TRYPTOPHAN PRODUCTION

Abstract: A multi-stage microbial process for the production of tryptophan is described where the biocatalyst production and bioconversion stages are separated. In the biocatalyst production stage bacterial host cells are transformed with a vector containing a DNA sequence coding for tryptophanase wherein the expression of the tryptophanase gene is directly controllable; the transformed host cells are induced to synthesise tryptophanase; and then in a subsequent bioconversion stage the reaction substrates for tryptophan synthesis are added and the tryptophan which accumulates in the reaction mixture is optionally isolated.

Description: TRYPTOPHAN PRODUCTION Field of the Invention This invention relates to a process for the production of tryptophan. In particular, the invention relates to a multi-stage microbial process for the production of tryptophan. Background to the Invention L-Tryptophan, hereinafter referred to as tryptophan, is an aromatic amino acid which is essential for human and animal nutrition. It is widely used in the pharmaceutical and food industries and is of potential use as an additive to animal feeds. In attempts to try to satisfy the demand for tryptophan a number of different fermentation based production methods have been developed. To date, however, there is no satisfactory process for the large scale production of tryptophan that produces tryptophan in high yield and at a high rate. Bacterial synthesis of tryptophan is catalysed by a series of enzymes which are expressed by a cluster of genes which make up the tryptophan operon. The last stage in the process is catalysed by tryptophan synthase which in the bacterial cell catalyses the reaction of L-serine and indole glycerol phosphate to yield tryptophan. Tryptophan synthase is also able to catalyse the reaction of indole and L-serine for tryptophan production i_n vitro. In order to drive the reaction in vitro in the anabolic direction i.e. towards tryptophan synthesis, it is necessary to use a high concentration of one of either serine or indole. Serine, however, is an expensive raw material and is thus unsuitable if the process is to be economic. The other enzyme substrate indole is toxic to bacteria at low concentrations and it is therefore disadvantageous to use the indole concentration as the driving force for the synthetic reaction in bacteria. There also exists in the bacterial cell the catabolic enzyme tryptophanase which catalyses the breakdown of L-tryptophan to indole, pyruvate and ammonia. Ammonia is an inexpensive raw material, the concentration of which can be used to drive the reaction in the reverse direction i.e. towards tryptophan synthesis. There are, therefore, potential economy advantages in using tryptophanase as the enzyme to catalyse the production of tryptophan. There are, however, practical difficulties associated with operating a process which uses tryptophanase. The use of bacteria producing high levels of tryptophanase is disadvantageous: the commonly used and inexpensive bacterial growth media contain tryptophan, which is broken down in the presence of tryptophanase to yield indole. However, as indole is toxic to bacteria at low concentrations, any increase in indole concentration is thus extremely undesirable. In order to overcome the indole toxicity problem, known processes have used surfactant in the medium. The surfactant acts to "partition" the indole from the growth medium, thereby minimising the concentration of indole to which the bacteria are directly exposed. Our work has, however, shown that the presence of surfactant may have an adverse effect on the tryptophanase. In addition the presence of surfactant leads to the establishment of a two phase system which makes the overall process, especially the downstream processing of the tryptophan produced, more difficult to operate. As an alternative to the commonly used bacterial growth media, tryptophan negative growth media are available in which bacteria producing tryptophanase can be grown. Such media are, however, expensive, and their use does not provide an economic process for the production of tryptophan. The process of the present invention overcomes the problems associated with known processes and leads to an economic process which produces tryptophan in good yields and at higher rates than previously obtained. Summary of the Invention Thus, according to a first aspect the invention provides a process for the production of tryptophan comprising the steps of: a) in a first, biocatalyst production stage, propagating bacterial host cells transformed with a vector containing a DNA sequence coding for tryptophanase wherein the expression of said tryptophanase gene is directly controllable; b) inducing said host cells to synthesise tryptophanase by inducing expression of the tryptophanase gene; c) in a subsequent bioconversion stage adding reaction substrates for tryptophanase to a reaction mixture containing said host cells induced for tryptophanase synthesis such that tryptophan accumulates in the reaction mixture; and d) optionally isolating the accumulated tryptophan The process of the invention separates the biocatalyst production and bioconversion stages. This has the practical advantage that in the biocatalyst production stage bacterial growth can be optimised and high levels of tryptophanase produced in the absence of indole. In the subsequent bioconversion stage the tryptophan synthetic reaction can be optimised without the need to consider effects on cell growth and without having to take measures to overcome indole toxicity. In this way, the process of the invention, unlike known processes, advantageously does not require the addition of surfactants to achieve high conversion rates. In the process of the invention the level of tryptophanase is controlled such that expression of the tryptophanase gene and hence production of tryptophanase can be switched on, for example by a change in temperature or a change in the concentration of a metabolite or metabolite analogue, when the bacteria have reached the required stage of cell growth. A particularly efficient way of achieving such control is by use of host cells containing a dual origin vector system as described in UK Patent No. 2136814B. In the dual origin vector system, host bacteria transformed with a vector containing a DNA sequence coding for tryptophanase are grown at one temperature at which replication of the vector is controlled from a stable low copy number origin of replication. When the bacteria have reached a desired stage of growth, the temperature is raised and replication of the vector is controlled from a second high copy number origin of replication. The increase in the copy number results in an increase in the level of tryptophanase produced. Other controllable systems may also be used, for example the runaway replication vectors described in European Patent No. 3062B. The expression of tryptophanase may also be controlled by the use of controllable promoters, for example the lac promoter/operator system. Tryptophanase is a tetrameric protein composed of four identical subunits encoded by the tnaA gene. Active tryptophanase (holoenzyme) requires pyridoxal-5*-phosphate to be associated with the protein (apoenzyme). The DNA sequence coding for tryptophanase may be obtained from any suitable bacterial source such as for example a strain of Escherichia coli or Proteus rettseri using conventional methods. We have found that tnaA DNA sequence from plasmid pMD.6 (Deeley and Yanofsky (1981) J. Bacteriol 1A7 (3) 787-796) to be particularly suitable. We have further found that incorporation of the tryptophanase DNA sequence into the dual origin vector pMG196 (see UK Patent No. GB 2136814B) to give plasmid pIMS1015 enables high level controlled expression of tryptophan to be achieved. Dual origin expression vectors containing a tryptophanase DNA sequence such as plasmid pIMS1014 and plasmid PIMS1015 form a further feature of this invention as do host cells transformed with such vectors. Suitable hosts for expression of tryptophanase may be any gram-positive or gram-negative bacterial cell. The use of a gram-negative host such as E. coli is preferred. E. coli MD33 [Deeley, M.C. and Yanofsky, C. , J. Bacteriol. 147. (3), 787-796 (1981)] is a particularly preferred host. Other particularly useful hosts include strains of E. coli that are capable of over-producing compounds of the vitamin B - type. Such strains include E. coli o WG497 and, in particular, E. coli AT1024 [Dempsey, W.B. and Acrement, L.T. (1971) J. Bacteriol. 107., 580-582]. The biocatalyst production and bioconversion stages may be carried out in separate reaction vessels. Thus, at the end of the biocatalyst production stage the bacteria are preferably transferred to a second bioconversion stage reaction vessel, to which the reaction substrates are added. In between the biocatalyst production stage and the bioconversion stage the bacteria may, if desired, be transferred to at least one holding tank. The provision of at least one holding tank in which the bacteria induced for tryptophanase production are held prior to the bioconversion stage is advantageous since it allows the process to operate with maximum efficiency since the second bioconversion stage may be operated continuously. The bioconversion stage may alternatively be operated as a batch or fed batch process. The bacteria used in the process of the invention may if desired be immobilised using conventional techniques such as, for example, entrapment in a polyacrylamide gel matrix. In this case the bacteria are preferably immobilised after tryptophanase expression has been induced and after transfer to a holding tank. The reaction substrates for tryptophanase, added at the bioconversion stage, include indole, pyruvic acid or a salt thereof, e.g. sodium pyruvate, and ammonia or a salt thereof, e.g. ammonium acetate. Alternatively, indole and L-serine or a salt thereof may be used. Preferably, the reaction substrates are indole, pyruvic acid or a salt thereof and ammonia or a salt thereof. We have found that the rate of tryptophan production is improved by dissolving any indole reaction substrate in a water miscible organic solvent to a high concentration and then pumping this solution into the reaction vessel at a controlled rate such that the indole concentration in the vessel does not exceed lOmM. The water miscible organic solvent may be, for example, an alcohol such as a C. . alkanol, e.g. methanol, ethanol or propanol; or a ketone, e.g. dimethyl ketone. The indole is dissolved in the water miscible organic solvent at as high a concentration as is possible such that the concentration of the water miscible solvent in the reaction mixture does not adversely affect the tryptophanase activity. Suitable concentrations of indole may be determined empirically. For ethanol, we have found that the concentration of indole in ethanol which may be used may be from 0.4M up to the solubility limit for indole in ethanol. The use of 5M indole in ethanol is particularly suitable. Thus, in a preferred embodiment, the invention provides a process for the production of tryptophan comprising the steps of: a) in a first biocatalyst production stage propagating host bacterial cells transformed with a vector containing a DNA sequence coding for tryptophanase wherein the expression of said tryptophanase gene is directly controllable; b) inducing said host cells to synthesise tryptophanase by inducing expression of the tryptophanase gene; c) in a subsequent bioconversion stage adding indole and other reaction substrates for tryptophanase to a vessel containing a reaction mixture comprising said host cells induced for tryptophanase synthesis such that tryptophan accumulates in the reaction mixture, the indole, before addition, being dissolved in a water miscible organic solvent to a high concentration and then being pumped into the vessel at a controlled rate, such that the indole concentration in the vessel does not exceed lOmM; and d) optionally isolating the accumulated tryptophan. The indole is preferably dispersed throughout the liquid in the bioconversion vessel at a substantially uniform concentration. This, together with the concentration of the indole ensures that the majority of the bacterial population is exposed to an optimum level of indole such that the activity of the tryptophanase, and hence the specific production rate of the process, is maximised. The exact concentration of reaction substrates used in the process according to the invention may be varied according to the amount of tryptophanase present and the reaction conditions used and can be determined empirically beforehand using small-scale tests in accordance with conventional practice. In general, however, where indole, pyruvate and ammonia are in use, the ammonia will be present in excess quantity to that of the other substrates. The concentration of ammonia or a derivative thereof such as a salt, e.g. ammonium acetate, may for example be from 0.1M to 7M, preferably from 0.5M to 2.5M. In the process according to the invention, the host cell, e.g. E. coli MD33, is initially transformed with a plasmid encoding tnaA, e .g. plasmid pIMS1015, and is then grown in a suitable vessel under conditions which maintain the plasmid in a low copy number state e.g. at a temperature of around 30 C . Any suitable bacterial growth medium may be used in accordance with conventional practice. Once the desired cell density is reached, the culture conditions are altered e.g. the temperature of the medium is increased to 37 C or above, to induce an increase in the copy number of the plasmid, with a concomitant rise in the amount of the tryptophanase produced. Growth may then be continued into the late exponential phase, whereupon the bacteria may, if desired, be harvested, e.g. by centrifugation. Depending on the organism and plasmid used, this protocol may result in the synthesis of tryptophanase at levels of at least 15% up to 90% total cell protein. Where the bacterial cells containing tryptophanase have bee harvested, a suspension of these, or an extract thereof, are adde to a further vessel containing substrates, e.g. indole, sodiu pyruvate and ammonium acetate, together with any necessary enzyme cofactors, e.g. ρyridoxal-5*-phosphate, at a pH in the range pHδ.O to ρHll.0 e.g. around pH8.5, at a temperature in the range 10 -60 C e.g 20 -50 C. In a preferred alternative method, however, the indole is added to the reaction mixture containing enzyme, pyruvate and ammonia at a controlled rate throughout the bioconversion process, for example as described above. A substantially similar procedure may be followed when the bacterial host cells are not harvested, except that substrates and co ctors are added as appropriate to the initial fermentation broth. The production of tryptophan during the process according to the invention may be monitored using appropriate small scale tests, for example by the method of Nkonge, C. and Ballance, G.K. in Anal. Bioche . 122. 6-9 (1982) as described hereinafter. The tryptophan produced according to the invention may be recovered by conventional means such as, for example, by centrifugation, dialysis or ultrafiltration. Brief Description of the Drawings The invention is further illustrated in the following non-limiting examples and with reference to the accompanying drawings in which Figure 1 shows plasmid restriction maps and indicates the DN manipulations which were used to prepare an intermediate plasmid pIMS1008; Figure 2 shows plasmid restriction maps and indicates the DN manipulations which were used to prepare an intermediate plasmid pIMS1007; Figure 3 shows plasmid restriction maps and indicates the DNA manipulations which were used to prepare a plasmid pIMSlOll for use in the process of the invention, Figure 4 shows plasmid restriction maps and indicates the DNA manipulations which were used to prepare a dual origin expression vector plasmid pIMS1014 according to the invention; and Figure 5 shows plasmid restriction maps and indicates the DNA manipulations which were used to prepare a further dual origin expression vector plasmid pIMS1015 according to the invention. Description of Specific Embodiments Example 1 Construction of plasmids for the expression of tnaA in E.cσli 1) Plasmid IMSlOll comprising the tnaA gene under the control of the hybrid trp/ ac promoter, carrying an ampicillin resistance gene and having a ColEl-type origin of replication was constructed as follows: a) Construction of PIMS1008. Plasmid pAT153 [Ref. Bolivar et al. (1977) Gene 2. 95-113] was digested to completion using the enzyme Cla I. The resulting 5' terminal extensions were converted to blunt ends by treatment with the large (Klenow) fragment of DNA polymerase I in the presence of all four deoxyribonucleotides. The pAT153 was then ligated to a Bglll linker with the sequence 5ΑGATCT3', digested to completion with BRIII and purified by electroelution from an agarose gel to remove unincorporated Bglll linker. Following self ligation using T4 DNA ligase and transformation of Escherichia coli DH1, ampicillin resistant transformants were obtained. Plasmid DNA was purified from those transformants and restriction enzyme analysis confirmed the structure of pIMS1008 shown in Figure 1. b) Construction of pIMS1007. The source of the tnaA gene, encoding tryptophanase, was the plasmid p D.6 [Ref. Deeley, M.C. and Yanofsky, C. J. Bacteriol. 12 (3) 787-796 (1981)] shown in Figure 2. pMD.6 DNA was digested to completion with BamHI and Sphl and a DNA fragment of approximately llOObp carrying the promoter and start of the tnaA gene was isolated by electroelution from an agarose gel. This fragment was then further digested with Sau3a and the resulting mixture of fragments ligated to pIMS1008 DNA which had previously been digested with Bglll and Sphl. Following transformation of E.coli DH1 and restriction analysis of plasmid DNA isolated from the ampicillin resistant transformants a plasmid with the structure of pIMS1007, shown in Figure 2 was obtained. This plasmid carries the first 99bp of the tnaA structural gene. Further analysis of pIMS1007 DNA unexpectedly revealed the presence of approximately lOObp more DNA than predicted fro the published DNA sequence. This extra DNA was located near the start of the tnaA structural gene. The discrepancy has not been analysed further. c) Construction of pIMSlOll. Plasmid pIMS1007 was digested to completion with EcoRI and Sphl and an approximate 230bp fragment containing the start of the tnaA gene isolated b electroelution from an agarose gel. Plasmid pMD.6 was digested with Sphl and Hindlll and a fragment of approximatel 2050bp encoding the distal portion of tnaA isolated. Equimolar amounts of these fragments were ligated to the expression vector pKK223-3 (purchased from Pharmacia, Catalogue No 27-4935-01) which had previously been digested with EcoRI and Hindlll. Following transformation of a strain of E.coli DH1 over-expressing the laci gene and restriction analysis of the ampicillin resistant transformants obtained, a plasmid with the structure of pIMSlOll, shown in Figure 3, was obtained. 2) Plasmid pIHS1014 comprising the tnaA gene under the control of the hybrid trp/lac promoter, carrying an ampicillin resistance gene and having a constitutive pSClOl type origin of replication and a thermoinducible ColEl type origin of replication was constructed as follows: The dual origin vector ρMG196 [Wright, E. et al Gene 4_9 311-321 (1986)] was digested to completion with BamHI and Pstl and the largest subsequent fragment, carrying both origins of replication and part of the ampicillin resistance gene, was isolated by electroelution from an agarose gel. A second sample of pMG196 was digested with Hindlll and Pstl and in this case the small fragment carrying the T7 terminator and part of the ampicillin resistance gene was isolated. The plasmid pIMSlOll was digested with BamHI and Hindlll and a fragment containing the tnaA gene under the control of the hybrid trp/lac promoter was isolated from an agarose gel. Equimolar amounts of the three fragments were ligated together and following transformation of E.coli DH1 and selection on ampicillin medium at 30 C a plasmid with the structure of pIMS1014, shown in Figure 4 was obtained. 3) Plasmid pIMS1015 comprising the tnaA gene under the control of the trp promoter, carrying an ampicillin resistance gene and having a constitutive pSClOl type origin of replication and a thermoinducible ColEl type origin of replication was constructed as follows: The dual origin vector pMG196 was digested to completion with BamHI and EcoRI and a small fragment carrying the trp promoter was isolated following agarose gel electrophoresis. Plasmid pIMSlOll DNA was digested with EcoRI and Hindlll and an approximate 2050bp fragment carrying the tnaA structural gene but lacking a promoter was isolated by electroelution from an agarose gel. The two fragments were ligated to the BamHI to Pstl and Hindlll to Pstl fragments isolated during the construction of pIMS1014. Following transformation of E.coli DH1 and selection on ampicillin medium at 30 C a plasmid with the structure of pIMS1015, shown in Figure 5 was obtained. Example 2 - The controllable expression of tryptophanase 1) From plasmid pIMS 1011 Plasmid pIMS 1011, constructed as in Example 1, was transformed into E. coli MD33. Two cultures were grown at 37°C in 25 ml of a medium containing 10 g/L Difco Bactotryptone, 5 g/L Difco Yeast Extract, 5g/L sodium chloride, lmL/L 1M sodium hydroxide and 0.1 g/L ampicillin. During early exponential growth 0.7 g/L lactose was added to one of the cultures to induce expression of tryptophanase. Samples were taken after a further two hours. These were assayed for their tryptophanase content by the method of Morino, Y. and Snell, E.E. (Methods in Enzymology, 17A, 439-446, 1970) except that the sample volume was raised to 160 uL by raising the concentration of the stock reagents. One unit (U) of tryptophanase as used in the Examples herein is defined as the amount of enzyme that produces one ymole of indole per minute under these conditions. The volumetric titre (U/mL) was 3.5 fold higher in the induced culture compared to the uninduced culture. 2) From plasmid pIMS 1015 (a) Total Enzyme Plasmid pIMS 1015, constructed as in Example 1, was transformed into E.coli strains MD33 and E103S. Two cultures of each transformed strain were grown at 30 C in 10 mL of the medium described above. During early exponential growth one culture of each strain was subjected to a temperature of 42 C, to induce expression of the tryptophanase, and returned to 37°C. The other cultures were maintained at 30 C. Final samples were taken after a further four hours, and their tryptophanase content was determined. The volumetric titres were 6.8 (MD33) and 2.7 (E103S) fold higher in the induced cultures compared to the uninduced cultures. Similarly, E. coli MD33 transformed with plasmid ρIMSl015 was o grown at 30 C in 40 πL of a medium containing 12 g/L Na HPO , 10 g/L cas-amino acids, 6 g/L KH„P0,, 4 g/L glucose, 2 g/L NH.C1, 1 2 4 4 g/L NaCl and 10 mL/L of a suitable trace element solution, and adjusted to pH 7.4 with a suitable quantity of 1M NaOH. During early exponential growth the culture was divided into two flasks, one of which was subjected to a temperature of 42 C, to induce expression of the tryptophanase, and returned to 37 C. The second culture was maintained at 30 C. After a further 3τ hours the tryptophanase content of the cultures was determined. The volumetric titre was 14.8 fold higher in the induced culture compared to the uninduced culture. Similarly, E. coli MD33 containing plasmid pIMS 1015 was grown at 30 C in several lots of 75 ml of the medium described above (Example 2, Case 1). After growth overnight, these were introduced into fermenters containing a suitable growth medium (consisting of yeast extract, glycerol, ammonium sulphate, sodium dihydrogen orthophosphate, antifoam and a suitable trace element solution) . Tryptophanase was induced by raising the temperature from 34 C to 38 C. The amount of tryptophanase was less than 2 units/mL for un-induced cultures, whereas it was 39-66 units/mL after induction. (b) Holotryptophanase E. coli strains WG497 and AT1024 (obtained from Dr. W. Dempsey, Microbial Genetics Unit, Veterans Admin. Hospital) and MD33 were transformed with plasmid pIMS 1015, constructed as in Example 1. o AA ccuullttuurree ooff eeaacchh ttrraannssffoorrmmeedd ssttrraaiinn wwaass ggrroowwnn at 30 C in 20 mL of the medium described above (Example 2, Case 1) Tryptophanase expression was induced as described above (Example 2, Case 2a) and the total amount of tryptophanase was determined as described above. Furthermore, to determine the amount of holotryptophanase present, the assay was performed in the absence of exogeneous pyridoxal-5*-phosphate. The percentages of total tryptophanase expressed as holo-tryptophanase were 6 and 12 times higher for strains WG497 and AT1024 respectively compared with strain MD33. Example 3 - Bioconversion Bacteria containing a high amount of tryptophanase, produced as described in Example 2, were recovered by centrifugation, resuspended in 8.5g/L KCL solution, and stored prior to use. 1. Batch Reaction a. Indole concentration lml of the resuspended bacteria was added to 9ml of an aqueous co-substrate solution, to give final concentrations of 9U/ml tryptophanase, 870mM ammonium acetate, 620mM sodium pyruvate, 22mM potassium dihydrogen orthophosphate, 6.8mM pyridoxal-5*- phosphate, 2.2M ethanol and 7mM indole. The solution was stirred vigorously at 30 C. Samples were taken at timed intervals and the reaction stopped by mixing with an equal volume of 6M KOH, followed by storage at -20 C prior to assay. The tryptophan concentration was determined by a modification of the method of Nkonge, C. and Ballance, G. M (Anal. Biochem. 122. 6-9, 1982). The modi ications reduced interference from compounds observed in some reactions and involved washing of the samples three times with a ten-fold excess of toluene, and reading of the absorbance of both samples and standards at 590nm. In this reaction tryptophan was produced at the rate of 63-81g tryptophan/L/h. The rates obtained in the presence of lower (e.g. 4 and 5mM) or higher (e.g. 9 and 15mM) concentrations of indole were less than those obtained at an indole concentration of 7mM (30-34, 51-63, 32-49 and 9-13g tryptophan/L/h, respectively). b. Cell extracts Bacteria were used as described in Example 3.1a either without further treatment, or as a total or soluble extract. Total extracts were prepared by disrupting the bacteria in a French Pressure Cell. Soluble extracts were prepared by centrifuging the total extract and discarding the pellet. The rates of indole utilisation were measured by sampling as in Example 3a and measuring the indole concentration by the method used in assaying tryptophanase (described in Example 2.1). The rates of indole utilisation (and by inference, tryptophan production) were 197% (total extract-interpolated) and 133% (soluble extract) compared to the rate obtained with whole bacteria (100%). c. Immobilised Bacteria Resuspended bacteria were immobilised in polyacrylamide gel by the method of Pines and Freeman [Pines G. & Freeman A. Eur. J. Appl. Microbial Biotech. 16 75-80 (1982)], with the modification that the polyacrylamide hydrazide was 'wetted* with 2-3ml of ethanol and pressed dry immediately prior to dissolving. When the immobilised bacteria were utilised in a procedure similar to that in Example 3.1b (except that the final volume was 200ml) the reaction proceeded at a rate 89% of that obtained with untreated bacteria. The immobilised bacteria were recovered by decanting of the liquor and used in successive reactions. In the eighth reaction cycle the rate was 70% of that obtained initially. d. Fed-Batch Reaction 50ml of the resuspended bacteria was added to 1350ml of a co-substrate solution, to give final concentrations of 9U/ml tryptophanase, 870mM ammonium acetate, 620mM sodium pyruvate, 22mM potassium dihydrogen orthophosphate, 0.7mM pyridoxal-5'-phosphate and 870mM ethanol, pH8.5, in a MBR 'Mini' Bioreactor (MBR Bioreactor AG, Wetzikon, Switzerland; 130mm internal diameter, 4 x 10mm vertical baffles). The solution was warmed to 30 C and stirred with a turbine impellor at 450rpm. A solution of 5M indole in ethanol was added continuously at a rate such that the concentration of indole in the vessel did not exceed lOmM. Samples were taken at timed intervals and mixed with an equal volume of 6M K0H. They were either analysed for their indole or tryptophan content. The indole concentration was measured by extracting into an equal volume of toluene and measuring the indole concentration by the method used in assaying tryptophanase (described in Example 2.1). The tryptophan concentration was calculated from the volume and the amount of indole utilised. The validity of this procedure was confirmed by comparing the calculated value of selected samples with that measured by the direct assay for tryptophan (described in Example 3.1a). After 75 minutes, the tryptophan concentration was 24g/L, produced at a rate of 19g tryptophan/L/h, with a fractional coversion of the indole of greater than 0.99. The bacteria were recovered by centrifugation (at 3000xg for 20 minutes) resuspended and re-utilised as before (in a final volume of 1.3L to allow for losses during recovery). After a further 70 minutes the tryptophan concentration was again 24g/L produced at a rate of 21g tryptophan/L/h with a fractional coversion of the indole of 0.94. By allowing the reaction to proceed for longer periods, higher concentrations of tryptophan can be accumulated, similar reactions having produced 25-31g/L. At these concentrations the tryptophan precipitates, and may be recovered by removing the liquor.

Claims: C L A I M S 1. A process for the production of tryptophan comprising the steps of: (a) in a first, biocatalyst production stage, propagating bacterial host cells transformed with a vector containing a DNA sequence coding for tryptophanase wherein the expression of said tryptophanase gene is directly controllable; (b) inducing said host cells to synthesise tryptophanase by inducing expression of the tryptophanase gene; (c) in a subsequent bioconversion stage adding reaction substrates for tryptophanase to a reaction mixture containing said host cells induced for tryptophanase synthesis such that tryptophan accumulates in the reaction mixture; and (d) optionally isolating the accumulated tryptophan. 2. A process according to claim 1 wherein the reaction substrates for tryptophanase are indole, pyruvic acid or a salt thereof, and ammonia or a salt thereof 3. A process for the production of tryptophan comprising the steps of (a) in a first, biocatalyst production stage propagating host bacterial host cells transformed with a vector containing a DNA sequence coding for tryptophanase wherein the expression of said tryptophanase gene is directly controllable; (b) inducing said host cells to synthesise tryptophanase b inducing expression of the tryptophanase gene; (c) in a subsequent bioconversion stage adding indole and other reaction substrates for tryptophanase to a vessel containing a reaction mixture comprising said host cells induced for tryptohanase synthesis such that tryptophan accumulates in the reaction mixture, the indole before addition, being dissolved in a water miscible organic solvent to a high concentration and then being pumped into the vessel at a controlled rate, such that the indole concentration in the vessel does not exceed lOmM and (d) optionally isolating the accumulated tryptophan 4. A process according to any of the preceding claims wherein the host for expression of tryptophanase is a gram-positive or gram-negative bacterial cell. 5. A process according to claim 5 wherein the host for expression of tryptophanase is E.coli/ 6. A process according to any of the preceding claims wherein the host bacteria are immobilised. 7. Tryptophan when produced by a process according to any of the preceding claims. 8. A vector system including a DNA sequence coding for tryptophanase, and a controllable replication system comprising a first, stable low copy number, origin of replication and a second, high copy number, origin of replication. 9. A host organism transformed by a vector system according to claim 8. 10. Plasmids pIMS1014 and pIMS1015

Inventors: FROUD STEPHEN JAMES, LILLEY GERALD, LITTLE STEPHEN JOHN, ROBINSON MARTYN KIM, FROUD, STEPHEN, JAMES, LILLEY, GERALD, LITTLE, STEPHEN, JOHN, ROBINSON, MARTYN, KIM
Applicants: CELLTECH LTD, CELLTECH LIMITED
Application year: 1989
Main classification: C12P13
IPC codes: C12N1, C12N9, C12N15, C12P13, C12R1