EP0217862A1 - $i(IN VITRO) SYNTHESIS OF L-TRYPTOPHAN - Google Patents
$i(IN VITRO) SYNTHESIS OF L-TRYPTOPHANInfo
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- EP0217862A1 EP0217862A1 EP86902176A EP86902176A EP0217862A1 EP 0217862 A1 EP0217862 A1 EP 0217862A1 EP 86902176 A EP86902176 A EP 86902176A EP 86902176 A EP86902176 A EP 86902176A EP 0217862 A1 EP0217862 A1 EP 0217862A1
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- Prior art keywords
- plasmid
- serine
- transformed
- tryptophan
- tryptophanase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/227—Tryptophan
Definitions
- the present invention relates to the enzymatic synthesis of L-tryptophan from gly ⁇ ine, formaldehyde and indole.
- L-Tryptophan j_ s an essential amino acid in man and, therefore, is a valuable component of hyperalimentation solutions and other nutritional formulations.
- the amino acid L-tryptophan is a valuable commercial pro ⁇ duct that is used, for example, as an intermediate or starting material in certain synthetic processes. There is thus a demand for L-tryptophan, and various processes have been developed for producing it, including fermentation, chemical syntheses and enzymatic syntheses.
- a common shortcoming of the fermentation methods is that the concentration of L-tryptophan produced in the broth and recovered is relatively low, even after long periods of fermentation. Chemical syntheses of tryptophan often produce trypto ⁇ phan as a racemic mixture of the D and L optical isomers, or as the less preferred D-isomer. Methods of resolving the D, L-mixtures add to the cost of the product.
- Enzymatic synthesis of L-tryptophan has heretofore been accomplished by reacting L-serine with indole in the presence of tryptophan synthetase or tryptophanase.
- Living organisms utilize tryptophan synthetase in the production of tryptophan from serine, whereas tryptophanase is utilized in organisms to decompose tryptophan to form indole.
- synthesis of L-tryptophan is accomplished by reacting glycine, formalde ⁇ hyde and indole in the presence of biocatalytic amounts of the enzyme serine hydroxymethyltransferase, the cofactor tetrahydrofolic acid, and the enzyme tryptophan synthetase or tryptophanase.
- the serine hydroxymethyltransferase, or tryptophan synthetase or tryptophanase are preferably provided to the reaction medium in the form of expression products of transformed microorganisms containing expression vectors which code for serine hydroxymethyl- transferase, and tryptophan synthetase or tryptophanase. Tryptophan is thus synthesized in a single process without using serine as a starting material.
- the process takes advantage of the fact that tryptophan has limited solubility in water (11.4 g/ ⁇ at 25°C; Merck Index, 3rd Ed., p. 1256, 9458). Thus, tryptophan precipitates out of the reaction mixture and can easily be recovered.
- FIG. 1 is a schematic representation of the preparation of plasmids pGX2236 and pGX2237 which direct the production of SHMT for use in the process of the invention.
- Fig. 2 is a schematic representation of the preparation of pGX2208, a cloning vector which contains the trpBA genes on an EcoRI-Sall segment.
- Fig. 3 is a schematic representation of the preparation of plasmid pGX2213, an expression vector for tryptophan synthetase, in which expression of the trpBA gene is under the control of the trp/lac promoter.
- Fig. 4 is a schematic representation of the preparation of plasmid pGX2214, an expression vector for . ' tryptophan synthetase, in which expression of the trpBA gene is under the control of the lambda P L promoter.
- Fig. 5 is- a schematic representation of the preparation of plasmid pGX150, an expression vector for tryptophan synthetase, in which the trpBA gene is under the control of the trp promoter.
- Fig. 6 is a schematic representation of the preparation of pGX2302, a plasmid which directs the production of SHMT and lambda phage endolysin enzymes .
- Fig. 7 is a schematic representation of the preparation of pGX2308, a plasmid which directs the production of tryptophanase and lambda phage endolysin enzymes.
- the present invention relates to a method of synthe ⁇ sizing L-tryptophan by reacting glycine, formaldehyde and indole in the presence of serine hydroxymethyltransferase (hereafter referred to as SHMT), tetrahydrofolic acid (hereafter referred to as THF) , and tryptophan synthetase (hereafter referred to as TS) or tryptophanase (hereafter referred to as TASE).
- SHMT serine hydroxymethyltransferase
- THF tetrahydrofolic acid
- TS tryptophan synthetase
- TASE tryptophanase
- the enzymes SHMT, TS and TASE are important in the metabolism of microorganisms and higher organisms; thus, there are many potential sources for the enzymes.
- Serine hydroxymethyltransferase may be used in the form of whole cells containing the enzyme, a crude extract, or as a purified enzyme. If whole cells are used, tetrahy ⁇ drofolic acid may be obtained from the same source as SHMT, since THF is found in microorganism cells containing SHMT. If desired, additional THF may be added to increase the concentration of THF.
- the SHMT may be obtained from microorganisms that have been modified using conventional genetic engineering tech ⁇ niques to produce it in high yields. See Stauffer, G., et al.. Gene 15:63 ⁇ 72 (1981).
- the SHMT gene glyA may ' be ' isolated and cloned into an expression vector which then * can be used to transform suitable host cells resulting in high level SHMT expression.
- the vector may be a phage or a plasmid, but plasmids are preferred.
- Suitable hosts include, for example, Klebsiella, Salmonella and Escher-ichia. Mutant microorganisms which have been modified in their methionine metabolism also will overproduce SHMT . See Stauffer, G.V., and Brenchley, J.E., Genetics 88:221 (1978) and Stauffer, G.V., and Brenchley, J.E., J. 3acteriol. 129:740 (1977).
- the enzyme activity has been increased as much as twenty fold, and and the enzyme concentration can represent more than ten percent of the soluble protein of the cell.
- the SHMT gene is taken from a natural source, it can be modified by random mutagenesis or site directed rautagenesis to produce an enzyme with improved stability.
- the gene can also be completely chemically synthesized with multiple changes to improve the enzyme's stability during the dis ⁇ closed process.
- a suitable modified microorganism for use as an SHMT source is an E_ ; _ coli strain (GX1703) transformed with pGX122, a plasmid derived from pBR322 containing the SHMT (glyA) gene. This transformant has been deposited at the Northern Regional Research Laboratories in Peoria, Illinois as NRRL No. B-15215.
- a Klebsiella aerogenes strain (GX1704) transformed with a similar but smaller plasmid with an alteration caus ⁇ ing high copy number, pGX139, has been deposited with the American Type Culture Collection in Rockville, Maryland, as ATCC No. 39214 and a Salmonella typhimurium strain (GX1682) transformed with pGXl39 has been deposited as ATCC No. 39215.
- the pre- ferred vectors comprise plasmids which contain both the trp operon (trpEDCBA) operably linked to its associated trp promoter-operator and the SHMT (g_lyA) gene operably linked to its associated promoter-operator. Construction of the preferred expression vectors can be carried out in the following manner (see Fig. 1).
- a vector containing the trpEDCBA operon e.g., pGX110 or pEP392 is cleaved at an endonuclease restriction site outside the trp operon and regulatory region to linearize the plasmid.
- Plasmid pEP392 is described in Enger-Valk, B.E. et al-. , Gene _9:69-85 (1980).
- Plasmid pGX110 is essentially identical to pHP12, also described in this reference. In the case of pGX110 and pEP392, a single Xhol restriction site, upstream of the promoter for the trp operon, serves as a convenient cleavage site.
- a second vector, carrying the SHMT (glyA) gene and its associated regulatory sequence, e.g., pGX139, is also linearized by cleavage at an endonuclease restriction site outside the SHMT (g_lyA) gene and regulatory region.
- pGXl39 a single Sail restriction site, just downstream of the SHMT (glyA) gene, serves as a convenient restriction site.
- the two linearized plasmids are then ligated to produce a recombinant plasmid which contains both the trp operon, operably linked to its regulatory region and the SHMT (glyA) gene, operably linked to its regulatory region.
- the "sticky" ends can be filled in by reaction with dATP, dCTP, dGTP and dTTP in the presence of DNA poly- merase I (Klenow fragment) to produce blunt ended linear ⁇ ized plasmids, which can be blunt-end ligated at high DNA concentration.
- the two plasmids each contain a second restriction site, outside the trp operon and the glyA gene, which can be employed to form unit length ligation products that are circularized to form the desired recombinant plasmid removing unnecessary DNA.
- each of the plasmids contains an EcoRI site conveniently positioned such that the ligation mixture can be secondarily digested with EcoRI before final ligation (circularization) to remove substantial segments of unwanted DNA to produce a replicable plasmidic expression vector containing both a functional glyA gene and a functional trp operon.
- pGX2236 and pGX2237 Two such expression vectors produced in this manner, the preparations of which are illustrated in Fig. 1, were designated pGX2236 and pGX2237. Because of the presence of an ampicillin resistance gene in pGX110, transformants containing the resulting recombinant pGX2236 are ampicillin resistant.
- Plasmid pGX2236 was modified by the insertion of lambda phage endolysin genes ( ⁇ RR z ) to create plasmid pGX2302 as illustrated in Figure 6.
- the expression of the lambda endolysin genes cause cell lysis by degradation of the cell wall through the lysozyme and endopeptidase action of the gene products, but only after the cell membrane has been ruptured to allow the R and R z gene products to contact the cell wall.
- Several types of chemical treatment can be utilized to gently disrupt the membrane and thus initiate cell lysis.
- detergents like NP40, Triton X-100, cetyl-pyridiniu ⁇ fhydrochloride or solvents such as chloroform, methylene chloride or butanol may be used.
- the expression of the endolysin genes allows ready autolysis of the cells and release of enzyme which avoids the necessity of mechanical lysis and associated energy-requiring process ' steps.
- • chloroform to cause lysis of cells expressing ⁇ RR z genes is well known in the art and has been used for many years for preparation of lambda phage from ⁇ S gene mutants (Harris, A.W., Mount, W.A., Fuerst, C.R. and Siminovitch, L., Virology, 32:553, 1967).
- Endolysin genes have also been placed on plasmids to effect cell lysis (Garret, J. et al., Mol . Gen. Genet , 182:326-331 , 1981; Crabtree, S. and Cronan, J.E., J. Bact., 158:354-356, 1984; Simons, 0. et al., Gene, 28:55-64, 1984).
- the construction of plasmid pGX2302 is illustrated in Figure 6.
- An EcoRI - Clal fragment of ⁇ cI857 Sam7 DNA base pairs 44973-46441 of the ⁇ genome, Sanger, F. et al. , J. Mol.
- the endolysin genes were subsequently moved from pGX2298 to the lyA plasmid pGX2236 again using the EcoRI site and the PstI site of the pGX1066 portion of pGX2298 to create plasmid pGX2302.
- the transcription regulation regions of the Sam, R and R z genes have been removed in the course of these plasmid constructions, there is sufficient low level expression of the R and R z genes on the plasmids to provide the lysis function.
- the lambda S gene also present has an amber mutation and thus does not produce a functional gene protein.
- Plasmids pGX2236, pGX2237 and pGX2302 contain both the glyA gene for. SHMT overproduction and a complete normally regulated tryptophan operon to complement a tryptophan operon mutation in the host. This provides a stabilization mechanism for the plasmid when tryptophan is left out of the media. Plasmid pGX2302 additionally contains the lambda RR Z genes confering autolysis capability.
- the preferred expression vectors can be used to trans ⁇ form microorganisms, which are preferably a trp mutant, to produce transformants which efficiently express SHMT and are a preferred source of SHMT for use in the process of the invention.
- a K. aerogenes strain (Gx1705), an L- serine deaminase (lsd) mutant which is also a trp mutant was transformed with pGX2236 and deposited at the American Type Culture Collection, Rockville, Maryland as ATCC No. 39408. The same strain was transformed with pGX2237 and deposited at the American Type Culture Collection, Rockville, Maryland as ATCC No. 39407.
- a nucleotide sequence coding for tryptophan synthetase or tryptophanase may be inserted into a plasmid containing the SHMT gene. Microorganisms transformed with such a plasmid will produce the enzymes for use according to the present invention.
- TS or TASE may be obtained separately from microorganisms that have been ⁇ modified using conventional genetic enginee ' ring techniques for high yield production of the respective enzyme.
- the nucleotide sequence coding for TS (trpBA) or TASE (tnaA) may also be isolated and cloned into vectors which can then be used to transform suitable host cells resulting in high level TS or TASE expression.
- the vectors may be phages or plasmids, but plasmids are preferred. Suitable hosts include, for example, Klebsiella, Salmonella and Escherichia.
- the TS or TASE may be used in the form of whole cells containing the respective enzyme, a crude extract, or as purified enzyme. In preferred embodiments, the SHMT and TS or TASE are from separate sources.
- the enzymes may be used in nonim- mobilized form, or immobilized either separately or together.
- the cofactor THF may also be in nonimmobilized or immobilized form, as it retains its activity when immobil ⁇ ized. Immobilizing THF by attaching it to a support which can be retained inside a bioreactor used for carrying out the reaction is advantageous, for it enhances repeated use of the cofactor durinq the process of the invention.
- THF can be immobilized with soluble polymers, such as dextran, polyethylene glycol, or polyethyleneimine. Immobilization takes place by means of covalent attachment with dextran and polyethylene glycol and by ionic interaction with polyethyleneimine. Covalent attachment generally occurs through the carboxy groups of the THF with an amino group of the support. Alternatively, using similar attachment methods, the THF can also be attached to an insoluble support.
- THF degrades rapidly in the presence of oxygen. It is therefore preferred that the process of the invention be carried out under anaerobic conditions, such as in a nitrogen atmosphere. It is preferred to add a reducing agent to the reaction mixture to help prevent oxidation of THF. Any reducing agent may be employed which does not deleteriously affect the reaction process.
- Preferred reducing agents are thioglycolate, ascorbic acid and thioalcohols, including mercaptans, e.g., 1,3- dimercaptopropanol, 1 ,2-dimercaptoethane, dithiothreitol , dithioerythritol, and mercaptoethanol, L-thiazolidine-4- carboxyli ⁇ acid as well as aminoethylisothiouronium bromide.
- Particularly preferred reducing agents are mercaptoethanol, dithiothreitol, and thioglycolate. 2- mercap ' toethanol is most preferred.
- the concentration of the reducing- agent in the reaction mixture is preferably in the range of from about 0.01M to about 0.2M, most preferably from 0.02 to 0.1M.
- the reactions necessary to produce L-tryptophan from glycine, formaldehyde and indole take place in a stirred tank reactor, as agitation of the reaction mixture facilitates the reaction process.
- the enzymes serine hydroxymethyltransferase and tryptophan synthetase or tryptophanase are preferably separately prepared in individual fermentation tanks by expression in cells of microorganisms transformed with expression vectors carrying the respective nucleotide sequences coding for
- the reaction process can be conducted in the presence of any non-deleterious solvent.
- solvents include ethanol, methanol, isopropanol and dioxane.
- the substrates, i.e., glycine, formaldehyde and indole, the enzymes SHMT and TS or TASE, and the cofactor THF may be reacted together in a variety of ways.
- the enzymatic pathway believed to account for the synthesis of L- tryptophan from glycine, formaldehyde and indole is shown below: formaldehyde + THF > methylene-THF SHMT methylene-THF + glycine > L-serine + THF
- Microorganisms which are preferably L-serine deaminase mutants and trp mutants, are transformed with a plasmid such as pGX2236, pGX2237 or pGX2302, containing both the SHMT gene and the trp operon (which is under the control of its associated trp promoter-operator) .
- Transformants are selected and grown in a tryptophan deficient medium in a fermentation tank under SHMT producing conditions to high cell density.
- the cells from strains such as GX1705 are then preferably concentrated by centrifuging off the fermentation broth, and the concentrated cells are placed in a stirred tank reactor. It is preferred that the cells not be viable and have ruptured cell membranes. This may be accomplished by a mechanical means or by contacting the cells with a detergent such as cetyl-pyridinium'hydro- chloride, Triton X-100, Tween 100, NP40 and the like or a solvent such as toluene. Crude extract from mechanically lysed cells is preferred over whole cells.
- Air contact with the contents of the reactor is pre ⁇ ferably prevented by providing a layer of inert gas (such as N 2 , Argon, etc.) above the contents of the reactor and reducing reagent is introduced to reduce the dissolved oxygen in the reaction mixture.
- THF is added to the mixture in the reactor to a concentration dependent upon the conditions of the reaction.
- these conditions gener ⁇ ally include a reaction temperature of from about 4°C to about 60°C and a pH in the range of about 5 to about 10.
- the preferred reaction conditions include carrying out the reaction at a temperature of from about 20 to about 45°C and a pH of about 6 to 8.5.
- the reaction time is slowed considerably, and if the temperature rises above about 60°C the enzyme can be inactivated.
- the enzyme can be inactivated.
- THF may be added to reach a concentration greater than 50 mM.
- the preferred concentration is between 0.1 mM and 5 mM.
- THF is easily oxidized under reaction conditions. It is therefore preferable to maintain a low redox potential in the reaction mixture, preferably about -300mV.
- a reducing agent is 2-mercaptoethanol (2-ME), which can be added to the reaction mixture with the THF to achieve the desired redox potential .
- the redox potential of the reaction mixture can be monitored throughout the reaction process by use- of a redox probe, and the desired redox potential main ⁇ tained by selective titration of the reducing agent into the reaction mixture.
- the desired pH is maintained while the THF is dissolv ⁇ ing in the reaction mixture. This can be accomplished by the selective titration into the reaction mixture of a basic solution which is non-deleterious to the reaction process.
- Preferred basic solutions are metal hydroxide solutions, with potassium hydroxide solution being most preferred.
- Serine ' hydroxymethyltransferase is easily inactivated by formaldehyde. Since THF reacts with the formaldehyde, forming methylene-THF and thus protecting the SHMT from ina ⁇ tivation, no more formaldehyde is added to the reaction mixture than is necessary to react with the amount of THF present therein, thus protecting against SHMT inactivation.
- pyridoxal-5'-phosphate may also be added to the reaction. If L-serine is synthesized in the reactor over an extended period of time, the pyridoxal phosphate may be lost or inactivated, in which case pyridoxal phosphate may be added.
- the concentration of pyridoxal-5'- phosphate added to the reaction can vary from 0 to about 20 mM, as needed, and is preferably about 0.1.mM to 1 mM.
- the rate of serine production is reduced due to the accumulation of serine. This is because of possible product inhibition and/or the reverse reaction due to the accumulated serine.
- the rate of formaldehyde addition can be maintained constant by increasing SHMT concentration, or preferably, the formaldehyde addition rate can be reduced accordingly. This is done because excess formaldehyde in the solution will inactivate SHMT.
- the reaction is continued until a relatively high concentration of serine is produced, i.e., until the concentration of serine in the reaction medium exceeds 50 mM. Preferably, the reaction is continued until equilibrium is reached with a resulting serine concentration from 1M to qreater than saturation.
- microorganisms which are preferably trp mutants, are transformed with a plasmid, such as pGX2213 (ATCC 39388) containing the nucleotide sequence coding for tryptophan synthetase (the trpBA segment of the trp operon) .
- Transformants are selected and grown in a tryptophan deficient medium in a fermentation tank under TS producing conditions to high cell density. The cells are then concentrated by centrifuging off the fermentation broth. It is preferred that the cells be rendered non-viable and have ruptured membranes. This is preferably accomplished by contacting the cells with a detergent such as Tween 100, NP40, Triton X-100, cetyl- pyridinium*hydrochloride, and the like or a solvent such as toluene.
- a detergent such as Tween 100, NP40, Triton X-100, cetyl- pyridinium*hydrochloride, and the like
- the TS- ⁇ ontaining cells are then added to the reaction mixture containing a high concentration of serine and very little formaldehyde.
- Indole is mixed with a water mis ⁇ ible organic solvent, such as dioxane, ethanol, and the like, and slowly added to the reaction mixture.
- Indole tends to inactivate tryptophan synthetase. It is therefore preferable to keep the free indole concentration as low as possible in the reaction mixture.
- TS When TS is present in the reaction mixture as non-viable whole cells havinq ruptured cell membranes, (the preferred form), the TS tends to be protected from indole inactivation by its presence in the cells.
- cell lysates can also be used.
- tryptophanase is used instead of tryptophan synthetase to convert L-serine and indole to L-tryptophan.
- a source of trypto ⁇ phanase microorganisms are transformed with a plasmid, such as pGX2308, containing the tnaA gene, trpED genes for plasmid stabilization and lambda endolysin genes RR Z .
- plasmid pGX2308 The construction of plasmid pGX2308 is illustrated in Figure 7.
- the lambda R and R z genes were subcloned from plasmid pGX2294 (discussed above and illustrated in Figure 6) through use of a Rsal site located 58 base pairs 5' to the initiation codon of the R gene and the unique Hindlll site of pGX2294 just 3' of the lambda R z gene.
- the Rsal- Hindlll fragment was inserted in pGX1066 to create pGX2300.
- the lambda RR Z genes from pGX2300 were moved on an Xbal- Hindlll fragment to between the Xbal and Ncol sites of plasmid pGX2287 (NRRL B-15788) to create pGX2301.
- Plasmid PGX2301 designed for chymosin production has conveniently 1inked lambda RR Z genes and trpED genes that can be utilized for plasmid stabilization.
- Plasmid pGX2308 has the desirable characteristic of being stable in a trpED mutant host such as GX1734 (F ⁇ , ⁇ trpED102, tna2) when grown in media without tryptophan or indole.
- Cells containing plasmid pGX2308 overproduce tryptophanase. Any trpED mutant host cell with good growth characteristics would be suitable.
- the E. coli tryptophanase gene is regulated by catabolite repression (J.L. Botsford and R.D. DeMoss, j ⁇ Bacteriology, 105:303-312, 1971; D.F. Ward and M.D. Yudkin, J. gen.
- a detergent such as Tween 100, NP40, Triton X-100, cetyl- pyridinium'hydro ⁇ hloride, and the like or a solvent such as toluene, methylene chloride or chloroform.
- a detergent such as Tween 100, NP40, Triton X-100, cetyl- pyridinium'hydro ⁇ hloride, and the like or a solvent such as toluene, methylene chloride or chloroform.
- TASE-containing cells or cell lysate is then added to the reaction mixture containing a high concentration of serine and very little formaldehyde. Since tryphtophanase is quite stable in the presence of hiqh concentrations of indole, solid indole is directly mixed with serine solution. Indole can also be mixed 'with a water miscible organic solvent, such as dioxane, ethanol, and the like, and slowly added to the reaction mixture in a similar manner as described above in connection with the use of TS as catalyst.
- a water miscible organic solvent such as dioxane, ethanol, and the like
- indole When using TS as catalyst, indole is preferably added to the reaction mixture continuously, as it is used in the synthesis of L-tryptophan from serine. As the L-trvOtoohan is synthesized, it precipitates out of the reaction mixture. When very little serine remains.in the reaction mixture, indole addition is halted and the remaining indole is allowed to react. When using TASE as catalyst, the calculated amount of indole is directly charged into the reaction mixture.
- the L-tryptophan which has precipitated out of the reaction mixture and collected on the bottom of the reac ⁇ tion chamber can then be removed by any convenient means of separation.
- Glycine and formaldehyde may then be added to the reaction mixture as described above to begin the process of serine synthesis again (supplementation with additional SHMT enzyme and PLP and THF cofactor may be desirable) .
- Tryptophan synthetase and tryptophanase are less sensitive to formaldehyde inactivation than SHMT. The avoidance of excess formaldehyde in the reaction mixture by not adding more formaldehyde than can be reacted with the amount of THF present will protect the TS or TASE against formaldehyde inactivation during serine synthesis phase of the reactor.
- Plasmids pGX2236 and pGX2237 were produced as illustrated in Figure 1 and described below. Plasmid pEP392 is described in detail in Enger-Valk, B.E. et al. , Gene 9:69-85 (1980). Plasmid pGX110 is essentially identical to pHP12, described in the same reference.
- Plasmid pGx110 (containing the nucleotide sequence coding for the trpEDCBA genes regulated by the trp promoter, an Xhol restriction site upstream of the trp promoter, and an EcoRI restriction site upstream of the Xhol site) was linearized by cleavage with Xhol.
- Plasmid pGX139 (containing the nucleotide sequence coding for glyA, a Sail restriction site downstream from the glyA gene, and a EcoRI restriction site upstream of the glyA gene) was linearized by treatment with Sail, and ligated to the linearized pGX110 plasmid using DNA ligase at a high DNA concentration (approximately 1000 ⁇ .g/ml) . The resulting ligation mix was digested with ⁇ cjoRI producing short linear DNA ligation products. The ligation products were circularized using DNA ligase at low DNA concentration (approximately 10 ⁇ g/ l).
- Plasmid pGX139 was linearized by treatment with Sail and ligated to Xhol cut pEP392 using DNA ligase at high DNA concentration (approximately 1000 ⁇ g/ml). The resulting high molecular weight ligation polymer was digested with EcoRI producing short linear DNA ligation products.
- the ligation products were circularized using DNA ligase at low DNA concentration (approximately 10 ⁇ g/ml). In each case, the high concentration ligation at the first step produced long concatenates of the two plasmids . These were cut to the desired size by the secondary EcoRI digestion. The second ligation * at low DNA concentration promoted the formation of circles for efficient transforma- tion. Cells with a mutation in the trp operon were transformed with the ligation mix using a calcium shock procedure and selected for growth on media without tryptophan.
- the trp "1" transformants were then screened for the predicted increase in plasmid size and high specific activity SHMT production to distinguish between reclosure of the starting trp plasmids and the desired recombinants .
- Isolates from both ligations were identified and named pGX2236 and pGX2237. Plasmid pGX2236 also confers ampicillin resistance on the cells.
- the plasmids were then used to transform Klebsiella aerogenes GX1705.
- This strain is a tryptophan synthetase mutant created by nitrosoquanidine mutation of GX1704 (a Klebsiella aerogenes strain defective in L-serine deaminase) and screening for mutants able to grow with tryptophan supplementation but not with indole supplementation.
- Example II Construction of the plasmid pGX2213 In order to create a plasmid for the expression of the tryptophan synthetase (trpBA) gene, the trpBA gene was subcloned into a plasmid with convenient restriction sites, pGX2208 (see Fig. 2). Plasmid pGX110 was digested with Bglll and religated at low concentration (approximately 10 ⁇ g/ml) in the presence of T4 ligase to delete three DNA fragments including part of trpE, all of trpD and part of trpC.
- the resultant plasmid identified as pGX112, contained only functional trpBA genes that code for the beta and alpha subunits of tryptophan synthetase (TS) and retained the normal trp promoter and regulatory region.
- TS tryptophan synthetase
- Plasmid pGX112. was digested with Bglll .
- Plasmid pBR322 was digested with BamHI.
- BamHI and Bglll produce " complementary ends.
- the two linearized plasmids were ligated in the presence of T4 ligase at high DNA concentra- tion (approximately 1000 ⁇ g/ml) in 10 ⁇ l .
- the resultant ligation mixture was digested with Sail and then recircu- larized in the presence of T4 ligase at a concentration of approximately 10 ⁇ g/ml to produce plasmid pGx141, which contained a portion of the trpC gene and all of trpBA inserted between the BamHI and Sail sites of pGX2322. Plasmid pGXl41 was digested with Hindlll.
- the small fragment comprising all of trpC and the first 89 bases of trpB was deleted and the large fragment was then recircu- larized by ligation at a concentration of approximately 10 ⁇ g/ml in the presence of T4 ligase to produce plasmid pGX143, containing the 3' end of trpB and all of trpA.
- a 57 ⁇ g aliquot of plasmid pGX141 was digested within Haelll .
- the fragment containing the Hindlll recognition site was isolated on a 5% acrylamide gel.
- the resultant plasmid, pGX149 contained the trpBA gene and an EcoRI site immediately upstream of the gene at the original location of the last Haelll site in trpC.
- Plasmid pGX149 (5 ⁇ g) was digested with EcoRI.
- Two 10 base-pair synthetic oligodeoxynucleotides having the sequences 5'-GATCCTCGAG-3 ' and 5'-AATTCTCGAG-3 were phosphorylated with T4 polynucleotide kinase and ATP and then ligated to the EcoRI linearized plasmid in the presence of T4 ligase to produce the plasmid ⁇ GX2208 " .
- This plasmid pGX2208 contains the trpBA gene and several restriction sites .ordered EcoRI, Xhol, BamHI, Xhol, EcoRI just upstream of the gene created by the synthetic DNA.
- Plasmid pGX2208 was digested with BamHI. Plasmid pGX145 was also digested with BamHI . Plasmid pGXl45 (see Fig. 3) is a very close analog of pGXl34 (E. col ⁇ L strain GX1045 transformed with pGX134 is on deposit at the American Type Culture Collection as ATCC 39037). Plasmid PGX145 carries a trp/lac hybrid promoter similar to a described trp/lac promoter (DeBoer et _al. , in "From Gene to Protein:Translation into Biotechnology" , Miami Winter Symposium, Academic Press, New York (1982) pp. 209-327).
- the two linearized plasmids pGXl45 and pGX2208 were ligated in a volume of 20 ⁇ l at a concentration of approximately 1000 ⁇ g/ml in the presence -of T4 ligase and the resultant ligation mixture was digested with Sail to remove unwanted DNA sequences. The digested ligation mix was then recircularized at a concentration of approximately 10 ⁇ g/ml in the presence of T4 ligase to produce pGX2213.
- This plasmid which contains the trpBA gene under the control of a trp/lac hybrid promoter, can be employed to transform microorganisms which will serve as a source of TS in the process of the invention.
- Plasmid pGX2214 an expression vehicle for the trpBA gene, was prepared starting with pGX2208 (see Example II) and pGW7 (see Fig. 4). Plasmid pGW7 was constructed by first cloning base pairs 34500-39168 of ⁇ cI857 cro12 on an EcoRI-BamHI fragment into the EcoRI and BamHI sites of pBR322 then deleting base pairs 35712-38814 of the original ⁇ DNA by partial Bglll digestion and re-ligation causing a deletion in cro and 0 genes and all of the ell gene.
- Plasmid pGW7 was digested with Hpal.
- An 8 base-pair synthetic oligodexoynucleotide linker containing a BamHI recognition site was ligated to the linearized plasmid at low plasmid concentration (approximately 10 ⁇ g/ml) and high linker concentration in terms of DNA ends (5 ⁇ g/ml).
- a ligation mix was then digested with BamHI and recircularized by ligation in the presence of T4 ligase at low DNA concentration (approximately 1 ⁇ g/ml) removing the original Hpal-BamHI fragment leaving a unique BamHI site.
- the new plasmid was identified as pGX2204.
- Plasmid pGX2208 (see Fig. 2) (5 ⁇ g) and plasmid pGX2204 (5 ⁇ g) were digested with BamHI.
- the linearized plasmids were ligated in the presence of T4 ligase at high DNA concentration (approximately 1000 ⁇ g/ml), digested with Sail to remove unwanted DNA sequences, and religated at low concentration (approximately 10 ⁇ g/ml) to produce pGX2214.
- This plasmid codes for a fusion protein between a small remaining amino terminal portion of ⁇ N protein and the small remaining carboxy terminal portion of the trpC protein.
- Plasmid pGX2214 can be used to transform a host microorganism, which can serve as a source of TS in the process of the invention when transcription is induced from the ⁇ P L promoter.
- Plasmid pGXl50- containing the trpBA gene under the control of the trp promoter, was produced in the manner illustrated in Fig. 5.
- Plasmid pGX149 (5 ⁇ g), which is described in Example II, was digested with EcoRI and treated with bacterial alkaline phosphatase to prevent recircularization without insertion of the promoter sequence.
- trp promoter EcoRI fragment from pGX99 was then ligated to the linearized pGX149 in the presence of T4 ligase to produce pGX150.
- the structure of pGX150 was confirmed by endonuclease digestion analysis.
- Plasmid pGX150 was used to transform Klebsiella aerogenes GX1705.
- the tryptophan synthetase mutation in the GX1705 chromosome allows stable maintenance of the plasmid in cells maintained in growth in media without tryptophan.
- the transformation has been deposited with the
- Example V Production of L-tryptophan from glycine, formaldehyde, and indole in the presence of tetrahydrofolic acid, transformants containing pGX2236 and transformants containing pGX150
- Cells were collected by centrif gation and served as a source of serine hydroxymethyltransferase biocatalyst .
- reaction mixture containing 7.00 g glycine, 0.7 M pyridoxal-5*-phosphate, 10 mM tetrahydrofolic acid and Klebsiella aerogenes GX1705 (pGX2236) collected from 150 ml culture medium, a linear gradient of decreasing formaldehyde concentration (formed with 8.5 ml formaldehyde and 8.5 ml water and a gradient forming apparatus) was added at a flow rate of 0.4 ml per hour. The pH was controlled at 8.0 with 7.0 M KOH and temperature was maintained at 37°C.
- the reaction mixture contained 0.1 M 2-mercapethanol initially and 2- mercaptoethanol (6N) was added to the reaction mixture at a constant rate of 0.01 ml per hour. A flow of N 2 gas over the reaction mixture was used to exclude 0 2 from the reactor. Aliquots of the SHMT reaction were removed at 10 and 25 hours for initiation of the tryptophan synthesis phase of the reaction.
- the reaction where the tryptophan syntehsis phase was initiated at 10 hours demonstrates tryptophan can be synthesized even at a low serine to glycine ratio.
- the reaction where the tryptophan synthesis phase was initiated at 25 hours provides tryptophan at higher yield and concentration.
- Plasmid pBR322 DNA (9 ⁇ g) was digested with EcoRI in a 100 ⁇ l volume.
- a ligation (25 ⁇ l volume) with T h DNA ligase (total DNA 200 ⁇ g/ml and a molar ratio of 1:5 or 1:10 of EcoRI cut pBR322 DNA:EcoRI cut ⁇ DNA) was performed. An aliquot of the ligations was digested with Clal, then ligated again at a DNA concentration of approximately 0.2 ⁇ g/ml. The second ligation mixture was used to transform _ coli DH1 (F ⁇ , recAl , endA1 , gyrA96, thi-1 , hsd_R17, supE44, relAl , ⁇ ⁇ ) selecting for ampicillin resistance.
- Transformants with the proper sized plasmids and the predicted endonuclease digestion pattern with EcoRV, Hindlll and Narl for the insertion of the EcoRI-Clal fragment of ⁇ (bp 44973-46441 ; see Sanger et al. , J. Mol. Biol. , 162:729-773, 1982) into the EcoRI-Clal sites of pBR322 were selected.
- One such plasmid was named pGX2294.
- the lambda DNA fragment was further subcloned from pGX2294 into PGX1066 (ATCC 39955) " to create plasmid pGX2298.
- Both plasmids pGX1066 (10 ⁇ g) and pGX2294 (10 ⁇ g) were digested in 100 ⁇ l volume with EcoRI.
- An initial ligation contained 5 ⁇ g EcoRI cut pGx1066 DNA and 5 ⁇ g EcoRI cut pGX2294 DNA and ⁇ DNA ligase in a 20 ⁇ l volume.
- the ligation product was cut with BamHI, and 2.5 ⁇ g of the BamHI digested DNA was ligated in a 200 ⁇ l reaction.
- the ligation mixture was used to transform coli and ampicillin resistant colonies were selected.
- Cells with plasmids of approximately the correct size (5197 bp) for insertion of the EcoRI-BamHI fragment containing the RR Z genes from pGX2294 into the BamHI and EcoRI sites of pGX1066 were selected.
- Constructs of the desired type were verified by digestion with BamHI, EcoRI, EcoRV, Xbal and Hindlll, and one was labeled pGX2298.
- the unique EcoRI and PstI sites of pGX2298 were used to remove the ⁇ RR Z genes and insert them into pGX2236.
- EcoRI digets in a 200 ⁇ l volume with 3.15 ⁇ g of pGX2236 and 7.5 ⁇ g of pGX2298 were performed.
- a ligation with 2.5 ⁇ g of EcoRI digested pGx2236 and 2.5 ⁇ g of EcoRI digested pGX2298 in 20 ⁇ l volume was performed.
- the ligation product was digested with PstI and 1 ⁇ g of the PstI digest product was ligated in a volume of 150 ⁇ l to allow recircularization.
- the ligation mixture was used to transform E.
- coli AE-1 ( ⁇ trpEDCBA) and transformants able to grow on minimal medium without tryptophan were selected.
- the plasmid in one such transfor ant was named pGX2302.
- the plasmid pGX2302 was transformed into Klebsiella aerogenes GX1705 (lsd, trp) and deposited with the American Type Culture Collection, Rockville, Maryland (ATCC 53052)..
- the transformants are the preferred sources of SHMT for use in the process of the invention.
- Example VII Construction of plasmid pGX2308 The construction of plasmid pGX2308 is illustrated in Figure 7. The objective was to provide trpED stabilization to a plasmid containing the tryptophanase gene (tnaA) as well as the lambda endolysin genes ( ⁇ RR z ). ' In order to describe exactly how this was performed, the consruction of two precursor plasmids are first described. The first, pGX2300, is a plasmid containing only lambda RR Z genes surrounded by convenient restriction endonuclease sites .
- Plasmid pGX2294 (illustrated in Figure 6 and described in Example VI) has five Rsal sites, and one of these sites is 58 base pairs 5' of the ATG initiation codon of the R gene within the S gene. Since the S gene has an amber mutation and S gene function is not desired in the plasmid, the Rsal site was used to subclone the RR Z genes into pGX1066 (present in strain GX1186, ATCC 39955). Plasmid pGX2294 (15 ⁇ g) was digested with Rsal in a 75 ⁇ l reaction volume and pGx1066 DNA (10 ⁇ g) was digested with Smal in a 50 ⁇ l reaction volume. A ligation was performed using Tj.
- DNA ligase in 20 ⁇ l with 2.5 ⁇ g of Rsal digested pGX2294 and 2.5 ⁇ g of Smal digested pGXl066. Both Rsal and Smal endonucleases produce blunt-ended DNA fragments and thus the different DNAs readily ligated together.
- the ligation product was digested with Hindlll , then ligated at low DNA concentration (less than 10 ⁇ g/ml) to allow recircularization of the plasmid DNA. The ligation mixture was used to transform E_ ⁇ coli and colonies resistant to ampicillin were selected.
- Both DNAs (7.8 ⁇ g Ncol digested pGX2287 DNA; 3.6 ⁇ g Hindlll digested pGX2300 DNA) were treated with 25 units of E ⁇ coli DNA polymerase I (Poll) in a reaction volume of 200 ⁇ l containing 0.25 ⁇ M dATP, dTTP, dGTP and dCTP for 30 minutes at room tempera ⁇ ture to fill in the single-stranded DNA ends left by Ncol and Hindlll digestion.
- E ⁇ coli DNA polymerase I Polymerase I
- a high DNA concentration ligation was performed containing T ⁇ DNA ligase, 3.9 ⁇ g of the NcoI digested-PolI treated pGX2287 and 1.55 ⁇ g of the Hindlll digested-PolI treated pGX2300 DNA in a 30 ⁇ l volume. All the resulting ligation product was digested with Xbal in a 100 ⁇ l volume, then ligated at a relatively low DNA concentration (200 ⁇ l volume) to cause recircularization. The ligation mixture was used to transform E ⁇ coli GX1731 3 U
- the tryptophanase gene (tnaA) from E_ ⁇ coli has been cloned and sequenced (M.C. Deeley and C. Yanofsky, J. Bacteriology, 147:787-796, 1981) and is available on plasmid pMD6.
- the tnaA gene from pMD6 was inserted into plasmid pGX2301 in order to obtain a composite plasmid with the tnaA, trpED and ⁇ RR Z genes.
- Plasmid pMD6 (5.0 ⁇ g) and plasmid pGX2301 (4.9 ⁇ g) were each digested with Pv l in a 100 ⁇ l reaction volume.
- the DNAs were ligated (2.5 ⁇ g of Pvul digested pGX2301, 1 ⁇ g of Pvul digested pMD6) with T[. ligase in a 20 ⁇ l reaction volume.
- the ligation product was digested with BamHI.
- Some of the resultant DNA (2.1 ⁇ g) was ligated in a volume of 200 ⁇ l to allow recirculari ⁇ zation of the DNA.
- the ligation mix was used to transform E ⁇ coli GX3021 ⁇ F ⁇ - , tna2, nadA: :Tn10, AtrpEDCBA [ ⁇ cI857
- Klebsiella aerogenes strain GX1705 (pGX2236), ATCC 39408 and GX1705 (pGX2302), ATCC 53052 were grown following the procedures described in Example V.
- Strain GX1705 (pGX2302) has endolysin genes on the plasmid and autolysis of this strain was demonstrated in the following experiment.
- organic solvent CHC1 3 or CH 2 C1 2
- SHMT activity in the supernatant after removal of cell debris by centrif gation was determined after 8 hours incubation.
- the crude SHMT extract , f rom E . coli GX1 705 ( pGX2302 ) which was incubated for 8 hours in the presence of 2% CHC1 3 were collected after removing cell debris by centrifugation and was used to produce serine following the procedure described in Example V. After 25 hours reaction, 2.2 M serine with remaining 0.6 M glycine was produced.
- the crude SHMT extract from E ⁇ _ coli GX1705 (pGX2302) which was incubated for 8 hours in the presence of 2% CH 2 C1 2 was prepared as described above and was used to produce serine following the procedures described in Example V. After 24 hours reaction, 2.3 M serine with a remaining 0.55 M glycine was produced.
- Example IX Serine hydroxymethyltransferase (SHMT) was prepared as described in Example V. L-serine was produced in a reaction using the SHMT biocatalyst as in Example V. The serine-producing reaction was run for 25 hours.
- SHMT Serine hydroxymethyltransferase
- Tryptophanase was prepared by the following method .
- One colony of E. coli strain GX1734 (pGX2308) was inoculated into 200 ml of culture medium III described below and was shaken at 37°C overnight. Cells were collected by centrif gation and served as a source of tryptophanase biocatalyst.
- Culture Medium III K ⁇ PO ⁇ 1 0 , . 5 g/i KH 2 PO ⁇ 4. . 5 g/i (NH ⁇ JgSO ⁇ 1 g/i
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Abstract
Procédé de synthèse de L-tryptophane à partir de glycine, formaldéhyde et indole, utilisant des quantités biocatalytiques de sérine hydroxyméthyltransférase, d'acide tétrahydrofolique et de tryptophane synthétase ou tryptophanase. La sérine hydroxyméthyltransférase et la tryptophane synthétique ou tryptophanase sont ajoutées au milieu de réaction de préférence sous la forme de produits d'expression de microorganismes contenant des vecteurs d'expression codant pour la sérine hydroxyméthyltransférase et la tryptophane synthétase ou tryptophanase.Process for the synthesis of L-tryptophan from glycine, formaldehyde and indole, using biocatalytic amounts of serine hydroxymethyltransferase, tetrahydrofolic acid and tryptophan synthetase or tryptophanase. Serine hydroxymethyltransferase and synthetic tryptophan or tryptophanase are added to the reaction medium preferably in the form of expression products of microorganisms containing expression vectors coding for serine hydroxymethyltransferase and tryptophan synthetase or tryptophanase.
Description
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US71261285A | 1985-03-18 | 1985-03-18 | |
US712612 | 1985-03-18 |
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DE3882394T2 (en) * | 1987-03-03 | 1993-12-16 | Light Oil Utilization Res Ass | Plasmids. |
GB8819135D0 (en) * | 1988-08-11 | 1988-09-14 | Celltech Ltd | Tryptophan production |
WO2007095682A1 (en) * | 2006-02-24 | 2007-08-30 | The Australian Wine Research Institute | Methods and micro-organisms for modulating the conversion of non-volatile sulfur compounds to volatile thiol compounds |
CN111961656B (en) * | 2020-09-03 | 2022-07-22 | 中国科学院上海高等研究院 | Mutant of serine hydroxymethyl transferase and application thereof |
CN115261365B (en) * | 2022-06-22 | 2024-01-30 | 南通紫琅生物医药科技有限公司 | Tryptophan synthase mutant and application thereof |
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FR2263302A1 (en) * | 1974-03-05 | 1975-10-03 | Bofors Ab | |
GB2048266A (en) * | 1979-05-09 | 1980-12-10 | Mitsui Toatsu Chemicals | Enzymatic preparation of l-tryptophan |
GB2130216A (en) * | 1982-11-19 | 1984-05-31 | Genex Corp | Enzymatic synthesis of L-serine |
-
1986
- 1986-03-17 EP EP19860902176 patent/EP0217862A4/en not_active Withdrawn
- 1986-03-17 JP JP50167986A patent/JPS62502934A/en active Pending
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FR2263302A1 (en) * | 1974-03-05 | 1975-10-03 | Bofors Ab | |
GB2048266A (en) * | 1979-05-09 | 1980-12-10 | Mitsui Toatsu Chemicals | Enzymatic preparation of l-tryptophan |
GB2130216A (en) * | 1982-11-19 | 1984-05-31 | Genex Corp | Enzymatic synthesis of L-serine |
Non-Patent Citations (3)
Title |
---|
BIOTECHNOLOGY & BIOENGINEERING, vol. 26, no. 4, April 1984, pages 308-314, John Wiley & Sons, Inc., New York, US; U. BEHRENDT et al.: "The production of L-serine with a methylotrophic microorganism using the L-serine pathway and coupling with an L-tryptophan-producing process" * |
JOURNAL OF GENERAL MICROBIOLOGY, vol. 130, 1984, pages 3091-3100, SGM, GB; G.S. SKOGMAN et al.: "Factors affecting the biosynthesis of L-tryptophan by genetically modified strains of Escherichia coli" * |
See also references of WO8605515A1 * |
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