GB2196333A - Continuous bioconversion process for the preparation of phenylalanine - Google Patents

Abstract

L-Phenylalanine is produced continuously in bioreactors utilising whole microbiol cells containing a specific phenylalanine dehydrogenase. The reaction requires reducing equivalents (NADH) to aminate the phenylpyruvic acid precursor and these are supplied continuously by in vivo generation using a suitable oxidisable substrate (eg. formate, lactate or pyruvate) and the respective dehydrogenase reaction. By direct coupling, an in vivo phenylalanine and formate (or lactate or pyruvate) dehydrogenase system enables cells to be used in a bioconversion medium devoid of all the growth-promoting components. Cells can be immobilised by entrapment and manipulated for prolonged periods of time to produce phenylalanine in high yields by continuously regenerating reducing equivalents via directly-coupled oxidisable substrates. The latter facilitates simple and rapid recovery of the phenylalanine product.

Description

SPECIFICATION Bioconversion process FIELD OF THE INVENTION This invention relates to the production of L-phenylalanine from phenylpyruvic acid by reductive amination of the latter keto substrate using NADH. More specifically, this invention relates to continuous production of reducing equivalents, in cells resting or immobilised, which ensures prolonged reaction of dehydrogenase enzymes specific for phenylalanine production. The invention therefore enables rapid synthesis and high yields of phenylalanine to be achieved in a bioconversion process using a reduced cofactor-mediated catalyst as opposed to a fermentation procedure.

INTRODUCTION AND BACKGROUND OF THE INVENTION L-Phenylalanine is an essential amino acid used in a variety of applications, including food additives, nutritional supplements and intravenous feeding. The most important commercial application of this amino acid is its use in the sweetener ASPARTAME (N,L-aspartyl-L-phenylalanine1-methyl ester). The amino acid can be prepared by chemical synthesis, using benzaldehyde and glycine for example, but necessitates an optical resolution step to control the stereochemistry of the product formed.

Direct fermentation methods normally result in essentially the L-isomer being produced from carbon sources such as molasses, glucose etc. and microorganisms such as coryneforms, E. coli etc. However, the yields from such fermentations are often low and the product has to be recovered and purified from a complex mixture of medium components and fermenter byproducts.

L-phenylalanine has also been prepared by a variety of biotransformation procedures such as using yeast phenylalanine ammonia-lyase and trans-cinnamic acid; N-acetyl-D, L-phenylalanine and deacetylase enzymes; keto acids (phenylpyruvic acid) and amino donors using transamination reactions eg. by E. coli, Pseudomonas fluorescens. One such tranformation/fermentation procedure using phenylpyruvic acid and ammonium ions has been described (EP-A-O 140 503). The latter invention manipulated the normal metabolic pathways of cells (E. coli) growing slowly in a minimal medium, to generate reducing equivalents, such as NADPH, which in turn generate amino acids which enable transamination of phenylpyruvic acid to L-phenylalanine to take place during fermentation. The latter invention is not suitable for prolonged use in resting cells or in an immobilised reactor.

It is the object of the present invention to provide a novel and improved process for the continuous conversion of phenylpyruvic acid to L-phenylalanine using whole microbial cells.

It is a further object of the invention to provide a method for producing and regenerating reduced cofactor, NADH, pools in non-growing microbial cells.

It is the intention of the present invention to provide a process whereby fully induced cells can be reacted with phenylpyruvate, ammonia and oxidisable substrate such as formate or lactate, to produce L-phenylalanine.

In addition, it is the object of this present invention to produce phenylalanine in a medium devoid of growing cells, growth-promoting medium components, metal ions, and other sugars and amino acids.

It is a further object of the present invention to produce phenylalanine from whole cells by coupling of a specific phenylalanine dehydrogenase and an appropriately induced dehydrogenase capable of generating NADH from the oxidation of cheap substrates, such as format and lactate.

In addition, it is a further object of the present invention to use whole cells, immobilised in a bioreactor by entrapment or covalent attachment, in which reduced equivalents are continuously produced and regenerated by means of a simple reaction mixture consisting of not more than 4 components and resulting in not more than 2 products. From such a reaction mixture, the recovery of L-phenylalanine is rendered relatively simple.

SUMMARY OF THE INVENTION The present invention relates to a novel process for continuous conversion of phenylpyruvate to phenylalanine by in vivo coupling, and continuous regeneration, of reducing equivalents in microbial cells. The invention related to a process wherein microbial cells are not actively growing (dividing and synthesising DNA) but have attained maximum synthesis and expression of the two dehydrogenases requiring coupling and are manipulated in an essentially resting or immobilised stage.

The present invention further relates to a process of preparing phenylalanine by reacting fully induced cells in a buffered solution containing phenylpyruvate, ammonium ions and oxidisable carbon substrate. The present invention does not necessitate the use of a minimal, or rich, defined or undefined medium to support other aspects of microbial metabolism.

The present invention relates to a novel process for continuous phenylalanine production in accordance with a dehydrogenase reaction, utilising oxidisable substrate, generating the NADH reducing equivalents necessary to drive a second dehydrogenase reaction, utilising keto substrate and ammonium ions, to generate phenylalanine and oxidised cofactor NAD+. The NAD+ is subsequently utilised for oxidising further incoming substrate via the first dehydrogenase enzyme of- the sequence.

In addition, the present invention relates to an improved process for preparing phenylalanine, by a specific dehydrogenase reaction, in accordance with a constantly maintained and continuously replenished intracellular pool of reducing equivalents. The oxidisable cosubstrate, such as formate, lactate or pyruvate, can be added continuously along with the keto acid precursor, phenylpyruvate, or added periodically to mop up accumulating NAD+ and elevate levels of NADH.

The present invention further relates to an improved process for phenylalanine production in accordance with the use of resting or immobilised cells, packed in a bioreactor, which are reacted with a relatively simple solution of substrates. Selection of appropriate cosubstrates, eg.

formate, restricts the coproduct formation in spent reaction mixtures. The use of the present invention greatly enhances the ease of recovery and purification of phenylalanine from reactor medium.

DETAILED DESCRIPTION OF THE PREPARED EMBODIMENTS In accordance with the process of the present invention, L-phenylalanine is generated in high yields from whole cells of microorganisms in which two dehydrogenases are coupled. The organism referred to in this invention is Corynebacterium equi EVA 5 (deposited with ATCC under accession no. 53208 in connection with US Patent Application Serial no. 06 767 364 8/20/85) but is not limited to this strain as the invention can be applied to other microorganisms containing a phenylalanine dehydrogenase and capable of coupling to another appropriate dehydrogenase eg. lactate dehydrogenase.

in accordance with a perferred embodiment of the present invention, a microorganism capable of synthesising the appropriate dehydrogenases is grown in a suitable medium. The media required are well known to those of ordinary skill in the art and include, for example, glucose, yeast extract, peptone (NH4)2SO4, phosphate, magnesium salts, calcium salts, zinc, iron and vitamins. More specifically, media can contain amino acids such as D,L-phenylalanine, L-histidine, L-tyrosine, D,L-alanine, L-glutamate, L-aspartate or carbon sources such as lactate, pyruvate, formate, citrate, succinate and glycerol.

In accordance with a preferred embodiment of the present invention the carbon and nitrogen sources are selected according to the dehydrogenase required to be induced. For example, medium would contain amino acids such as phenylalanine and histidine to afford induction of a specific phenylalanine dehydrogenase. Lactate, pyruvate and formate can induce synthesis of their respective dehydrogenases capable of metabolising the inducers as a source of carbon whilst generating NADH as cofactor product: Lactate + NAD+ LDH Pyruvate + NADH

Formate + NAD+ FDH CO2 + NADH

NAD + NAD+ PDH Pyruvate + NAD PDH Acetyl-CoA + NADH

CoA Such dehydrogenases are normally present in low levels as part of central metabolic pathways in cells. Addition of suitable inducers results in increased synthesis of a specific dehydrogenase or one or more isoenzymes of increased specificity. The cells, once fully induced for the particular dehydrogenases requiring coupling, can be used directly in the growth medium (as described in EP-A-O 140 503) or removed and used in a bioreactor of choice.

It is in accordance with a preferred embodiment of the present invention that cells are fully induced for two complementary dehydrogenases such that the activity of one generates the reduced cofactor to stimulate the activity of the second dehydrogenase: the activity of the latter generates the oxidised cofactor to stimulate activity of the first dehydrogenase in the cycle.

Thus, in this particular embodiment, phenylpyruvic acid is reacted with ammonium ions in the presence of lactic acid. The lactic acid initiates the sequence of events by generating the essential reduced cofactor required to aminate the phenylpyruvate:

H I + CH3-C-C0OB + H CH3-C-COOB CH,-C-OOH + II OH NAD N:6 Lactate NADB 6 CI'.. ~~ oA (Acetyl-CoA) Ii 0 H2 H - C-C-COOH < < 9 C-C-COOH + NH4 + H Ho L-Phenylalanine Phenylpyruvate Thus, the reduced cofactor is continuously generated as long as lactate is supplied to the system.In this type of system, the oxidisable substrate used can supply excess reducing equivalents due to subsequent metabolism of the pyruvate product to acetyl-CoA, generating further NADH.

In accordance with a preferred embodiment of the present invention, format is used as a direct source of reducing equivalents due to the production of CO2 as coproduct which does not contaminate the reaction mixture. It is preferred to use the ammonium salt of formate to drive the synthesis of L-phenylalanine. Other oxidisable substrates could also be used so long as they are metabolised by a route which permits the electrons to be passed to NAD+. It is beneficial not to use such energy sources such as glucose, sucrose, citrate, or fructose because of the other accumulating intermediates and requirements for reaction. It is preferred to use direct sources of electrons, via single step reactions, which contribute NADH directly to the system of L-phenylalanine formation.

In accordance with a preferred embodiment of the invention, cells are not permeabilised by physical or chemical means so as to prevent leakage of cofactor pools from within whole cells.

It is preferred to react whole cells in the presence of osmoregulator fluids such as sorbitol, glycerol, mannitol, sucrose, polyethylene glycol, to enhance cellular integrity and limit loss of pools of intra-cellular cofactors. Thus, in a preferred embodiment, cells are removed from culture medium, containing detrimental fermentation products such as organic acids, and resuspended in osmoregulator solutions. Such cell suspensions are then preferably immobilised by entrapment in gels, such as alginate, agar, k-carrageenan or polyacrylamide, or foams, such as polyurethane, and cross linked and further stabilised by glutaraldelyde and polyethylene glycol treatment familiar to those skilled-in-the art.

In accordance with a preferred embodiment of the present invention, immobilised cells, fully induced for coupled dehydrogenases, are used in a packed or fluidised bed reactor in which reaction mixture is passed or percolated continuously, either in a single passage or is recycled.

The reaction mixture is of simple composition containing either minimal medium or, preferably, just buffer solution in which is dissolved the two main substrates and cosubstrate for the system. Typical reaction mixtures could contain 0.1M glycerol or sorbitol, 0.3M lactate or pyruvate or 0.5M formic acid. Such reaction mixtures greatly facilitate recovery and purification of L-phenylalanine as opposed to typical fermentation medium-based bioconversions.

The process of this present invention is, thus, best carried out by growing a suitable strain, or selected mutant with constitutively high expression of the appropriate dehydrogenases, in a complex medium which affords maximum synthesis of biomass and enzymes of interest, incurr ing little cost of added inducers and vitamins as normally used in a defined medium. When optimal activity of catalysts have been attained, the cells are removed by centrifugation and the paste washed prior to immobilisation in an appropriate gel. The stabilised cells are then continuously reacted with the oxidisable substrates which generate the reducing equivalents necessary to drive the aminating dehydrogenase reaction. The L-phenylalanine produced is easily recovered from the reaction effluent containing very low levels of fewer than 4 components after efficient conversion has taken place.

In a preferred embodiment of the present invention, L-phenylalanine is also prepared from acetamidocinnamic acid using strain Corynebacterium equi EVA5 or a mutant such as OAR1-16 derived from the latter parent strain by mutagenesis involving U.V. and chemical mutagens.

Strain OAR1-16 has been deposited with ATCC under accession no 53529. The cells cultivated in a growth medium containing acetamidocinnamic acid, phenylalanine and lactate to afford induction of the appropriate deaminase, to yield phenylpyruvate, and the two coupled dehydrogenases to generate the L-phenylalanine. In the case of using a mutant strain, such as OAR1-16, only lactate is required due to constitutive synthesis and high expression of the acetamidocinnamate deaminase and phenylalanine dehydrogenase in this strain.

The following non-limiting Examples are intended to further illustrate the presnet invention.

Example 1 An agar slope of Corynebacterium equi EVA-5 was used to inoculate 100 ml ACA-induction medium containing; 1% glucose, 0.5% yeast extract, 0.7% KH2PO4, 0.4% Na2HPO4, 0.2% (NH4)2SO4, basal salts pH 7.0 and 0.2% acetamidocinnamic acid as inducer. After 24h growth at 30"C the flask was used to inoculate 1 litre of the same medium and grown at 30"C for 32h.

The cells were harvested by centrifugation, washed twice in phosphate buffer and stored on ice until use. An aliquot of cell suspension (5ml) was mixed with 5ml of a solution containing acetamidocinnamic acid (ACA), 1591-1, and a solution containing ACA 1591-1 and Formic Acid 1591-'. These reactors were incubated at 30"C for 26h, the cells remove by centrifugation and added back to the same volume of fresh mixtures. This was continued for 8 consecutive reactions. The reactor containing ACA alone only produced phenylalanine for the first 2 reactions and then ceased; the reactor containing ACA and formate continued to operate with over 40% conversion efficiency for all 8 reactions. (N.B. The cells used in this experiment had only low levels of formate dehydrogenase as they were not induced prior to carrying out the transformations).

EXAMPLE 2 Cells of Corynebacterium equi EVA-5 were induced and prepared according to methods described in Example 1. An aliquot of cell suspension (20ml) was mixed with 20ml of a solution containing 100mM phosphate buffer pH6.5 and acetamidocinnamic acid 1291-'. An identical reaction mixture was set up but containing pyruvate at 2091-'. The mixtures were incubated at 30"C for 24h in 250ml conicals and shaken at 140rpm. After reacting 24h, cells were separated by centrifugation, washed twice in buffer and reacted in the same volume of fresh medium. The reactor containing acetamidocinnamate only produced 9.891-1 phenylalanine in 24h and a further 1.1gel~' on subsequent reaction with fresh substrate.The second reactor, containing pyruvate, produced 1291-1 phenylalanine in 24th and continued to synthesise this amino acid for over 20 days. The yield progressively fell to approximately 50% of the original and stabilised around this level. The results for 6 successive reactions are shown in Fig. 1.

EXAMPLE 3 Cells of Corynebacterium equi EVA-5 were prepared according to Example 1 and reacted as described in Example 2. Reactors contained in 100ml, 3691-1 cell dry weight in 100mM phosphate buffer pH6.5 with 1291-' acetamidocinnamic acid alone, with 20gl-' acetate or 2091-' lactate to supply reducing equivalents. Reactions were monitored for 8 days and it was found that phenylalanine production ceased in the control but continued with a yield of 32% in the presence of acetate and 47% in the presence of lactate.

EXAMPLE 4 Cells of Corynebacterium equi EVA-5 were grown and mutagenised using U.V. irradiation.

Mutants resistant to p-fluorophenylalanine were picked and screened for higher rates of production of L-phenylalanine from acetamidocinnamate. One such mutant was referred to as OAR1-16 (which has been deposited with ATCC under accession no. in connection with a British Patent Application of even date also in the name of Allelix under the title Mutant Microorganisms) and showed increased expression of ACA deaminase and phenylalanine dehydrogenase. This mutant was grown as described in Example 1. The harvested cells were immobilised in 2% alginate and entrapped in beads prepared in 0.5% CaCI2 solution. The beads were washed and packed into a beaker (500ml volume). Reaction mixture containing acetamido cinnamate 2091-1, pyruvate 3091-', (NH4)2SO4 1591-1 and in 100mM phosphate buffer pH6.8 was added to form a fluidised bed.The beaker was gently shaken so that the mass of beads were evenly and continuously dispersed. Initial yields were 1791- (100% molar conversion) for several changes of reaction mixture; after 9 days the yield equilibrated and stayed at around 45% thereafter.

EXAMPLE 5 Cells of EVA-5 mutant, OAR1-16, were grown as described in Example 1. The harvested cell paste was disrupted using a Dyno Mill so as to remove permeability barriers to cofactors. The cell slurry was mixed with 2% alginate and entrapped as described in Example 4. The immobilised extract in beads was split into 4 reactors. Two reactors contained a solution of acetamidocinnamate 2% and (NH4)2SO4 2% and the remaining 2 contained phenylpyruvate 2% and (NH4ì2SO4 2%. NADH was added to one of the reactors containing ACA and one containing phenylpyruvate at 6h intervals. All 4 reactors attained 100% conversion for the initial changes of reaction mixture but, after 5 days of continuous operation, only the 2 reactors with added cofactor continued to produce L-phenylalanine.Over 40% conversion was observed after 5 days and 15% conversion after 11 days of operation.

EXAMPLE 6 Corynebacterium equi EVA-5 was grown in a medium as described in Example 1 but containing 1% lactate, instead of glucose, and 0.2% D,L-phenylalanine, instead of (NH4)2SO4; acetamidocinnamate was also included at 0.2% v/v and an equivalent amount added again after 20h growth. The cells were harvested after 32h growth and washed in phosphate buffer. Cell suspensions were mixed with reaction mixtures to give a final cell concentration of 34gl-t dry weight. Cells reacted with 2%ACA only, gave an initial rate of phenylalanine production of 0.691-lh-' whilst reaction with 2% ACA+2 ,6 lactate+2% (NH4)2SO4 had a production rate of 1.6gl-1h-'. Reaction mixtures were changed every 24h and fresh substrates added.The control reactor containing ACA alone ceased production of phenylalanine after 48h whilst the second reactor continued phenylalanine synthesis for over 10 days.

EXAMPLE 7 Freshly induced cells of C.equi mutant OARl 1-16 were prepared as described in Example 1.

The cells were washed in 500mM Tris-buffer pH7.0 and resuspended in the same buffer containing 1.5M sorbitol. The cells were incubated with lysozyme for 3h at 300C . The protoplast-cell extract was washed in 25mM Tris-buffer pH7.0 and shaken at 30"C for 1h to lyse protoplasts. To this extract was added an aliquot of formate dehydrogenase, prepared in 20% glycerol from Candida boidinii. This mixture was diluted with glycerol and sorbitol to a final concentration of 0.2M of each polyhydric alcohol. An aliquot of concentrated NAD+ was added to the suspension and then mixed in a 1:1 ratio with 2% sodium alginate. Beads were formed in the usual manner by dropping into 0.8M CaCI2 (ice cold) and left to crosslink for 5h.The beads were washed free of CaCI2 and reacted with 500ml of a reaction mixture containing phenylpyruvate 2%, formate 1% and (NH4)2SO4 0.2M pH7.0. The immobilised extracts continued to produce L-phenylalanine, with over 40% conversion yield, provided fresh formate was supplied intermittantly to supply reducing equivalents.

EXAMPLE 8 Cells of Corynebacterium equi EVA-5 were grown up and mutagenised using U.V. For 30 secs and 500 uW/cm2. The mutagenised cells were spread on a range of media containing fluoroanalogues of phenylalanine. Mutants were picked and screened for phenylalanine production from acetamidocinnamic acid.The best mutants were compared for their ability to regenerate reduced cofactor by reacting with 2% ACA and 1% pyruvate at 34"C. The results are shown below in table 1: L-Phenylalanine (gl-') Strain 1st Run - 2nd Run 3rd Run EVA-5 7 6 3 179 15 4 2 183 11 5 3 WB18 12 11 7 WB21 15 10 6 WB22 14 13 5 WB48 10 9 6 W1374 15 11 5 APPENDIX 1 EVA-5 was initially isolated from samples of soil and sewage in the area of Orangeville, Ontario, Canada.

Characteristics: (i) Description of the novel isolate Corynebacterium EVA-5 In its characteristics, it is a gram positive rod of variable length, occurring mostly as short clubbed rods (almost coccoid) in palisades and 'v' forms. On testing with carbohydrate substrates, it is found that it ferments glucose and does not oxidise it. No acid was produced from any tested carbohydrate. The isolate strain grows between 18"C and 37"C, but grows very best at 25"C-30"C. It grows well at any pH from 4-9.

Accordingly the isolate probably belongs to the genus Corynebacterium and resembles the species Corynebacterium equi. It differs from the equi species, however, in that it does not reduce nitrate, its colonies are not pink in colour, it does not grow well above 30"C, it does not exist as long curved rods in culture, and it can utilize ACA as sole carbon and nitrogen source for growth. Accordingly, it shows many of the characteristics of a number of species belonging to the genus Brevibacterium, and may be classifiable as such.

Hence it is concluded that the novel isolate strain is probably but not certainly a new species of Corynebacterium or a variety of the species Corynebacterium equi. For convenience, it is herein designated Corynebacterium strain EVA-5.

As regards its growth characteristics, the strain herein designed Corynebacterium EVA-5 grows well on a range of carbon and nitrogen sources, salts and extracts. A typical medium contains glucose, sucrose, fructose or glycerol at 1-5% w/v; (NH4)2504, glutamate or asparagine, 0.2-1%1 KH2PO4, 0.2-1%; K2HPO4, 0.1-0.4%; Na2HPO4, 0.05-0.02%; MgSO4.7H20, 0.01-0.07%; NaCI, 0.1-1%; CaCI2.6H20, 0.0001-0.01%; FeC3. 7H20, 0.0001-0.0008%; ZnSO4.7H20, 0.0001%; MnCl2, CusO4, Coy12, in trace amounts; yeast extract, peptone or meat extract, 0.2-2%. Growth is stimulated by addition of various vitamins, particularly thiamine, biotin pyridoxal, nicotinic acid, pantothenate and p-aminobenzoic acid, 1-45 ugl-1. The strain can therefore be cultivated on a range of conventional laboratory and commercial media. The characteristics of Strain EVA-5 may be summarized as follows: Morphology: Rods, 0.5-1.0 by 1.0-1.5 um, gram positive. Almost coccoid, non motile, palli sade and "V" forms. Non spore forming colonies cream coloured, circular shiny and mucoid. Black colonies formed on potassium tellurite.

Physiology: Nitrate reduction Catalase + Oxidase Capsule + Spore Metachromatic granules + Acid Fast Hemolysin Starch hydrolysis Gelatin hydrolysis Urease Casein hydrolysis Lipolysis + Acid from: Maltose Galactose Lactose Glycerol Arabanose Fructose Rhamnose Xylose Glucose Melibiose Sucrose Erythrose Sorbitol Optimum temperature 25C Optimum pH 5-8 Deposit: A viable culture of the strain Corynebacterium EVA-5 as described herein has been deposited with ATCC for storage therein under conditions giving permanence to the culture in viable form, and has been allotted accession reference no. 53208.

APPENDIX 2 Production and Characteristics of C equi mutant strain OAR1-16 Production Cells of parent strain Corynebacterium equi EVA-5 (ATCC 53208) were used to derive the mutant OAR1-16 by the following procedure: An LB agar slant of C. equi EVA-5 was used to inoculate 20 ml of LB broth in a 250 ml flask and grown for 24 hours at 30"C with shaking 160 rpm.

Cell suspensions of exponentially growing EVA-5 were mutagenised as follows: Ethyl methane sulphonate (EMS) was added to a final concentration of 1% (v/v) and shaken vigorously to disperse the EMS. Samples (1 ml) were removed every 20 minutes and a kill curve determined.

After 90 minutes at 25"C the cells were harvested and washed twice in phosphate buffer containing 6%(wt/v) sodium thiosulphate followed by three washes in distilled water. Cells were resuspended in LB broth medium for 2 hours and 30"C to allow expression of mutations.

The mutagenised stock was dispensed in 100 ul aliquots onto basal M9 medium agar containing 100 ug ml-' of p-fluorophenylalanine. Plates were incubated at 30"C for 5 days. Control plates of LB agar were also inoculated with serial dilutions of mutagenised stock so as to determine the concentration of colony forming units and frequency of resistant colonies arising.

Colonies arising on M9 analogue plates were picked and transferred to a second selective M9 analogue (PFP) plate. The latter plates were incubated at 30"C for 4 days. The largest colonies were transferred to 50 ml shake flasks containing 10 ml sterile ACA induction medium and incubated at 30"C for 48 hours and 165 rpm shaking. Cells were harvested by centrifugation washed and resuspended 50 mM phosphate buffer pH 7.0 to 50 mg cell dry weight per ml.

Reactions contained 0.2 ml cells suspension and 0.2 ml ACA 2% w/v in 100 mM phosphate buffer pH 6.8. Phenylalanine production was monitored. One particular strain isolated in this manner was found to produce more than 9 gl-1 L-phenylalanine and was designated PFP 26.

A colony of PFP 26 grown was re-mutagenised using EMS. The second generation mutants were screened as described above, and one mutant, designated AR1, was found to produce more than 11 gl-' L-phenylalanine from 2% ACA. A cell suspension of this mutant was further mutagenised using NTG as follows: The cell suspension was transferred to a 50 ml screw cap plastic centrifuge tube to which was added a saturated solution of N-methyl-N'-nitro-N-nitrosoguanidine (NTG, Sigma) to one tenth the initial concentration. The tube was shaken gently and incubated at room temperature for 45 minutes. Samples (1 ml) were removed at intervals and a kill curve determined.Mutagenised cells were washed with sterile water ten times to remove NTG and finally resuspended in 20 ml of LB broth and incubated for 2 hours at 300C to allow mutations to be expressed.

Mutants were selected on agar plates containing o-fluorophenylalanine 10 mM. Colonies exhibiting resistance were restreaked on selective agar prior to screening as follows: The plates were incubated for 4 days and a single colony from each plate inoculated into a flask of medium (ACA-inducation) containing: 1% glucose, 0.5% yeast extract, 0.7% KH2PO4, 0.4% Na2HPO4, 0.2% (NH4)2SO4, 0.1% MgSO4.7H20, trace salts (Ca2+, Fe3+, Zn2+) and adjusted to pH 7.0. Acetamidocinnamate was added as inducer at a final concentration of 2 gI-1. Cultures were grown for 24 hours at 30"C after which cells were removed by centrifugation, washed twice in 50 mM phosphate buffer pH 7.0 and resuspended in the equal volume of the same buffer. To this suspension (usually 10 ml) was added 1 ml reaction mixture containing 2% ACA in 100 mM phosphate pH 7.0.Samples (50 ul) were removed periodically for 24 hours and assayed using paper chromatography and copper nitrate in methanol at Or505. Mutants were assessed for activity on the basis of rate of phenylalanine production and total yield achieved.

One mutant was shown to produce more than 17 gl-1 L-phenylalanine from 2.5% acetamidocinnamate and was designated OAR 1-16. A viable culture. of this strain was deposited with ATCC for storage therein under conditions giving permanence to the culture in viable form and has been allotted accession reference number 53529.

Characteristics The strain designated Corynebacterium OAR1-16 is a phototrophic derivative, produced by mutagenesis, of Corynebacterium strain EVA-5. Accordingly, the mutant strain exhibits most of the characteristics of the patent strain EVE5. It differs from EVA-5, however, in that growth occurs in the presence of the phenylalanine analogues p-fluoro-D, L-phenylalanine or o-fluoro-D, L-phenylalanine at concentrations up to 10 mM. In addition, the mutant stain OAR1-16 exhibits an improved ability to produce L-phenylalanine for ACA.

As regards its growth characteristics, the strain herein designated Corynebacterium OAR1-16 grows well on a range of carbon and nitrogen sources, salts and extracts. It can utilize ACA as sole carbon and nitrogen source for growth.

A typical medium contains glucose, sucrose, fructose or glycerol at 1-5% w/v; (NH4)2SO4, glutamate or asparagine, 0.2-1%; KH2PO4, 0.2-1%; K2HPO4, 0.1-0.4%; Na2HPO4, 0.05-0.2%; MgSO4.7H20, 0.01-0.07%; NaCI, 0.1-1%; CaCI2.6H20, 0.0001-0.01%; FeCI3.7H20, 0.0001-0.0008%; ZnSO4.7H20, 0.0001%; McCl2, CuSO4, Coy12, in trace amounts; yeast extract, peptone or meat extract, 0.2-2%. Growth is stimulated by addition of various vitamins, particularly thiamine, biotin, pyridoxal, nicotinic acid, pantothenate and p-aminobenzoic acid, 1-45 ugl-1.

The strain can therefore be cultivated on a range of conventional laboratory and commercial media.

The characteristics of Strain OAR1-16 may be summarized a follows: Morphology Rods, 0.5-1.0 by 1.0-1.5 um, gram positive. Almost coccoid, non motile, pallisade and "V" forms. Non spore forming colonies cream coloured, circular shiny and mucoid. Black colonies formed on potassium tellurite.

Physiology: Nitrate reduction Catalase + Oxidase Capsule + Spore Metachromatic granules + Acid Fast Hemolysin Starch hydrolysis Gelatin hydrolysis Urease Casein hydrolysis Lipolysis + Acid from: Maltose Galactose Lactose Glycerol Arabinose Fructose Rhamnose Xylose Glucose Melibiose Sucrose Erythrose Sorbitol Optimum temperature 25"C Optimum pH 5-8 OAR1-16 cells are constitutive for conversion of acetamidocinnamate to L-phenylalanine, unlike the patent EVA-5 which is inducible. Thus mutant OAR1-16 can be grown in a wide range of media in the absence of ACA inducer and the resultant cells are capable of transforming ACA to L-phenylalanine in high yieid., The nature of the constitutivity of OAR1-16 is at the level of the normally induced ACA permease and deaminase system.Both these enzymes were synthesised by OAR 1-16 in the absence of ACA in culture medium. As a result, ACA could be rapidly taken up and deaminated by cells of OAR1-16 grown in a simple glucose/salt medium. A further property of mutant OAR1-16 is the increased expression of the permease and deaminase thus facilitating a more rapid initial conversion of ACA substrate. Furthermore expression of the phenylalanine dehydrogenase is also partially constitutive and is markedly higher in OAR1-16 than in EVA-5. The phenylalanine dehydrogenase of OAR1-16 is easily detected in cell-free extract, there is increased synthesis of this enzyme and it is not subject to regulation by in vivo proteolytic modification as found with the parent EVA-5.

Furthermore, mutant OAR1-16 is not sensitive to end product feedback inhibition or ACA substrate inhibition as observed with EVA-5. Cells of OAR1-16 are able to produce L-phenylalanine at maximal rates in the presence of ACA 1-5% (w/v) and also in the presence of Lphenylalanine 1-3% (w/v). Mutant OAR1-16 also has significantly higher activity of the deacetylase enzyme catalysing conversion of N-acetyl-L-phenylalanine to L-phenylalanine in high yields.

Claims (14)

1. A process for producing L-phenylalanine from phenylpyruvic acid using resting or immobilised microorganisms wherein the intracellular pool of reducing equivalents is replenished by supplying oxidisable carbon substrate.

2. A process according to claim 1 comprising reacting fully induced cells in a buffered solution containing phenylpyruvate, ammonium ions and oxidisable carbon substrate.

3. A process according to claim 1 or claim 2 wherein the carbon substrate is formate, lactate or pyruvate.

4. A process according to any preceding claim wherein the carbon substrate is supplied continuously or intermittently.

5. A process according to any preceding claim wherein the microorganism contains phenylalanine dehydrogenase and another hydrogenase which is capable of coupling with phenylalanine dehydrogenase by generating NADH from NAD+ and the oxidisable substrate.

6. A process according to claim 5 wherein NADH, phenylpyruvate and ammonium ions are used by a phenylalanine dehydrogenase producing phenylalanine and NAD+, the NAD+ is used by the other dehydrogenase to oxidise the oxidisable carbon substrate thus producing NADH for use by the phenylalanine dehydrogenase.

7. A process according to claim 5 or claim 6 wherein the microorganism is of the genus Corynebacteria or Escherichia.

8. A process according to claim 7 wherein the microorganism is a strain of Corynebacterium equi or Escherichia coli.

9. A process according to claim 8 wherein the microorganism is C. equi EVA-5 (ATCC 53208) or a mutant thereof.

10. A process according to claim 9 wherein the microorganism is C. equi mutant strain OAR1-16 (ATCC 53529) or a mutant thereof.

11. A process according to any preceding claim wherein the microorganisms are entrapped in gels or foams percolated continuously with reaction media.

12. A process according to any preceding claim wherein phenylalanine is also produced by supplying the microorganism with acetamidocinnamic acid.

13. A process for producing L-phenylalanine substantially as hereinbefore described with reference to any one of the Examples.

14. L-phenylalanine whenever produced by a process according to any preceding claim.