters linked to aroG.

A 2-nucleotide shift in the arcG locus of pD2351 was introduced by Clal restriction endonuclease digestion in the presence of Klenow Poll and dNTP's, followed by treatment with T4 ligase. The frane shift renders pD2351 incapable of expressing DAHP synthetase, the peptide product of aroG, yet conserves practically the whole arcG nucleotide sequence. The plaanid with the frane shift is used to transform E. coli strain MAR13 (Held and Smith, J. Bacteriol., 101:202-208 (1970)), a strain carrying chromosomal aroG activity which is feedback resistant to inhibition by phenylalanine and hence resistant to aromatic anino acid inhibition. In this transformant, low frequency homologous exchange of DNA between the Mar13 chromosomal arcG and the plaanid pD2351 (frane- shifted) aroG gene occurs. Plasmid DNA is isolated from this strain by conventional methods and is used to transform E. coli strain C531 which is a spontaneous

Gal^rTet^r derivative of NK6969 (Roberts, D.) and carries a complete deletion of the aroG gene. Colonies are selected for growth on minimal medium containing nicotinanide, chloramphenicol, tyrosine and tryptophan. Colonies that grow under these conditions are screened individually by enzyme assay [Doy and Brown, Bicchanica et Biophvsica. Acta., 104:377389 (1965)] to identify those which contain a feedback resistant DAHP synthetase (arcG^fbr) activity. The plaanid in one such isolate was designated pD2422.

Plasmid PD2434 in which the arcG^fbr activity is brought under lac operator control is constructed from pD2422 as follows: a sample of plasmid pD2422 is digested with Bglll restriction endonuclease, Klenow Pol I and dNTP's to form an 8.5 kb fragment having blunt ends. The 8.5 kb υlunt-ended fragment is digested with Xhol restriction endonuclease to form a 7.4 kb fragment having one blunt end and one Xhol end.

Another sample of pD2422 is digested with Ddel restriction endonuclease and Klenow Pol I with dNTP's to form multiple fragments with blunt ends. These blunt ended Ddel fragments are then digested with Xhol restriction endonuclease to form a 1.0 kb fragment having an Xhol end and a blunt end. The 7.4 kb and 1.0 kb fragments are purified, mixed and ligated using T₄ ligase, to form plaanid pD2434.

Plasmid pD2625 was constructed from pD2434 and pD2537 in the following manner. pD2537 was digested with Clal and Nrul restriction endonuclease to yield a 1.85 kb fragment having a Clal end and a blunt end. A sample of pD2434 was digested with Clal and Bglll restriction endonuclease to yield a 7.7 kb fragment with a Bglll site adjacent to and downstream of the lac promoter and a Clal site in the arcGfbr gene.

A second sample of pD2434 was digested with XmnI and BallI restriction endonuclease to yield a 1.4 kb fragment having an XmnI end and a Bglll end. The 7.7 kb fragment of pD2434, 1.85 kb fragment of pD2537 and 1.4 kb fragment of pD2434 were ligated together with T₄ ligase and were used to transform JC158 (J.C. Clark via B. Bachmann) to Cam^rSer⁺. One of these transformants yielded plaanid pD2625.

A plasmid pD2624 in which the serA fragment is inserted into pD2434 in the opposite reading sense from that of pD2625 was also constructed by digesting a sample of pD2434 with Xhol and Bglll to yield a 7.4 kb fragment having Xhol and Bglll ends, and digesting a second sample of pD2434 with Xhol and Bglll as before. Plasmid pD2537 was digested with SaIl and MstI to yield a 2.0 kb fragment with a Sall end and MstI blunt end. The fragments were ligated together with T4 ligase as above. The SaIl end of the serA-containing fragment from pD2537 and Xhol end of the large pD2434 fragment are compatible as these two endonucleases produce compatible cohesive termini.

Construction of Plaanid pGM3207 Plaanid pGM3207 (shown in Figure 2) carrying serB and serC was constructed in the manner described immediately below in Sections A-C.

A. Construction of serB-Containing Donor Plaanid

An E. coli serine auxotroph was constructed by transducing strain CGSC #5409 (argI61, argF58, serB28, purA54, thr-25, tonA49, relAl, spoT1; obtained from B. Bachmann) to PurA⁺ with P1 phage grown on strain W3110. One Pur⁺ transductant was purified and designated G3004. A nucleotide sequence ( λ 1059 serB) derived from an E. coli K12 genomic clone bank (Karn et al., Proc. Nat' 1. Acad. Sci., 77, 9: 5172-5176 (1980); clone bank obtained from M. Eenedik, Stanford University) was isolated, digested with BanHI restriction endonuclease and ligated into BamHI-digested pB3339 under conditions favoring the formation of hybrid molecules. E. coli serine auxotroph G3004 was then transformed to Kan^r and serine independence using aliquots of the hybrid molecules. Plasmid pGM3147 was isolated as a serine independent Kan^r, tet^s transformant of G3004.

B. Construction of serC-Containing Donor Plasmid A nucleotide sequence ( λ) 1059 serC) derived from an E. coli K12 genomic clone bank (supra) was isolated, digested with Ncol restriction endonuclease and ligated into Ncol-digested pACYC184 under conditions favoring formation of hybrid molecules. E. coli serine auxotroph CGSC #4297 (K.B. Low via B. Bachmann) was transformed to tet^r and serine independence using aliquots of the hybrid molecules. Plaanid pGM3134 was isolated as a serine independent tet^r transformant of CGSC #4297.

C. Construction of pGM3207 From Donor Plasmids

Plasmid pGM3134 was digested with Bglll and Xhol restriction endonucleases, to yield a 5.3 kb Bglll-Xhol fragment containing serC.

A sanple of plaanid pGM3147 was digested with Bglll and Sall restriction endonucleases to yield a 9.9 kb Bglll-Sall fragment containing serB and a gene specifying kanamycin resistance.

The pGM3134 5.3 kb Bglll-Xhol fragment was ligated to the pGM31479.9 kb Bglll-Sall fragment with T₄ ligase. The products of this ligation mixture were used to transform CGSC #4297 (serC auxotroph) to Kan^r, Ser⁺. Plaanid DNA purified from one of these transformants was used to transform G3004 (CGSC #5409 derivative and serB auxotroph, see above in Section A) to Kan^r, Ser⁺. The transforming plaanid was designated pGM3207. Construction of Host Strain B1238 [W3110 F- (argF-lac)^Δ U1 69, ^Δ(gal-bio), ∅ (trp-lac) W205, ∅(trp-^Δ 61-intc-226), trp^R] (Benedik et al., Gene, 19:303-311 (1982)) was transduced to Gal⁺ with P1 phage grown on strain W3110. The resulting strain designated S1238GB was subjected to ethyl methane sulfonate mutagenesis and was plated on glucose minimal median containing 2000 mg/l anthranilate. One anthranilate resistant colony was selected and was designated A103.

A103 was transduced to tetracycline resistance with a P1 lysate of E. coli strain TST 1 (T.J. Silhavy via B. Bachmann). TST 1 is known to carry a Tn10 insertion in the malE gene (malE52:Tn10) which is closely linked to the gene ceding for phosphoglucose isomerase (pgi). Tet^r colonies were selected on LB agar median with 10 mg/l tetracycline and one of these was designated A103T. A nunber of spontaneously-occurring fusaric acid resistant derivatives of A103T were isolated on fusaric acid plates. Fusaric acid resistant isolates were screened on glucose tetrazolion plates and those isolates which formed white colonies on this medium were tested further. One of these was found to be auxotrophic for lysine in the presence of methionine and threonine. Since the genes lysC and malE flank the pgi gene it was assumed that the lysine auxotroph had a complete deletion of the pgi locus. This isolate was designated CT10 and was assumed to be ( lysC-pgi-malE)^Δ .

A derivative of CT10 which was additionally deficient in the activity of tryptophanase (tna), was made by P1 transduction of CT10 to Sal⁺ (growth on salicin plates) with lysates of strain C537. These transductants were screened for tryptophanase activity and one Tna- isolate was selected and named C536. C537 mentioned above had been constructed by P1 transduction of MV17 (D. Helinski to Sal⁺ using lysates of E. coli strain N1624 (M. Gottesman via B. Bachmann) and selecting one transductant that was still Tna-. C534, a Tna- derivative of A103, was constructed in a manner analogous to that described above for C536.

Strain D2307, which carries a mutation in the tyrR gene ceding for tyrosine repressor which controls the prcduction of shikimate kinase, was made bv P1 transduction of E. coli strain JP2144F (A.J. Pittard via B. Bachmann) to Trp⁺ with lysates of E. coli strain W3110. D2307 was picked as a 3-fluorotyrosine resistant strain on minimal median containing 3-fluorotyrosine.

Strain D2316, a strain deficient in the prcduction of phenylalanine caused by a deficiency in the pheA gene which cedes for the first enzyme of the phenylalanine biosynthetic pathway, was made by conjugation of strain C536 and strain KA197 (H. Qkestra via B. Bachmann). D2316 was selected as a Trp⁺ phenylalanine auxotrophic transconjugant by replica plating on median either containing or lacking phenylalanine.

Strain D2139 was made by P1 transduction of strain MV17 (D. Helsinki, supra) with lysates of MB82 (Benedik et al. Virology, 126:658668 (1983)). Strain D2316 was made Tet^r by P1 transduction with lysates of strain D2139, which contains a Tn10 insertion in the trpB gene (trpB::Tn10). A Tet^r Trp- transductant was isolated and designated D2317. D2317 was PI transduced to 3-fluorotyrosine resistance with lysates of D2307 described above. D2318 characterized as tryptophan independent, 3-fluorotyrosine resistant and phenylalanine-requiring was one of the transductants obtained.

D2318 was P1 transduced using lysates of strain AT2471 (A.L. Taylor via B. bachmann), a tyrA mutant strain. Phenylalanine-independent colonies were selected by growth on median containing tryosine but lacking phenylalanine. Phenylalanine-independent colonies were screened for tyrosine dependence and a Phe⁺Tyr- strain was isolated and designated D2320.

A strain having feedback resistant anthranilate synthetase was constructed from strain D2320 by first making D2320 Tet^r by P1 transduction with lysates of D2319. The trpB::Tn10 sequence was introduced into D2320 in a manner analogous to that used in making D2317. One Tet^r transductant (D2324) was P1-transduced using lysates of a spontaneously occurring derivative of W3110 having anthranilate synthetase resistant to feedback inhibition by tryptoph.an. A tryptophan independent, 7-methyltryptoρhanresistant transductant that was auxotrophic for tyrosine and sensitive to tetracycline was selected and designated D2325. Enzyme assays (according to Ito and Crawford, Genetics, 52: 1303-1316 (1965)) in the presence of 2 mM L-tryptophan confirmed that D2325 specified a feedback-resistant anthranilate synthetase.

A strain designated D2327 which is auxotropic for phenylalanine and tyrosine independent was made by P1 transduction of D2325 to Tyr⁺ using lysates of strain KA197. (Hoekstra via B. Bachmann).

Strain D2327 was made Tet^r by P1 transduction with lysates of an E. coli strain (C541) having a Tn10 insertion in the nadA locus and an adjacent deletion removing nadA, aroG, gal. C541 was made by P1 transduction of MV17 (D. Helsinki, supra) with lysates of strain C531. The Tet^r derivative of D2327 was then transduced using P1 lysates of strain MAR13 (Held and Smith, supra). Marl 3 carries an arcmatic anino acid feedback resistant DAHP synthetase activity. Transduced cells were isolated as Nad⁺, Phe-, tetracycline sensitive strains. In one transductant, feedback resistance of DAHP synthetase to phenylalanine was determined by enzyme assay in the presence of 1 mM phenylalanine. This strain was designated D2346. A phenylalanine independent strain of D2346 was made by P1 transduction of D2346 using lysates of AT2471 (A.L. Taylor, supra) and selecting for phenylalanine independence on median containing tyrosine. One Phe⁺Tyr- transductant was designated D2402.

B1364 is a trp⁺lac⁺ transductant of B1363 (Mascarenhas et al., Virology, 124:100-108, (1983)) using P1 lysates of W3110. B1364 thus has genetic information for β-galactosidase, lactose transacetylase, and lactose permease activity all fused to and under the control of the tryptophan operon (∅ trp-lac W205). Strain D2402 was made Tet^r by P1 transduction using a lysate of D2139 as above; a Tet^r derivative of D2402 was then transduced to Trp⁺ac⁺ with P1 lysates of strain B1364. One Trp⁺Lac⁺Tet^s 7-methyl-tryptophan resistant (7MT^r) isolate was designated D2432.

D2432 was made Tet^r by P1 transduction using P1 lysates of strain RS162 (J. Wechsler via B. Bachnann) . The resulting Tet^r strain (D2548) has a Tn10 insertion in the zjb locus and a temperature sensitive mutation dnaB252. A spontaneous isolate of this strain capable of utilizing hydroxyphenyl pyruvate was isolated and designated D2549. D2549 was P1-transduced using lysates of strain EG30 (D, Gelfand via B. Bachmann), a strain lacking transaninase A (tyrB-). Transductants were isolated by growth on LB agar at 42°C (loss of dnaB252 mutation) and screened for inability to grow on plates containing p-hydroxyphenylpyruvate. One such isolate was designated D2550.

A spontaneous tetracycline sensitive (fusaric acid resistant) derivative of D2550 designated D2618 was selected on fusaric acid plates. D2618 was then made Tet^r by P1 transduction using lysates of NK6024 (N. Kleckner via B. Bachmann), a strain having a Tn10 insertion in the pheA locus. Tetracycline resistant colonies were selected and one Phe-Tyr- transductant was designated D2636. Spontaneous fusaric acid resistant (Tet^s) derivatives of D2636 were selected on fusaric acid plates and checked for phenylalanine and tyrosine requirements. One such isolate (Phe-Tyr-Tet^s) was designated D2637.

D2637 was transduced to Tet^r (Ser-) using P1 lysates of strain 123A1, which contain a Tn10 insertion linked to the serC locus in strain strain KL282 (K. B. Low via B. Bachmann). One Tet^rSer- transductant, designated D2638, was transduced to Ser⁺ with P1 lysates from DG30 (D. Gelfand via B. Bachmann) which contains an aspC mutation and is auxotrophic for aspartic acid. These transductants were screened for aspartic acid auxotrophy. One aspartic acid auxotroph that was serine independent and Tet^S was designated D2639.

Serine deaninase-deficient derivatives of D2637 were constructed as follows: Strain MEW191 and a P1-sensitive derivative of strain 1K15-5 (both strains received from E. Newman, Concordia University) are believed to carry mudX ( Cam,Amp) insertions in regulatory and structural genes for L-serine deaminase, respectively. A P1 lysate of each of these strains was used to transduce D2637 to Cam^rMp^r. Single transductants from each cross were designated D2711 and D2713 respectively, and were deemed to carry the mudX (Cam, Mp) insertions (regulatory:D2711, structural:D2713) present in their respective parents. Spontaneous Cam^SAmp^s derivatives of D271 1 and D271 3 were isolated and designated D2714 and D2715 respectively. Enzyme assays confirmed that both D2714 and D2715 were deficient in L-serine deaminase activity.

Improved Tryptophan prcduction in Pgi- host compared to Isogenic Pgi⁺ host C534 and C536 are isogenic E. coli strains except that the pgi gene has been deleted in C536. C534 was made by PI transduction of A103 using lysates grown on C537 and screening for Tna- colonies. C536 was described above. These strains were cured of their resident plasmids by isolation of Amp^s segregants in each case. These cured derivatives were then transduced to Tet^R with P ₁ grown on D2636 (tyrA4 pheA18: :Tn10 ) . Single transductants were then cured of the transposon by selection of spontaneous fusaric-acid-resistant segregants on fusaric acid plates. One of these, D2704, was Pgi⁺Tyr-Phe-Tet^s (derived from C534 ) . D2705, which is Pgi-, was derived from C536 in a way similar to D2704 and is otherwise isogenic to D2704.

D2704 and D2705 were each transformed sequentially with pD2634, pD2643 and pD2625 - by selection for Amp ^R, Tet^R and Cam^R, respectively, using conventional calcium shock to render the host competent for transformation. These plasmids carry the genes for the key enzymes of tryptophan biosynthesis and were described hereinafccve. One triple transformant in each case was used for nuclear magnetic resonance (NMR) analysis.

¹³C-glucose NMR spectroscopy of D2704 and D2705 derivatives

Single colonies of each strain were inoculated into minimal median (5 mis) containing 1 . 5% glucose, 0. 4% NZ-amine, 10mM M g SO₄, 1 ug/ml each FeSO₄ and MnSO₄, and anpicillin, tetracycline and chloranphenicol. These cultures were grown overnight at 37 °C, then diluted 1 : 12 into the sane median plus 1 mM isopropyl- β -D-thicgalactopyranoside (IPTG) and 0.5% w/V ¹³C-glucose ( labelled in the C6 position) . This culture was transferred to an NMR tube and used directly for N MR spectroscopy at 28 °C using a Varion Associates XL200 NMR spectrcmetry system. Aeration of the culture was achieved by rotating the tube and pumping air through the culture during the run. Spectra were acquired at 3-hour intervvls. Analysis of results

Table II summarizes the efficiency of conversion of glucose to tryptophan by the strains described immediately above. Fran the intensity of the peaks of the NMR spectra corresponding to C6-glucose and C6-tryptophan, one can calculate the increase in tryptophan relative to the decrease in glucose (after making allowance for the molecular weights of the two compounds).

The results suggest that the pgi block in D2705 serves to substantially increase efficiency of conversion of glucose to tryptophan, when compared to D2704.

*Correction factor 1.133 applied for molecular weight difference

Effect of Chromosomal Mutations on Tryptophan Production Twenty milliliter cultures of the strains listed in Table III below were grown in side-arm flasks at 30°C on a rotary shaker at 280 rpm. The growth median was MINIMAL MEDIt-M #3 described hereinabove supplemented with 1% gluccse and 0.4% casamino acids (Difco). Culture supernatants were assayed for tryptophan after 3 days, by the method of Spies and Chamber (Anal. Chem. 20:30, 1947). w.t. = wild type;

* all strains are derivatives of W3110F- and carry, in addition, the following markers: (argF-lac) U169, tna 2, bglR6, (lysC-pgi-malE) trpR, (anthranilate-resistance)

Effect of Transaninase A activity on Tryptophan Accumulation Cultures used in Table IV were grown and assayed exactly as described in the legend to Table III except that the growth median was MINIMAL MEDKSM supplanented with 2% glucose, 1% NZ-amine, 1mM IPTG, chloranphenicol (20 mg per ml) and ampicillin (100 mg per ml).

** These strains also carry the following markers: (argF-lac) U169, arcG^FBR, ∅(trp-lac) W205, trpE^fbr31, tyrR366, tyrA4, tna2, bglR6, (lysC-pgi-malE) , trpR, (anthranilate-resistance).

Effect of Plasmid Genes on Tryptophan Prcduction Fifty milliliter cultures of the strains listed in Table V below were grown in 500 ml baffled flasks, in triplicate, for 44 hours on a rotary shaker (280 rpm) at 30ºC. The growth mediun was MINIMAL MEDIUM supplanented with 5% glucose, 1% NZ-amine, 1mM IPTG, tetracycline (5mg/ ml), ampicillin (100 mg/ml) and chloranphenicol (20 mg/ml). Culture supematants were assayed by the method of Spies and Chanber (Anal. Cham. 20: 30, 1947). The genotype of the host strain, D2618, is as follows: W3110 F-( argF-lac) U169, aroG^PBR, ∅ (trp-lac) W205, trpE^FBR31, tyrR366, tyrA4, tna2, bglR6, (lysC-pgi-malE) , tyrB507, zjb, trpR, (anthranilate-resistance).

Tryptophan Prcduction in Serine-Deaninase Deficient Strain

Cultures indicated in Table VI were grown and assayed exactly as described in the legend to Table V above except that the incubation period was 43 hours and each value is the average of 2 flasks only. D2715 is derived from D2618 by introducing deletions into the pheA gene and into the structural gene for serine deaminase.

Deposited Microorganisms

The following microorganisms have been deposited in the American Type Culture Collection.

Strain D2715 in which are cloned pD2625, pD2634, pD2643 and pGM3207 is not only deposited under ATCC Accession Number 53064 but is also deposited in a security deposit to which the United States Patent and Trademark Office has irrevocable access. (Security Deposit No. 799.)

It will be readily apparent that the inventor has provided, in addition to the unique bacterial hosts and plaanids, a method for increasing the production of tryptophan in a host of the strains of Escherichia, particularly E. coli. The general method according to the invention comprises the steps of transforming an E . coli host deficient in tryptophanase with plasmid-borne genetic information to control tryptophan prcduction divided between at least two plaanids, growing the transformed host in an appropriate nutrient median for a period of time sufficient to produce tryptophan as exemplified above, and producing tryptophan by the transformed host. Tryptophan from the culture broth may be removed by any conventional technique. The tryptophan so obtained may be further refined or purified as desired. Preferably the host, in addition to being Tna-, will be deficient in the activity of phosphoglucose isomerase (pgi-). The host, in addition to or instead of being pgi-, may also have DAHP synthetase activity resistant to aromatic anirio acid inhibition, anthranilate synthetase activity resistant to tryptophan innibition, deficiencies in the activity of lac repressor, aromatic transaminase A, L-serine deaninase either singly or in any combinations thereof.

The inventor has also provided a method for increasing the production of tryptophan in a host of the genus Escherichia, particularly E. coli by transforming an E.coli host deficient in the enzyme tryptophanase with a plaanid carrying plaanid-borne genetic information to control tryptophan production, a portion of said information including lac operator/promoter control, growing the transformed host in an appropriate median as exemplified above and prcducing tryptophan by the transformed host.

Preferably the E. coli host will have the additional characteristics described above with respect to pgi-, DAHP synthetase, anthranilate synthesis, lac repressor activity, aromatic transaminase and L-serine deaminase.

In a most preferred embodiment, the preferred E. coli host is transformed with plasmid-borne genetic information to control tryptophan production divided between at least two plaanids, a portion of the information to control tryptophan prcduction being under lac operator/promoter control.

The foregoiηg description of the invention is intended to provide a guide, to those ordinarily skilled in the arts to which the invention pertains, to the means to attain and practice the invention as claimed hereinbelow. It will be readily apparent to those ordinarily skilled in the art to which the invention pertains that one may depart from the exact description of the invention contained herein without departing from the scope of the invention as claimed.

Claims

WHAT IS CLAIMED IS:

1. A bacterium comprising a host of the genus Escherichia deficient in the enzyme tryptophanase carrying plasmid-borne genetic information to control tryptophan production divided between at least two replication-compatible plaanids.

2. A bacterium comprising a host of the genus Escherichia deficient in the enzyme tryptophanase carrying plasnid-borne genetic information to control tryptophan prcduction divided between at least two plaanids , a portion of said information under lac operator/promoter control.

3. The bacteriun of Claim 2 having information to control serine biosynthesis including serA, serB and/or serC not under lac operator/promoter control.

4. The bacterium of Claim 1 wherein at least one of said plaanids carries information controlling biosynthesis of the common precursors of the aromatic amino acids.

5. The bacterium of Claim 4 wherein at least one of said plasmids carries genetic information for DAHP synthetase activity resistant to inhibition by aromatic amino acids under lac promoter control.

6. The bacterium of Claim 2 wherein said plasmid borne genetic information for control of tryptophan prcduction includes trpA-E genes wherein trpA-D are under lac operator/promoter control , said plasmid carries a deletion in the trp attenuator region and wherein trpE cedes for anthranilate synthetase resistant to inhibition by tryptophan.

7. The bacteriun of Claim 2 wherein said plasmid-borne genetic information further includes a region for constitutively produced lac repressor activity.

8. The bacterium of Claim 2 wherein said host additionally has activities selected from the group consisting of DAHP synthetase resistant to aromatic anino acid inhibition, anthranilate synthetase resistant to tryptophan inhibition, deficiency in activity of: lac repressor ; phosphoglucose isomerase ; arcmatic transaminase A; L-serine deaminase ; tyrA; pheA; trpR and combinations thereof.

9. A method for the production of tryptophan comprising growing the bacteriαn of Claim 1 in an appropriate culture medium for a period of time suitable to produce tryptophan, and raneving said tryptophan from the culture medium.

10. A method for increasing the production of tryptophan by an E. coli bacterium comprising transforming the host of Claim 1 with at least two plaanids, said plasmids being divided therebetween genetic information to control tryptophan prcduction, and growing said transformed host in culture median appropriate for tryptophan production by said transformed host.