Classifications

• • 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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/42—Hydroxy-carboxylic acids
• • 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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

Definitions

• the invention relates to the field of industrial microbiology. More specifically, methods for the enhancement of yield and rate of production of aromatic carboxylic acids such as para-hydroxycinnamic acid and cinnamic acid are disclosed.
• pHCA para-hydroxycinnamic acid
• CA cinnamic acid
• pHCA para-hydroxycinnamic acid
• cinnamic acid CA
• these aromatic compounds have application as monomers for the production of liquid crystalline polymers, and in the production of resins, elastomers, coatings, adhesives, automotive finishes and inks. Chemical synthetic methods for producing these aromatic compounds are known.
• 2003/007925 describe methods for producing CA and pHCA using recombinant microorganisms comprising at least one gene encoding a tyrosine ammonium lyase (TAL) activity and at least one gene encoding a phenylalanine hydroxylase (PAH) activity.
• TAL tyrosine ammonium lyase
• PAH phenylalanine hydroxylase
• a problem encountered with the biological production of these aromatic compounds is end-product inhibition, which limits product yield.
• the fermentation is typically run at a pH that is not optimal for the tyrosine ammonium lyase activity, required to convert tyrosine to pHCA, or the phenylalanine ammonia lyase (PAL) activity, required to convert phenylalanine to CA.
• PAL phenylalanine ammonia lyase
• the pH optimum for these enzymes is in the alkaline pH range, typically about pH 8.5 (see for example Hodgins, J. Biol. Chem. 246:2977-2985 (1971)).
• Evans et al. Enzyme and Microbial Technology 9:417-421 (1987J; Appl. Microbiol. Biotechnol. 25:399-405 (1987); and Journal of Industrial Microbiology. 2:53-58 (1987) describe methods for the biotransformation of trans-cinnamic acid to phenylalanine using whole yeast cells, which have phenylalanine ammonia lyase activity, at a pH of 9.0 to 12.0.
• the invention provides methods for the enhanced production of aromatic carboxylic acids such as pHCA and CA by fermentation under the influence of a tyrosine ammonia lyase or phenylalanine ammonia lyase enzyme where the catalytic event takes place at alkaline pH.
• the invention provides a method for the production of para-hydroxycinnamic acid comprising: (i) providing a microbial production host cell which : a) makes tyrosine when grown with a fermentable carbon substrate; and b) comprises a gene encoding a polypeptide having tyrosine ammonia lyase activity operably linked to suitable regulatory sequences; (ii) contacting the host cell of (i) with a fermentable carbon substrate in a growth medium at physiological pH for a time sufficient to allow tyrosine to accumulate in the growth medium; and (iii) raising the pH of the growth medium to a pH of about 8.0 to about 11.0 for a time sufficient to allow para- hydroxycinnamic acid to accumulate; and (iv) optionally recovering said para-hydroxycinnamic acid.
• the invention provides a method for the production of cinnamic acid comprising: (i) providing a microbial production host cell which : a) makes phenylalanine when grown with a fermentable carbon substrate; b) comprises a gene encoding a polypeptide having phenylalanine ammonia lyase activity operably linked to suitable regulatory sequences; (ii) contacting the host cell of (i) with a fermentable carbon substrate in a growth medium at physiological pH for a time sufficient to allow phenylalanine to accumulate in the growth medium; and (iii) raising the pH of the growth medium to a pH of about 8.0 to about 11.0 for a time sufficient to allow cinnamic acid to accumulate; and (iv) optionally recovering said cinnamic acid.
• the invention provides a method for the production of para- hydroxycinnamic acid comprising the sequential steps of : (i) providing a microbial production host cell which makes tyrosine when grown with a fermentable carbon substrate; (ii) contacting the production host cell of (i) with a fermentable carbon substrate in a growth medium at physiological pH for a time sufficient to allow tyrosine to accumulate in the growth medium; (iii) contacting the growth medium of (ii) with tyrosine ammonia lyase; (iv) raising the pH of the growth medium to a pH of about 8.0 to about 11.0 for a time sufficient to allow para- hydroxycinnamic acid to accumulate in the growth medium; and (iv) optionally recovering said para-hydroxycinnamic acid.
• the invention provides a method for the production of cinnamic acid comprising the sequential steps of : (i) providing a microbial production host cell which makes phenylalanine when grown with a fermentable carbon substrate; (ii) contacting the production host cell of (i) with a fermentable carbon substrate in a growth medium at physiological pH for a time sufficient to allow phenylalanine to accumulate in the growth medium; (iii) contacting the growth medium of (ii) with phenylalanine ammonia lyase; (iv) raising the pH of the growth medium to a pH of about 8.0 to about 11.0 for a time sufficient to allow cinnamic acid to accumulate in the growth medium; and (iv) optionally recovering said cinnamic acid.
• the invention provides a method for the production of para-hydroxycinnamic acid comprising the sequential steps of : (i) providing a microbial production host cell which makes tyrosine when grown with a fermentable carbon substrate; (ii) contacting the production host cell of (i) with a fermentable carbon substrate in a growth medium at physiological pH for a time sufficient to allow tyrosine to accumulate in the growth medium; (iii) isolating the tyrosine produced in (ii) from the growth medium; (iv) contacting the isolated tyrosine of (iii) with a source of tyrosine ammonia lyase in a solution having a pH of about 8.0 to about 11.0 for a time sufficient to allow para- hydroxycinnamic acid to accumulate; and (v) optionally recovering said para-hydroxycinnamic acid.
• the invention provides a method for the production of cinnamic acid comprising the sequential steps of : (i) providing a microbial production host cell which makes phenylalanine when grown with a fermentable carbon substrate; (ii) contacting the production host cell of (i) with a fermentable carbon substrate in a growth medium at physiological pH for a time sufficient to allow phenylalanine to accumulate in the growth medium; (iii) isolating the phenylalanine produced in (ii) from the growth medium; (iv) contacting the isolated phenylalanine of (iii) with a source of phenylalanine ammonia lyase in a solution having a pH of about 8.0 to about 11.0 for a time sufficient to allow cinnamic acid to accumulate in the growth medium; and (iv) optionally recovering said cinnamic acid.
• the invention provides a method for the production of para-hydroxycinnamic acid comprising the sequential steps of : (i) providing a microbial production host expressing a gene encoding a polypeptide having tyrosine ammonium lyase activity; and (ii) contacting said microbial production host of (i) with tyrosine at a pH of about 8.0 to about 11.0 wherein para- hydroxycinnamic acid is produced.
• the invention provides a method for the production of cinnamic acid comprising the sequential steps of : (i) providing a microbial production host expressing a gene encoding a polypeptide having phenylalanine ammonium lyase activity; and (ii) contacting said microbial production host of (i) with phenylalanine at a pH of about 8.0 to about 11.0 wherein cinnamic acid is produced.
• Figure 2 shows the growth and pHCA production in a fermentation wherein the pH is changed to 8.4 after 24 hours.
• the following sequences conform with 37 C.F.R. 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures - the Sequence Rules") and consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions).
• the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1.822.
• SEQ ID NO:1 is the nucleotide sequence of the gene encoding the phenylalanine-tyrosine ammonia lyase (PAL/TAL) enzyme from Rhodotorula glutinis.
• SEQ ID NO:2 is the amino acid sequence of the phenylalanine- tyrosine ammonia lyase (PAL/TAL) enzyme from Rhodotorula glutinis.
• SEQ ID NOs:3-6 are the nucleotide sequences of primers used to construct E. coli strain DPD4009.
• SEQ ID NOs:7 and 8 are the nucleotide sequences of primers used to confirm the successful construction of E. coli strain WS158.
• SEQ ID NOs:9 and 10 are the nucleotide sequences of primers used to amplify the pal gene from Rhodotorula glutinis.
• SEQ ID NOs:11 and 12 are the nucleotide sequences of primers used to construct plasmid pLH276. DETAILED DESCRIPTION OF THE INVENTION The invention relates to methods for improving the yield and rate of production of para-hydroxycinnamic acid (pHCA) and cinnamic acid (CA).
• pHCA para-hydroxycinnamic acid
• CA cinnamic acid
• Microbes having the ability to produce either tyrosine or phenylalanine are grown in the presence of enzymes having tyrosine ammonium lyase (TAL) acitivity or phenylalanine ammonium lyase (PAL) activity for the production of para-hydroxycinnamic acid (pHCA) and cinnamic acid (CA), respectively.
• TAL tyrosine ammonium lyase
• PAL phenylalanine ammonium lyase
• Production yields and rates of the aromatic carboxylic acid product is enhanced at pH's of about 8.0 to about 11.0.
• the production of para-hydroxycinnamic and cinnamic acid are useful as monomers in a number of industrial applications including the production of resins, elastomers, coatings, adhesives, automotive finishes and inks.
• LCP Liquid Crystal Polymers
• pHCA para-hydroxycinnamic acid
• CA cinnamic acid
• LCPs are polymers that exhibit an intermediate or mesophase between the glass-transition temperature and the transition temperature to the isotropic liquid or have at least one mesophase for certain ranges of concentration and temperature. The molecules in these mesophases behave like liquids and flow, but also exhibit the anisotropic properties of crystals. LCPs are used in liquid crystal displays, and in high speed connectors and flexible circuits for electronic, telecommunication, and aerospace applications.
• LCPs are used in medical devices, and in chemical and food packaging.
• PAL Phenylalanine ammonia-lyase
• TAL Terosine ammonia-lyase
• pHCA pHCA
• TAL activity refers to the ability of a protein to catalyze the direct conversion of tyrosine to pHCA.
• PAL activity refers to the ability of a protein to catalyze the conversion of phenylalanine to cinnamic acid.
• paf represents a gene that encodes an enzyme with PAL activity.
• taf represents a gene that encodes an enzyme with TAL activity.
• PAL/TAL activity or "PAL/TAL enzyme” refers to a protein which contains both PAL and TAL activity.
• Such a protein has at least some specificity for both tyrosine and phenylalanine as an enzymatic substrate.
• protein and “polypeptide” will be used interchangeably.
• Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
• Native gene or “wild type gene” refers to a gene as found in nature with its own regulatory sequences.
• Chimeric gene refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature.
• a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
• Endogenous gene refers to a native gene in its natural location in the genome of an organism.
• a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.
• Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
• Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance.
• transgenic or “recombinant” or “transformed” organisms.
• vector or "vector” and “cassette” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double- stranded DNA molecules.
• Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
• Transformation cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.
• Expression cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
• Suitable regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream
• Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
• a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
• promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. Promoters which are only turned on in response to an inducing stimulus are referred to as “inducible promoters”.
• the inducing stimulus may include a variety of agents including chemicals, the presence of common metabolic end products as well as physical stimuli such as heat. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
• operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
• a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
• Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
• expression as used herein is intended to mean the transcription and translation to gene product from a gene coding for the sequence of the gene product.
• RNA complementary RNA
• RNA complementary RNA
• the thus transcribed messenger RNA is translated into the above-mentioned gene product if the gene product is a protein.
• the term "fermentable carbon substrate” refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, carbon dioxide, methanol, formaldehyde, formate, and carbon-containing amines, and/or mixtures thereof.
• production host refers to a microorganism having the ability to produce tyrosine or phenylalanine at high levels (over-producer). Additionally, production hosts of the invention may comprise genes encoding enzymes having either TAL or PAL activity.
• Over-producing strain refers to a recombinant microorganism that produces a gene product at a level that exceeds the level of production in normal or non-transformed microorganisms.
• over-producing strain as used herein will typically refer to those microbial strains that overproduce either tyrosine or phenylalanine.
• accumulation when applied to an aromatic amino acid means that the aromatic acid concentration within the growth medium increases over time.
• the term “rate” means amount of product made per unit time, wherein the amount of product will be expressed as a concentration i.e. g/L, or mM, for example.
• concentration may be defined as the final titer of product, or final concentration of product at the end of a fermentation run.
• physiological pH or “physiological conditions” refers to the pH range at which the production host retains good growth and active metabolism. Although most bacterial production hosts operate optimally at a pH of about 6.5 to about 7.5, there are those that operate optimally outside that range and their "physiological pH” would correspond to those subjective conditions.
• alkaline pH means a pH equal to or above 8.0.
• Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter "Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L.
• the invention relates to a method for the production of pHCA or CA in a fermentation process.
• the production of cinnamic acid follows from the contacting of phenylalanine with an enzyme having PAL activity.
• pHCA The production of pHCA follows from the contacting of tyrosine with an enzyme having TAL activity. [TAL] tyrosine pHCA
• the process of the invention proceeds in two stages.
• the first stage comprises providing a microbial production host having an enhanced ability to produce the aromatic amino acids tyrosine or phenylalanine (an over-producer). These cells are grown at physiological pH to a point where the amino acid is accumulated in the growth medium. At this point it may be optimal to isolate the tyrosine or phenylalanine from the growth media before further processing, however it is not necessary to do so.
• the cells are contacted with a source of either TAL (in the case of the tyrosine producing cells or PAL (in the case of phenylalanine producing cells) at a pH of about 8.0 to about 11.0.
• the process again comprises two stages; however, the microbial production host is now provided with the ability to produce the aromatic carboxylic acid.
• the overproducing production host will additionally have the ability to express either TAL or PAL.
• cells are grown at physiological pH to accumulate aromatic amino acid and in the second stage, the pH is increased to about 8.0 to about 11.0 to maximize the conversion of aromatic amino acid substrate to the corresponding aromatic carboxylic acid product.
• Sources of TAL and PAL The present invention makes use of the enzymes having either tyrosine ammonium lyase (TAL) activity or phenylalanine ammonium lyase (PAL) activity. Enzymes having these activities are ubiquitous and easily obtained by the person of skill in the art. Phenylalanine Ammonium Lyase (PAL), and Tyrosine Ammonium
• Lyase Genes encoding PAL are known in the art and several have been sequenced from both plant and microbial sources (see for example EP 321488 [R. toruloides]; WO 9811205 [Eucalyptus grandis and Pinus radiata]; WO 9732023 [Petunia]; JP 05153978 [Pisum sativum];
• WO 9307279 [potato, rice]).
• the sequence of PAL genes is available (see for example GenBank AJ010143 and X75967).
• the gene encoding the PAL enzyme from Rhodotorula glutinis is given as SEQ ID NO:1.
• the wild type gene may be obtained from any source including but not limited to, yeasts such as Rhodotorula sp., Rhodosporidium sp.
• Rhodotorula sp. Rhodosporidium sp., Rhodotorula glutinis, and Sporobolomyces sp. are preferred.
• genes isolated from the organisms Rhodotorula sp., Rhodosporidium sp., Rhodotorula glutinis, and Sporobolomyces sp. are preferred.
• Several of the PAL enzymes mentioned above have some substrate affinity for tyrosine.
• genes encoding TAL activity may be identified and isolated concurrently with the PAL genes described above.
• the PAL enzyme isolated from parsley (Appert et al., Eur. J. Biochem. 225:491 (1994)) and corn ((Havir et al., Plant Physiol. 48:130 (1971)) both demonstrate the ability to use tyrosine as a substrate.
• the PAL enzyme isolated from Rhodotorula glutinis, (Hodgins DS, J. Biol. Chem. 246:2977 (1971)), given as SEQ ID NO:2 also may use tyrosine as a substrate.
• Such enzymes will be referred to herein as PAL/TAL enzymes or activities.
• genes isolated from maize, wheat, parsley, Rhizoctonia solani, Rhodosporidium, Sporobolomyces pararoseus and Rhodosporidium may be used as discussed in Hanson and Havir, The Biochemistry of Plants; Academic: New York, 1981 ; Vol. 7, pp 577-625, where the genes isolated from Rhodosporidium sp. Rhodotorula glutinis, Trichosporon cutaneum, Rhodobacter sphaeroides, and Rhodobacter capsulatus are preferred.
• sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR)).
• PCR polymerase chain reaction
• LCR ligase chain reaction
• genes encoding homologs for anyone of the mentioned activities could be isolated directly by using all or a portion of the known sequences as DNA hybridization probes to screen libraries from any desired plant, fungi, yeast, or bacteria using methodology well known to those skilled in the art.
• oligonucleotide probes based upon the literature nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of or full-length of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
• two short segments of the literature sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
• the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the literature sequences, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding bacterial genes.
• the second primer sequence may be based upon sequences derived from the cloning vector.
• the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the literature sequences. Using commercially available 3' RACE or 5' RACE systems (Invitrogen, Carlsbad, CA), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
• the TAL or PAL enzymes may used in the present invention as being expressed in the microbial production host or in some other microbial cell. Methods for the transformation of host cells with the appropriate genes are discussed below.
• partially purified or purified enzyme may be added to the fermentation culture at a point where the pH is in the alkaline range, thereby effecting the production of the desired aromatic carboxylic acid product. Methods for obtaining purified or partially purified enzyme are common and well known.
• TAL or PAL may be isolated from microbial cells in the following manner. The cells are separated from the culture medium using known methods including, but not limited to centrifugation or filtration.
• the cells are washed and then disrupted using a French press, an ultrasonic disrupter, a homogenizer, a Dyno Mill, or other means known in the art, to obtain a cell-free extract.
• the cell-free extract is centrifuged to remove cell debris.
• the cell- free extract is used as the source of enzyme activity.
• the TAL or PAL enzyme is purified from the cell-free extract using methods known in the art, including but not limited to ammonium sulfate precipitation, anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, electrophoresis and the like.
• the culture medium may be treated in the same manner as described for the cell-free extract to obtain the purified enzyme.
• the enzyme may be immobilized prior to use.
• Methods of cell and enzyme immobilization are well-know in the art (see for example, Weetal, Methods in Enzymology, Vol. XLIV, K. Mosbach, ed., Academic Press, New York (1976), Bickerstaff, Immobilization of Enzymes and Cells, Methods in Biotechnology Series, Humana Press, Totowa, NJ (1997), and Taylor, Protein Immobilization: Fundamentals and Applications, BioProcess Technology, vol. 14, Marcel Dekker, New York (1991 )).
• the enzyme source having TAL or PAL activity may be immobilized by entrapment in a polymer gel, adsorption onto a solid support, covalent crosslinking using a bifunctional reagent, or covalent binding to an insoluble matrix, such as glass, polystyrene, nylon, or polyacrylic acid derivatives.
• a cell-free extract or the purified enzyme is immobilized by covalent. attachment to oxirane acrylic beads, available from Sigma Chemical (St. Louis, MO).
• wildtype or recombinant host cells are immobilized by entrapment in calcium alginate beads, as described by Bickerstaff, supra.
• the entrapped cells may be cross-linked by polyethyleneimine and glutaradehyde or other suitable crosslinking agents known in the art.
• Microbial Production Hosts The invention provides microbial production hosts which have the ability to produce the aromatic amino acids, tyrosine and phenylalanine.
• a number of microbial host cells are suitable for this purpose including, but are not limited to Escherichia, Methylosinus, Methylo onas, Pseudomonas, Streptomyces, Corynebacterium, and Rhodobacter.
• the preferred host cells of the instant invention are Escherichia coli and Pseudomonas putida.
• the most preferred host cells of the instant invention are mutant strains of these bacteria that overproduce either phenylalanine or tyrosine.
• Tyrosine overproducing strains are preferred for the production of pHCA using an enzyme having TAL activity.
• phenylalanine overproducing strains are preferred for the production of CA using an enzyme having PAL activity.
• Tyrosine overproducing strains are known and include, but are not limited to Corynebacteria, Brevibacteria, Microbacterium, E. coli, Arthrobacter, Candida, Citrobacter, Pseudomonas and Methylomonas.
• Particularly useful tyrosine overproducing strains include but are not limited to Microbacterium ammoniaphilum ATCC 10155, Corynebactrium lillium NRRL-B-2243, Brevibacte um diva ⁇ catum NRRL-B-2311 , Arthrobacter citreus ATCC 11624, and Methylomonas SD-20.
• Other suitable tyrosine over-producers are known in the art, see for example Microbial production of L-tyrosine: A Review, T. K. Maiti et al, Malawistan Antibiotic Bulletin, vol 37, 51-65, 1995. Additionally an example of an
• Escherichia tyrosine overproducing strain that may be used is E. coli TY1 , available from OmniGene Bioproducts, Inc. Cambridge, MA.
• Phenylalanine overproducing strains are known and include but are not limited to E. coli, Microbacterium Corynebacteria, Arthrobacter, Pseudomonas and Brevibacteria. Particularly useful phenylalanine overproducing strains include, but are not limited to Microbacterium ammoniaphilum ATCC 10155, Corynebactrium lillium NRRL-B-2243, Brevibacterium divaricatum NRRL-B-2311 , E. coli NST74 and Arthrobacter citreus ATCC 11624.
• phenylalanine overproducing strains are known and a review may be found in Maiti et al, supra and Metabolic Engineering For Microbial Production Of Aromatic Amino Acids And Derived Compounds, J. Bongaertes et al., Metabolic Engineering vol 3, 289-300, 2001.
• a phenylalanine overproducing strain that may be used is E. coli NST74, available as strain ATCC No. 31884 from the American Type Culture Collection, Manassas, VA.
• the production host may additionally contain genes that encode enzymes having TAL or PAL activity.
• the TAL and PAL enzymes may be expressed in a host that is not the production host but is simply suitable for the expression of these enzymes.
• methods of introducing the genes into the appropriate host cell using appropriate vectors and transformation techniques are well known and protocols are commonly available (see Maniatis, supra).
• Vectors or cassettes useful for the transformation of suitable host cells are well known in the art.
• the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
• Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination.
• both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
• Initiation control regions or promoters which are useful to drive expression of the instant TAL or PAL gene in the desired host cell are numerous and familiar to those skilled in the art.
• any promoter capable of driving these genes is suitable for the present invention including, but not limited to: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, are, tet, trp, IP L , IP R , T7, tac, and trc (useful for expression in Escherichia coli) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus.
• Termination control regions may also be derived from various genes native to the preferred hosts.
• a termination site may be unnecessary; however, it is most preferred if included.
• cells would be grown at physiological pH for a time sufficient to accumulate the aromatic amino acid in the growth medium.
• an inducer could be added to the culture that would stimulate the expression of the TAL or PAL gene concurrently with the raising of the pH to the alkaline range. In this manner a single cell could serve as the source of both the aromatic amino acid as well as the enzyme.
• Inducible promoters are common and well known in the art (see Maniatis, supra) Once the vectors are constructed, host cells may be transformed by the common techniques of electroporation or conjugal mating (see Maniatis, Supra)
• a production host having the ability to over-produce tyrosine or phenylalanine is grown at physiological pH (typically, about 6.5 to about 7.5) for a time sufficient to allow the accumulation of the aromatic amino acid.
• physiological pH typically, about 6.5 to about 7.5
• cells in this stage are grown to stationary phase.
• the culture is contacted with a source of enzyme having either TAL or PAL activity depending on the nature of the aromatic amino acid produced by the host cell.
• the pH of the system is raised to a pH of about 8.0 to about 11.0, where a pH of about 9.5 to about 10.5 is preferred.
• the production host is no longer viable, however the rate and yield of production of the aromatic carboxylic acid product are enhanced at this pH.
• About a four-fold improvement in yield has been seen over single stage cultures where the pH was maintained at physiological conditions.
• the present invention may be practiced in two stages where, in the first stage the production host comprises the genes encoding enzymes having either TAL of PAL activity.
• the production host will not only have the ability to overproduce the appropriate aromatic amino acid but will also be equipped with the enzyme suitable for its conversion to the desired aromatic carboxylic product.
• a tyrosine over-producing host will be transformed with a gene encoding an enzyme having TAL activity and a phenylalanine host will be transformed with a gene encoding an enzyme having PAL activity.
• Production hosts are grown under physiology conditions (pH of about 6.5 to about 7.5, for industrially useful bacteria and for yeasts about 3.5 to about 7.5) in the first stage.
• the pH is raised to about 8.0 to about 11.0 to effect the enhanced rate and yield of production of pHCA or CA.
• the presence of ammonia in the culture may have an inhibitory effect on the rates and yield of the TAL or PAL catalyzed reaction.
• removal of ammonia may enhance the rates and yields of this reaction.
• Removal of ammonia may be accomplished by means well known in the art. For example aeration or the addition of specific sorbents, such as the mineral clinoptilolite or ion exchange resins are typically suitable means.
• sorbents such as the mineral clinoptilolite or ion exchange resins are typically suitable means.
• Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof.
• the growth medium used is not critical, but it must support growth of the microorganism used and promote the enzymatic pathway necessary to produce the desired product.
• a conventional growth medium may be used, including, but not limited to complex media, containing organic nitrogen sources such as yeast extract or peptone and a fermentable carbon source; minimal media and defined media.
• Suitable fermentable carbon sources include, but are not limited to monosaccharides, such as glucose or fructose; disaccharides, such as lactose or sucrose; oligosaccharides and polysaccharides, such as starch or cellulose; one-carbon substrates such as carbon dioxide, methanol, formaldehyde, formate, and carbon-containing amines and/or mixtures thereof.
• the growth medium must contain a suitable nitrogen source, such as an ammonium salt, yeast extract or peptone; minerals, salts, cofactors, buffers and other components, known to those skilled in the art (Bailey et al. supra).
• Extractants useful for this purpose are water immiscible organic solvents and may includem but are not limited to, diisopentyl ether, n-propyl benzoate, 2-undecanone, dibenzyl ether, 2-tridecanone, 2-decanone, 1-pentanone 1-phenyl, methyl decanoate, 1-undecanol, diisobutyl DBE-IB and mixtures thereof.
• the pHCA or CA is dissolved in the extractant and removed from the medium.
• the pHCA or CA may then be recovered from the extractant by well known means such as distillation, adsorption by resins, or separation by molecular sieves.
• the pHCA or CA may be recovered by acidification of the growth medium to a pH below 2.0, followed by crystallization.
• EXAMPLES The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
• the LB culture medium used in the Examples contains the following per liter of medium: Bacto-tryptone (10 g), Bacto-yeast extract (5 g), and NaCI (10 g).
• E. coli strain DPD4009 is a tyrosine-overproducing, plasmid-Iess, phenylalanine auxotroph, which was derived in several steps from E.
• coli TY1 DGL430
• OmniGene Bioproducts, Inc. OmniGene Bioproducts, Inc.
• TY1 was cured of the plasmid it was carrying to yield a tetracycline-sensitive strain called TS5.
• TS5 was the recipient in a P1 -mediated transduction using E. coli strain CAG12158, which carries pheA18::Tn10 (Coli Genetics Stock Center, Yale University, #7421), as the donor.
• One tetracycline-resistant transductant was called BNT565.2.
• E. colistrain WS158 was constructed using the two PCR fragments integration method described by Suh in copending and commonly owned U.S.
• Patent Application No. 10/734936 incorporated herein by reference, via /l-Red recombinase system.
• a first linear DNA fragment (1581 bp) containing a kanamycin selectable marker flanked by site-specific recombinase target sequences (FRT) was synthesized by PCR from plasmid pKD4 (Datsenko and Wanner, Proc. Natl. Acad. Sci. 97:6640- 6645 (2000)) with primer pairs, T-kan(tyrA) (5'-
• AATTCATCAGGATCTGAACGGGCAGCTGACGGCTCGCGTGGCTTAAC GTCTTGAGCGATTGTGTAG-3' (SEQ ID NO:3) which contains a homology arm (underlined, 46 bp) chosen to match sequences in the upstream region of the aroF stop codon and a priming sequence (20 bp), and B-kan(trc) (5'-
• a second linear DNA fragment (163 bp) containing a Ptrc promoter comprised of the -10 and -35 consensus sequences, lac operator (lacO), and ribosomal binding site (rbs) was synthesized by PCR from plasmid pTrc99A (Invitrogen, Carlsbad, CA) with primer pairs, T-trc(kan) (5'- CTAAGGAGGATATTCATATTCGTGTCGCTCAAGGCGCACT-3') (SEQ ID NO:5) that contains a homology arm (underlined, 18 bp) chosen to match sequences in the downstream region of the kan open reading frame and a priming sequence (22 bp), and B-trc(tyrA) (5'- CGACTTCATCAATTTGATCGCGTAATGCGGTCAATTCAGCAACCATG GTCTGTTTCCTGTGTGAAA-3') (SEQ ID NO:6) that contains a homology arm (underlined, 46 bp) chosen to match sequence
• the underlined sequences illustrate each respective homology arm, while the remainder is the priming sequences for hybridization to complementary nucleotide sequences on the template DNA for the PCR reaction.
• Standard PCR conditions were used to amplify the linear DNA fragments using the MasterAmp Extra-Long PCR kit (Epicentre, Madison, Wl) as follows.
• the PCR reaction mixture contained 1 ⁇ L of plasmid DNA, 25 ⁇ L of 2X PCR buffer #1 , 1 ⁇ L of the 5'-primer (20 ⁇ M), 1 ⁇ L of the 3'-primer (20 ⁇ M), 0.5 ⁇ L of MasterAmpTM Extra-Long DNA polymerase, and 21.5 ⁇ L of sterilized, deionized H 2 0.
• the PCR reaction conditions were: 94 °C for 3 min; 25 cycles of 93 °C for 30 sec, 55 °C for 1 min, and 72 °C for 3 min; followed by 72 °C for 5 min.
• the PCR products were purified using the Mini-elute QIAquick Gel Extraction Kit TM (QIAGEN Inc. Valencia, CA).
• the DNA was eluted with 10 ⁇ L of distilled water by centrifuging twice at high speed.
• the concentration of the isolated PCR product was about 0.5-1.0 ⁇ gl ⁇ L.
• coli MC1061 strain carrying a ⁇ -Red recombinase expression plasmid was used as a host strain for the recombination of PCR fragments.
• This strain was constructed by transformation with a ⁇ -Red recombinase expression plasmid, pKD46 (amp R ) (Datsenko and Wanner, supra) into E. coli strain MC1061 (Coli Genetics Stock Center, Yale University, #6649).
• the ⁇ -Red recombinase in pKD46 is comprised of three genes exo, bet, and gam, expressed under the control of an arabinose-inducible promoter.
• E. coli MC1061 strain carrying pKD46 were prepared as follows. E. coli MC1061 cells carrying pKD46 were grown in SOB medium (Hanahan, DNA Cloning: A Practical Approach, D.M. Glover, ed., IRL Press, Washington, DC, 1985, pp. 109-125) with 100 ⁇ g/mL ampicillin and 1 mM L-arabinose at 30 °C to an OD 60 o of 0.5, followed by chilling on ice for 20 min.
• SOB medium Hanahan, DNA Cloning: A Practical Approach, D.M. Glover, ed., IRL Press, Washington, DC, 1985, pp. 109-125
• Bacterial cells were centrifuged at 4,500 rpm using a Sorvall ® RT7 PLUS (Kendro Laboratory Products, Newton, CT) for 10 min at 4 °C. After decanting the supernatant, the pellet was resuspended in ice-cold water and centrifuged again. This process was repeated twice and the cell pellet was resuspended in 1/100 volume of ice-cold 10 % glycerol. Both the kanamycin marker PCR products ( ⁇ 1 ⁇ g) and Ptrc promoter PCR products ( ⁇ 1 ⁇ g) were mixed with 50 ⁇ L of the competent cells and pipetted into a pre-cooled electroporation cuvette (0.1 cm) on ice.
• Electroporation was performed using a Gene Pulser System (Bio-Rad Laboratories, Hercules, CA) set at 1.8 kV, 25 ⁇ F with the pulse controller set at 200 ohms. SOC medium (1 mL) was added after electroporation. The cells were incubated at 37 °C for 1 h. Approximately one-half of the cells were spread on LB plates containing 25 ⁇ g/mL kanamycin. After incubating the plate at 37 °C overnight, six kanamycin resistant transformants were selected. The chromosomal integration of both the kanamycin selectable marker and the Ptrc promoter in front of the tyrA gene was confirmed by PCR analysis.
• a colony of transformants was resuspended in 25 ⁇ L of PCR reaction mixture containing 23 ⁇ L of SuperMix (Invitrogen), 1 ⁇ L of 5'-primer T-ty(test) (5'-CAACCGCGCAGTGAAATGAAATACGG-3') (SEQ ID NO:7) and 1 ⁇ L of 3'-primer B-ty(test) (5'-
• test primers were chosen to amplify regions located in the vicinity of the integration region. PCR analysis with the T-ty(test) and B-ty(test) primer pair revealed the expected size fragment, i.e., 1 ,928 bp on a 1 % agarose gel. The resultant recombinant is designated herein as E. coli WS158. Strain BNT565.2, prepared as described above, was then used as the recipient in another P1 -mediated transduction with phage grown on E.
• E. coli Strain DPD4512 was constructed by transformation of E.
• Plasmid pCA16 was prepared as follows.
• Rhodotorula glutinis mRNA was reversed transcribed according to the Perkin Elmer (Norwich, CT) GeneAmp Kit instructions without diethylpyrocarbonate (DEPC) treated water.
• the primers used were the random hexamers supplied with the kit.
• Primers used to amplify the pal gene included the upstream primer 5'-
• E. coli DPD4515 Tyrosine producing strain E. coli DPD4515 was constructed by transformation of E.
• E. coli strain DPD4009 using plasmid pCL101 EA, which carries E. coli aroEACBL genes in pCL1920 (obtained from Central Bureau for Fungal Cultures, Baarn, The Netherlands), and selection for spectinomycin resistance.
• the pCL101 EA plasmid was constructed as described by Valle et al. in U.S. Patent Application Publication No. 2002/0155521 (in particular Example 7), which is incorporated herein by reference.
• Construction of E. coli Strain DPD5040 E. coli strain DPD5040 was constructed by transformation of E. coli
• E. coli strain DPD5013 was constructed by transformation of E. coli Strain DPD4009 with pCL.PAL. Plasmid pCL.PAL was constructed by isolating the Ptac promoter-R. glutinis PAL gene cassette from pCA16 plasmid via BamHI and HindlW digestion, and subcloning into the BamHI and Hind ⁇ restriction sites in the polylinker of plasmid pCL1920. pCL.PAL carries a spectinomycin resistance gene, and it allows the expression of the R.
• E. coli Strain DPD5041 was constructed by transforming E. coli tyrosine producing strain DPD4515 with plasmid pLH276. Plasmid pLH276 was constructed from plasmid pLH273 as follows. Plasmid pLH273 contains a bacteriophage T5 promoter cassette cloned into plasmid pACYC184 (GenBank/EMBL Ace. No. X06403, available from New England Biolabs, Inc., Beverly, MA).
• the T5 promoter cassette was isolated from pQE70 (Qiagen, Valencia, CA) by Xho ⁇ -Nhe ⁇ digestion. This DNA fragment containing the T5 promoter followed by a multiple cloning site was ligated into plasmid pACYC184 digested by the compatible Sa/I and Xba ⁇ restriction enzymes to give plasmid pLH273. Plasmid pLH273 is a low copy stable expression vector with p15A origin of replication for the expression of proteins in E. coli.
• Plasmid pLH276 was generated by PCR amplification of the pal gene from pCA16 using the 5' PCR primer of 5'- CTCGCTCGACTCGATCTCGCACTCGTTCGCAAACG-3' (SEQ ID NO:11 ) and the 3' PCR primer of 5'- TTGAACTCGAACTCGATCGCGCGCAAGTCG-3' (SEQ ID NO:12) by Pfx DNA polymerase (Invitrogen, Carlsbad, CA). This PCR fragment was digested by Sph ⁇ and Hind ⁇ restriction enzymes, and ligated into the Sph ⁇ , Hind ⁇ digested pLH273 vector, to give pLH276. Plasmid pLH276 is an expression vector for R.
• E. coli Strain DPD5047 was constructed by transforming E.coli strain BL21 (DE3) [genotype: recA1 endA1 hsdR17 supE44 thi-1 gyrA96 re!A1 f ⁇ OlacZ dM15 d(lacZYA- argF)U169 (DE3)], obtained from Stratagene (La Jolla, CA) with plasmid pCA16, which is described above. This strain expresses the PAL enzyme from R. glutinis in an E. coli B host.
• the samples were run using a flow rate of 1.0 mL/min with an eight-minute solvent gradient from 5% acetonitrile/water, containing 0.1 % trifluoroacetic acid, to 80% acetonitrile/water, containing 0.1% trifluoroacetic acid.
• a diode-array detector set at 210, 260, and 280 nm was used to detect the various compounds at their optimum absorbance wavelength.
• the aqueous samples were diluted 1 :3 with water before injection of 50 ⁇ L of the sample into the HPLC.
• a standard mixture of the analytes was prepared by mixing equal portions of solutions of the individual components, each at a concentration of 1 mg/mL.
• Spectrophotometric Assay for PAL and TAL Activity The method used to determine tyrosine ammonia lyase activity and phenylalanine ammonia lyase activity is based on the method reported by Abell and Shen (Methods Enzymol. 142:242-248 (1987)).
• To determine PAL activity the reaction was initiated by adding cell free extract to a solution containing 1 mM L-phenylalanine in 50 mM Tris-HCI (pH 8.5) buffer. The reaction was followed spectrophotometrically by monitoring the absorbance of the product, cinnamate, at 290 nm using a molar -1 extinction coefficient of 9000 cm .
• the assay was run over a 3 min period using an amount of enzyme that produced a change in absorbance in the range of 0.0075 to 0.018 /min.
• One unit of activity is defined as the amount of enzyme that deaminates 1 ⁇ mol of phenylalanine to cinnamate per min.
• TAL activity was measured similarly with tyrosine replacing phenylalanine in the reaction solution.
• the absorbance of the para- hydroxycinnamate produced was followed at 315 nm with an extinction coefficient of 10,000 cm .
• One unit of activity is defined as the amount of enzyme that deaminates 1 ⁇ mol of tyrosine to para-hydroxycinnamate per min. With the R.
• EXAMPLES 1-12 The Effect of pH on the Production of pHCA in Two-Stage Fermentations The purpose of these Examples was to demonstrate the effect of increasing the pH during fermentation on the production of pHCA by various recombinant strains. In these Examples the fermentation was carried out at an initial pH of 6.5 to 7.0 and the pH was changed at a time during the fermentation to a pH from 6.1 to 9.7.
• the strains used in these Examples were E.coli DPD5013, E. coli DPD4512, and E.coli DPD5040, constructed as described above. These strains are recombinant E.
• the pre-seed culture was a frozen culture of the desired recombinant E. coli strain.
• the seed culture was grown for approximately 15 h in a 2 L flask with 500 mL of seed medium, incubated in a gyratory shaker (New Brunswick Scientific, Edison, NJ), at 300 rpm and 35 °C.
• the seed medium consisted of KH2PO4 (1 g/L), Na2HP04 (3 g/L),
• the fermentation medium consisted of the following in a 7.5 L volume: KH 2 P0 4 (7 g), Na 2 HP0 4 (17 g), MgS0 4 -7H20 (4 g), (NH 4 ) S0 4 (8 g), thiamine (8 mg), phenylalanine (320 mg), and Mazu DF204 Defoamer (8 mL) with the pH adjusted to 6.5.
• KH 2 P0 4 7 g
• MgS0 4 -7H20 (4 g)
• (NH 4 ) S0 4 8 g
• thiamine 8 mg
• phenylalanine 320 mg
• Mazu DF204 Defoamer 8 mL
• the dissolved oxygen concentration was controlled at 25% of air saturation with a cascade of agitation from 400 to 1500 rpm and aeration followed from 2 to 10 SLPM.
• the temperature was controlled at 35 °C and the head pressure was 50 kPa.
• the pH was controlled at 6.5 or 7 with 20% w/v H3PO4 and 40% w/v NH4OH.
• the fermentation was done in a fed-batch mode with the addition of glucose (60% w/w).
• the glucose feed was initiated when the concentration dropped below 4 g/L.
• the following formula was used to calculate the glucose feed rate in g/min: OD550 x Ferm. wt. x 0.001762.
• the enhancement of pHCA production at higher pH may be due to mitigation of pHCA toxicity at pH 6.1 to 7.5, and to enhancement of TAL activity and improved Tyr accessibility to the enzyme at a pH above 7.8.
• These results demonstrate the enhanced production of pHCA by recombinant strains of E. coli in a two-stage fermentation wherein the pH is increased to alkaline values during the fermentation.
• EXAMPLES 13-17 Effect of pH on the Biocatalytic Conversion of Tyrosine to pHCA (Study #FL-2003- 16) The purpose of these Examples was to demonstrate the effect of pH on the biocatalytic conversion of tyrosine to pHCA using recombinant E. coli strains containing a TAL enzyme activity.
• coli DPD4515 The protocols for seed culture production and fermentation were similar to those described in Examples 1-12.
• the dissolved oxygen concentration was controlled at 25% of air saturation with a cascade of agitation that followed with aeration. Agitation varied from 500 to 1500 rpm and aeration from 2 to 8 SLPM.
• the head pressure was controlled at 50 kPa.
• the pH was maintained at 6.8 with the addition of NH4OH (40% w/v) initially, KOH (50% w/v) after 45 h, and acid titration with H3PO4 (20% w/v).
• Glucose feed was started with a 60% w/w glucose solution when the concentration fell below 4 g/L.
• feed rate g/min OD550 X Ferm. wt. X 0.001762.
• a lower glucose feed rate was used if glucose accumulated above 0.5 g/L. IPTG was added to a concentration of 1.0 mM when the culture reached an OD550 between 8 and 10. The fermentation was terminated at 72 h. Three fermentation runs as described above were run in parallel (pHCA212, -213, & -214).
• EXAMPLE 28 The Effect of Ammonium Ion on the Conversion of Tyrosine to pHCA (Experiment #FL-2004-11)
• the purpose of this Example was to demonstrate the inhibitory effect of ammonium ion concentration on the biocatalytic conversion of tyrosine to pHCA. These studies were done using 50 mL reaction mixtures in 250 mL baffled flasks. The reaction mixture contained 0.1 M CAPS buffer, 61 g/L of wet cells (E. coli DPD5047), 46.5 g/L tyrosine, obtained from Sigma Chemical Co. or J.T. Baker (Phillipsburg, NJ), and NH4CI at various concentrations. The pH was adjusted to 10.0 with KOH.
• the tyrosine is added back to the fermentor, which is filled with about 7.5 kg of water and the pH is adjusted to 10.0 using 50% w/w sodium hydroxide.
• Frozen cell paste from the fermentation of E. coli DPD5040, as described in Example 11 is thawed and 250 g is suspended to homogeneity in 0.5 L of cold 0.05 M potassium phosphate buffer at pH 10.0.
• the cell suspension is pumped into the fermentor.
• the fermentor is operated at 35 °C and 600 rpm and the pH is controlled at 10.0 by the addition of sodium hydroxide (50% w/w) and H3PO4 (20% w/v).
• the biocatalytic reaction is allowed to proceed for 16 h, producing pHCA, which accumulates in the reaction medium.
• the pHCA-containing medium is centrifuged and the solids are discarded.
• the supernatant is transferred to the fermentor, operated at 35 °C and 600 rpm.
• the pH of the solution is adjusted to 9.0 using 20% w/v sulfuric acid.
• 0.254 mL of Alcalase® which may be obtained from Novozymes, Krogshoejvej 36, 2880 Bagsvaerd, Denmark
• 0.134 g of Bromelain Acros Organics, which may be obtained from Fisher Scientific, Pittsburgh, PA
• the solution is titrated to pH 2.2 with 20% w/v to precipitate the pHCA.
• the resulting suspension is centrifuged and the pHCA is recovered as a wet cake.

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