WO2006134374A2

WO2006134374A2 - A method for production of l-ascorbic acid

Info

Publication number: WO2006134374A2
Application number: PCT/GB2006/002203
Authority: WO
Inventors: Robert Douglas Hancock; Gary Davidson Hunter; Jane Shaw; Roberto Viola
Original assignee: Scottish Crop Research Institute
Priority date: 2005-06-15
Filing date: 2006-06-15
Publication date: 2006-12-21
Also published as: WO2006134374A3; GB0512150D0

Abstract

There is provided a method for producing L-ascorbic acid (vitamin C) in which the enzymes tagatose epimerase and fucose isomerase are co-incubated with a micro-organism able to convert L-galactose to L- ascorbic acid. The micro-organism can be a yeast or a fungus. A recombinant polynucleotide comprising the sequence encoding for tagatose epimerase or fucose isomerase is also described, and the invention also includes a vector comprising such a polynucleotide and host cells transformed therewith.

Description

A Method for Production of L-Ascorbiα Acid

The present invention provides an improved method for the production of L-ascorbic acid.

L-Ascorbic acid (AsA) is a global commodity with applications in the pharmaceutical, cosmetic, food and beverage, agriculture and aquaculture industries (Hancock and Viola, 2002) . It has a global annual market in the region of US$ 500 million and is produced primarily in the USA, Europe and Asia where China has a global market share of 65% (Business Communications Co., 2004).

Industrial synthesis of AsA is currently undertaken via two principal methods. The Reichstein process is a chemical process in which L-sorbose, produced by bacterial fermentation is converted to AsA through a series of four chemical steps. Alternatively, L-sorbose is fermented to 2-keto-L- gulonic acid which is subsequently converted to AsA by chemical means. Significant investment in process improvement has resulted in relatively efficient syntheses with approximate AsA yields of 60% using either method. However both processes have significant limitations. The need for organic and inorganic solvents and heavy metal catalysts in the Reichstein process imposes a requirement for strict environmental controls resulting in significant waste disposal costs. Additionally, the high energy inputs required for many steps add to costs. Although many of these problems are absent in the fermentation process, this process does not result in the synthesis of AsA but the precursor 2- keto-L-gulonic acid which must be subsequently converted to AsA, thereby reducing overall process efficiency. One major barrier to the development of a process for the direct fermentation of AsA is the lack of commonly used industrial microorganisms that are capable of its synthesis. Previous work has demonstrated the utility of microalgae (Berry et al . , 2002). However these organisms are not commonly used in industrial processes and have the major limitation that such organisms grow only at low densities.

We have now found a novel methodology for the induction of the AsA biosynthetic pathway in any organism that has enzymes capable of converting L- galactose to AsA. In addition to plants (Wheeler et al., 1998) and algae (Running et al., 2003) such organisms include those that normally synthesise compounds that are structurally related to AsA. Examples include, but are not limited to, yeast such as Saccharomyces cerevisiae (Hancock et al., 2000) and Pichia pastoris that synthesise D- erythroascorbic acid from D-arabinose using the enzymes D-arabinose dehydrogenase and D-arabinono- 1,4-lactone oxidase and multicellular fungi such as Aspergillus nidulans, Basidiomycetes and Actinomycetes that additionally synthesise β-deoxy L-ascorbic acid (Okarαura, 1998).

In the method described herein, L-galactose is synthesised using the biological activity of bacterial enzymes. The first enzyme required is any enzyme capable of epimerisation of L-ketohexoses at the C3 position (e.g. tagatose epimerase) and specifically epimerisation of L-sorbose to L- tagatose. The second enzyme required is any enzyme capable of converting L-ketohexose to L-aldohexose and specifically L-tagatose to L-galacose (e.g. L- fucose isomerase; E. C. 5.3.1.25). Enzymes endogenously expressed in a suitable organism that contain appropriate enzyme cofactors then convert L- galactose to AsA. A putative mechanism is outlined in Fig. 1. An advantage of the methodology used is that L-AsA is produced using the inexpensive starting material L-sorbose.

Potential applications include but are not limited to: a) fermentation processes for the production of AsA; and/or b) a method for the production of AsA enhanced plants, algae, yeast and fungi. In one aspect, the present invention provides a method of producing L-ascorbic acid, said method comprising co-incubating a source of the enzymes tagatose epimerase and fucose isomerase with a micro-organism able to convert L-galactose to L- ascorbic acid.

In one embodiment the micro-organism able to convert L-galactose to L-ascorbic acid is yeast. One exemplary yeast is Saccharomyces cerevlsiae. Alternatives include fungus, for example Aspergillus nidulans.

In one embodiment the enzyme tagatose epimerase is obtained from Agrobacterium tumefaciens C58 (NCBI Accession No. AE008210) .

In one embodiment the enzyme fucose isomerase is obtained from E coli.

By "obtained from" we mean that the enzyme originates from the micro-organism specified. In one embodiment, the enzyme may be present in a purified or partially purified form, but this may not always be essential and a crude cell extract containing the enzyme may also be suitable in some circumstances. Use of the native genes encoding the enzyme from that organism to express the enzyme by genetic engineering means is also included.

Optionally the genes encoding these enzymes may be cloned and expressed in any suitable host micro- organism. Mention may be made of E coli or other bacteria such as Gluconobacter oxydans, Corynebacter±um sp. , Acetobacter liquifaciens; yeasts or fungi such as Saccharomyces cerevisiae, Pichia pastoris, Candida blankii, Aspergillus nidulans, as exemplary hosts micro-organisms. Optionally, the coding sequences for fucose isomerase and/or tagatose epimerase is optimised for expression in the expression in the selected host micro-organism.

In one embodiment, the enzymes expressed are transported to the outer surface of the host cell or are exported therefrom.

In one embodiment, the micro-organism able to convert L-galactose to L-ascorbic acid is itself transformed to additionally express one or both of tagatose epimerase and/or fucose isomerase, for example from recombinant genetic construct (s) . The present invention also provides a recombinant polynucleotide comprising a sequence as set out in SEQ ID Nos 1 or 3, or homologs thereof.

By the term "homologs" with reference to a polynucleotide, we refer to a polynucleotide modified by deletion, substitution or addition of nucleic acids to have at least 80% homology, preferably 85% homology, to the nucleotide sequence (s) as set out in the sequence listing. In one embodiment the homolog will have 90% or more homology, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, to the nucleotide sequence (s) as set out in the sequence listing and when assessed by direct sequence alignment and comparison sequence.

Sequence homology can be determined by direct best- fit sequence alignment and comparison, or by using any suitable homology algorithm, such as BLAST. BLAST is described by Altschul et al., in J MoI Biol 25:403 (1990). The score of an alignment, S, is calculated as the sum of substitution and gap scores. "Substantial homology" when assessed by BLAST refers to low Expectation (E) values. Expectation value is the number of different alignments with scores equivalent or better than S that are expected to occur in a database search by chance. The lower the E value, the more significant the score.

In particular, modifications to the nucleotide sequence which do not affect the amino acid expressed (due to redundancy in the genetic code) fall within the definition of "homolog". Additionally, the term "homologs" also includes a polynucleotide capable of hybridising to a polynucleotide comprising 15 contiguous bases from any one of SEQ ID Nos 1 or 3, preferably under stringent conditions. In one embodiment the polynucleotide hybridises to a polynucleotide comprising 20 or more contiguous bases (for example 25 to 50 contiguous bases) from any one of SEQ ID Nos 1 or 3, preferably under stringent conditions.

Stable hybridisation of polynucleic acids is a function of hydrogen base pairing. Hydrogen base pairing is affected by the degree to which the two polynucleotide strands in the duplex are complementary to each other and also the conditions under which hybridisation occurs. In particular salt concentration and temperature affect hybridisation. One of ordinary skill in the art would be aware that the effective melting temperature (E Tm) of the polynucleotide duplex is controlled by the formula. E Tm = 81.5 + 16.6(log M [Na⁺]) + 0.41(% G + C) - 0.72 (% formamide)

Where hybridisation is conducted under stringent conditions, only sequences having a high degree of complementary base pairs will remain in duplex form. As used herein the term "stringent conditions" with respect to hybridisation refers to IM Na⁺ at 65 to 68°C.

The polynucleotide can be DNA or RNA and can be single stranded or double stranded. Double stranded DNA (eg. cDNA) is usually convenient for most applications. The polynucleotide can be in the form of a vector, for example an expression vector.

The polynucleotides of the present invention can be isolated polynucleotides or can be incorporated into expression or cloning vectors. Such vectors can be used to transfect or transform host cells and the host cells cultured in conventional culture media according to methods described in the art.

Incorporation of cloned DNA into a suitable vector, transfection or transformation of host cells and selection of the transfected or transformed cells are all processes well known to those skilled in the art and numerous suitable methods are described in the literature (see, for example, Sambrook et al . , Molecular Cloning: A laboratory Manual, 2^nd edition, Cold Spring Harbor Laboratory Press, 1989) . Suitable host cells include bacterial, yeast, mammalian and plant cells. Generally the host cell will be selected to be compatible with the vector used.

In one embodiment, the present invention provides a polynucleotide comprising the nucleotide sequence as set out in SEQ ID No 1 or homologs thereof, and the protein expressed therefrom.

In one embodiment, the present invention provides a polynucleotide comprising the nucleotide sequence as set out in SEQ ID No 3 or homologs thereof, and the protein expressed therefrom.

In a further aspect, the present invention provides a protein comprising the amino acid sequence of SEQ ID Nos 2 or 4 or homologs thereof. By the term "homologs" with reference to a protein, we refer to a protein modified by deletion, substitution or addition of amino acids to have at least 80% homology, preferably 85% homology, to the amino acid sequence as set out in the sequence listing. In one embodiment the homolog will have 90% or more homology, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, to an amino acid sequence as set out in the sequence listing.

Additionally the present invention provides a protein expressed from a polynucleotide comprising the nucleotide sequence of SEQ ID Nos 1 or 3 or homologs of such polynucleotides.

In one embodiment the present invention provides a protein comprising the amino acid sequence as set out in SEQ ID No 2 or homologs thereof.

In one embodiment the present invention provides a protein comprising the amino acid sequence as set out in SEQ ID No 4 or homologs thereof.

For clarity the term "protein" is used herein to refer to a peptide and polypeptide and does not denote any particular size of the polymer.

A vector including such a recombinant polynucleotide is also encompassed by this invention. In a further aspect, the present invention provides a vector encoding tagatose epimerase from Agrobacterium tumefaciens.

In a further aspect, the present invention provides a vector encoding fucose isomerase from E coli.

Optionally the same vector may encode both enzymes.

The present invention also provides a host cell containing a vector as indicated above.

Optionally the host cell expresses the enzyme (s) in the presence of a micro-organism able to convert L- galactose^' to L-ascorbic acid, such that the provision of L-sorbose results in the production of L-ascorbic acid.

The L-ascorbic acid produced by the method of the invention may be purified and used as required. Alternatively, the L-ascorbic acid enriched biomass produced prior to purification of the L-ascorbic acid may itself find utility in specific applications.

Thus, in one aspect, the present invention provides a micro-organism (such as a microalgae, bacteria, plant cell culture or yeast) having an enhanced L- ascorbic acid content. In one embodiment the micro- organism is able to express tagatose epimerase and/or fucose isomerase (optionally from a recombinant genetic construct) and to convert L- galactose to L-ascorbic acid. Such micro-organisms are expected to be oxidatively stabilised and hence be of interest for the bioprocess industry. Additionally or alternatively the increased L- ascorbic acid content may give enhanced nutritional value when present in foodstuffs. Consequently the micro-organisms may find utility in the food or beverage industry.

The present invention will now be further described with reference to the following, non-limiting, examples and figures in which:

Figure 1 shows a putative mechanism for L-asorbic acid production;

Figure 2 gives the gene sequence of tagatose epimerase from Agrobacterium tumefaciens C58; underlined sections represent bases to which primers were generated in order to isolate and clone the gene.

Figure 3 is an SDS-PAGE gel showing the purification of recombinant tagatose epimerase; approximate MW (kDa) is shown on the left. Lane 1 - Crude protein extract, Lane 2 - Desalted protein extract, Lane 3 - Unbound protein, Lane 4 - 125 mM imidazole eluate, Lane 5 - 500 mM imidazole eluate.

Figure 4 gives the gene sequence of fucose isomerase from E coli Kl2; underlined sections represent bases to which primers were generated in order to isolate and clone the gene.

Figure 5 is an SDS-PAGE gel showing the purification of recombinant fucose isomerase; approximate MW (kDa) is shown on the left. Lane 1 - Crude protein extract, Lane 2 - Desalted protein extract, Lane 3 - Unbound protein, Lane 4 - 125 mM imidazole eluate, Lane 5 - 250 mM imidazole eluate, Lane 6 - 375 mM imidazole eluate, Lane 7 - 500 mM imidazole eluate.

Figure 6 shows HPLC analyis of L-sorbose incubated with tagatose epimerase and fucose isomerase; 20% L- sorbose was incubated for 3 days at 30⁰C with equal amounts of recombinant TE and FI. At the end of incubation, an aliquot was taken and the reaction stopped by boiling. After removal of precipitated proteins by centrifugation, reaction products were analysed by HPLC; and

Figure 7 shows HPLC analysis of L-sorbose incubated with tagatose epimerase, fucose isomerase and yeast. S. cerevisiae were cultured for 24 hours in a medium containing 2% L-sorbose that had been preincubated for 3 days with TE and FI. The lower trace shows authentic AsA and the upper trace shows clarified yeast extract. Insets shows the absorption spectra of the peaks indicated. Example 1

Materials and Methods

Growth and Maintenance of Micro-organisms Strains used are listed in Table 1.

Table 1 : Organisms and growth media

Growth media

1. LB broth

Peptone 10 g 1i-l, yeast extract 5 g 1 -1 NaCl 10 g I^"1, pH 7.5 with 5 M NaOH.

2. Overnight Express™ Instant TB Medium Instant TB powder 60 g I^"1 (VWR, Leicestershire, UK) , glycerol 10 ml I^"1, ampicillin 50 mg I^"1, pH 6.9 with 1 M NaOH or 1 M HCl. 3. Yeast complete medium Yeast nitrogen base (Sigma-Aldrich Co. Ltd., Dorset, UK) 6.7 g I^"1, glucose 20 g I^"1, complete drop-out powder 1.3 g I^"1.

4. Complete drop-out powder Adenine sulphate 2.5 g, L-arginine 1.2 g, L-aspartic acid 6.0 g, L-glutamic acid 6.0 g, L-lysine 1.8 g, L-methionine 1.2 g, L-phenylalanine 3.0 g, L-serine 22.5 g, L-threonine 12.0 g, L-tyrosine 1.8 g, L- valine 9.0 g, L-histidine 1.2 g, L-leucine 3.6 g, L- tryptophan 2.4 g, uracil 1.2g.

In all cases, media were supplemented with 15 g I^""1 bacteriological agar to produce solid media for plates.

All bacterial and yeast cultures were maintained on the appropriate solid medium at 4⁰C. The plates were routinely subcultured at monthly intervals and cultures grown at 37⁰C in the case of E. coli or 30⁰C in the case of A. tumefaciens, S. cerevisiae or P. pastoris. Glycerol stocks containing equal volumes of overnight culture and 80% glycerol were maintained at -80⁰C. In order to start cultures from glycerol stocks, cultures were streaked onto the appropriate solid medium. For growth in liquid media, cultures were shaken on a rotary shaker at 200 rpm in erhlenmeyer flasks. Cloning and Expression of Fucose Isomerase and Tagatose Epimerase Genes

Using pairwise alignment to the Pseudomonas cichorii tagatose epimerase gene (Ishida et al . , 1997), a gene encoding tagatose epimerase (TE) was identified in Agrobacterium tumefaciens C58 (NCBI Accesssion No: AE008210) . Similarly a gene encoding fucose isomerase (FI) was identified in Escherichia coli K12 (Accession No: NC 000913). Primers were designed according to the manufacturers instructions for the pENTR™ Directional TOPO Cloning Kit (Invitrogen, Paisley, UK) to facilitate cloning of the genes into the pENTR/D-TOPO entry vector. The TE primers were 5' CAC CAT GAA ACA CGG CAT CTA TTA TTC TTA CTG GG 3' (forward) and 5' GCC ACC AAG AAC GAA GCG GGA G 3' (reverse) and primers used to facilitate the cloning of FI were 5' CAC CAT GAA AAA AAT CAG CTT ACC G 3' (forward) and 5' TTA ACG CTT GTA CAA CGG ACC G 3' (reverse) . PCR products were generated and inserted into the entry vector following the manufacturer' s instructions. E. coli TOPlO chemically competent cells were transformed with the vector and transformants selected on LB containing kanamycin (50 μg ml^"1) . 5 clones were analysed by restriction digestion of plasmid DNA. The positive clones were sequenced using the Applied Biosystems Big Dye Terminator kit (Foster City, CA, USA) to check the integrity of the insert and to ensure it was cloned in the correct reading frame. The plasmid DNA of one positive clone of each gene was then selected and used for the LR Recombination Reaction into the His- tagged pDEST17 expression vector. The LR recombination reaction was performed as detailed in

TM the E. coli Expression System with Gateway Technology manual (Invitrogen, Paisley, UK) . E. coli DH5α chemically competent cells were transformed with the reaction as described within the manual and transformants selected on LB containing ampicillin (100 μgml^"1) . Clones were analysed by restriction digestion of plasmid DNA.

The TE and FI genes were functionally expressed in E. coli BL21 by transforming the chemically competent cells with plasmid DNA of the positive pDESTl7 clones. The transformed cells were transferred to 1 1 of Overnight Express™ Instant TB Medium to maximixe the expression of the proteins. The cultures were incubated at 3O⁰C on a shaker at 200 rpm until the cells reached stationary phase (-24-36 hrs) and then harvested by centrifugation (600Og, 10 minutes at 4°C) .

Purification of Recombinant Fucose Isomerase and Tagatose Epimerase

Cells were resuspended 2:1 (v/w) in either 50 mM Tris pH 7.5, 2 mM tris (2-carboxyethyl) phosphine HCl, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine hydrochloride, 0.5 mM PMSF (TE expressing cells) or 50 mM KPO₄ pH 7.6, 10 mM magnesium acetate, 1 mM EDTA, 10 mM β-mercaptoethanol (FI expressing cells) . Cells were homogenised in a hand homogeniser and passed through a one shot cell disrupter at 20 kPsi (Constant Cell Disruption Systems, Northampshire, UK) . Cell extracts were centrifuged (20000 g, 30 min, 1°C) and cleared lysates desalted through sephadex PD-IO columns (Amersham Biosciences, Buckinghamshire, UK) equilibrated with 2OmM NaPO₄, 0.5 M NaCl, 20 mM Imidazole pH7.4. Desalted protein extracts were loaded onto 1 ml His-Trap™ columns (Amersham Biosciences) at 1 ml min^"1 using a Kontron 420 pump. Columns were then washed with 2OmM NaPO₄, 0.5 M NaCl, 20 mM imidazole pH7.4 until the absorbance of the eluate measured at 280 nm returned to baseline levels as monitored with a Kontron 432 UV/vis detector. Bound proteins were eluted using step gradients up to a maximum concentration of 500 mM imidazole in 20 mM NaPO₄, 0.5 M NaCl pH 7.4. The protein concentration of each fraction was determined according to the method of Bradford (1976) using bovine serum albumin as a standard.

Protein Electrophoresis

Protein fractions collected from the purification of his_δ-TE and his₆-FI were analysed using the NuPAGE^® electrophoresis Bis-Tris buffer system under denaturing conditions (Invitrogen, Paisley, UK) . Approximately equal concentrations of total protein per sample were prepared as detailed in the NuPAGE^® Technical guide (Invitrogen, Paisley, UK) . Samples and SeeBlue Plus2 Prestained Standard (Invitrogen, Paisley, UK) were loaded and resolved on a Pre-Cast 4-12% NuPAGE^® Novex Bis-Tris gel. The gels were run in MES running buffer containing NuPAGE^® antioxidant in a Novex XCELL II™ Mini Cell under the recommended conditions (200 V, 35 min) using a Novex Powerease 500 power pack. The gels were removed and stained according to the protocol for Bis-Tris gels provided with the Colloidal Blue Staining Kit (Invitrogen, Paisley, UK) .

Determination of Fucose Isomerase and Tagatose Epimerase Activities

Tagatose epimerase activity was estimated in a reaction mixture containing 50 mM Tris pH 7.5, 100 mM of the appropriate sugar and an appropriate volume of enzyme extract in a final reaction volume of 100 μl. The reaction was allowed to proceed at 37°C and was stopped by boiling for 1 min.

Precipitated protein was removed by centrifugation (1600Og, I⁰C, 5 min) and the supernatant was diluted 100 times with distilled H2O prior to analysis of reaction products by HPLC as described below. Fucose isomerase activity was estimated in a reaction mixture containing 50 mM potassium phosphate buffer pH 7.6, 10 mM CH₃COOMg, 25 μM MnCl₂, 100 mM of the appropriate sugar and an appropriate volume of enzyme extract in a final reaction volume of 100 μl. The reaction mixture was incubated at 37°C and the reaction stopped by boiling for 1 min. After removal of precipitated protein, the mixture was diluted 100 times with distilled H₂O and the reaction products analysed by HPLC. Metabolite Analysis

Sugars were routinely extracted from microbial cells in 80% ethanol in sealed tubes at 80⁰C for 1 h using a 20:1 (v/w) dilution. After extraction, the samples were cooled on ice and cell debris removed by centrifugation (16000 g, 5 min, 1°C) . Samples were then evaporated to dryness at 40⁰C in a Savant SPD 131DDA centrifugal evaporator (Thermo LabSystems, Cheshire, UK) and resuspended in a known volume of water prior to analysis by HPLC.

Sorbose, tagatose and galactose were identified and quantified using a carbopak PA-I analytical column (4 X 250 mm) fitted with a PA-I guard column (4 X 50 mm) (Dionex UK Ltd. , Surrey, UK) . Mobile phase was 16 mM NaOH pumped at a flow rate of 1 ml min^'1 using a GP40 quarternary gradient pump (Dionex) with the post-column addition of IM NaOH using a Dionex pneumatic controller set to 80 psi. Sugars were detected and quantified by pulsed amperometry using an ED40 electrochemical detector with a quadruple waveform as recommended for the analysis of carbohydrates by Dionex. The column was regenerated after each 30 min analysis by washing for 10 min with 200 mM NaOH followed by 10 min equilibration to the start conditions. Under these conditions, galactose, tagatose and sorbose were baseline separated with retention times of 14.5, 16.2 and 21.8 min respectively. Organic acids were extracted from yeast by resuspending cells in a known volume of 5% (w/v) metaphosphoric acid containing 5 mM tris(2- carboxyethyl) phosphine hydrochloride. Cells were briefly vortexed then freeze-thawed three times and cell debris removed by centrifugation (1600Og, 5 min, 1°C) . AsA and erythroascorbic acid in the supernatant were analysed by HPLC using a Transgenomic Coregel 64H 300 X 5.7 mm column (Crawford Scientific, Glasgow, UK) maintained at 5O⁰C. Mobile phase was 8 mM H₂SO₄ at a flow rate of 0.6 ml min^"1 using a Gynkotech P580 pump and AsA and erythroascorbate were detected and quantified at 245 nm using a Gynkotech UVD340S diode array detector (Dionex UK Ltd., Surrey, UK). Under these conditions, the two compounds were baseline separated with retention times of 12.6 min and 14.4 min respectively.

Results

Identification, Cloning and Functional Expression of an Agrobacterium tumefaciens tagatose epimerase in Eschericihia coli

Previous reports have described a D-tagatose-3- epimerase from Pseudomonas cichorii ST-24 with broad substrate specificity (Ishida et al., 1997). The published gene sequence was used to perform a blast search in the European Bioinformatics Institute database and a gene annotated as tagatose epimerase with 27.4% identity at the nucleotide and 29% identity at the amino acid level was identified from A. tuiaefaciens . Primers were designed to the coding region of the gene (Fig. 2) and the gene cloned by PCR and sequenced to ensure correct reading of the template. The gene was then cloned into pDEST17 (Invitrogen, Paisley, UK) under the control of the T7 promoter and the plasmid introduced into E. coli strain BL21. Cells were grown to stationary phase in a medium suitable for gene induction (medium 2) and total protein extracted. Proteins were applied to a Ni⁺-containing metal chelate column and unbound protein collected. The column was washed with 125 mM imidazole in 20 πiM NaPO₄, 0.5 M NaCl pH 7.4 and eluting proteins collected. Finally, strongly bound proteins were eluted with 500 mM imidazole in 20 mM NaPO₄, 0.5 M NaCl pH 7.4. Each of the protein fractions were run on SDS-PAGE gels and stained with coomassie brilliant blue. Fig. 3 shows a strongly expressed protein of the predicted molecular weight (~ 33 kDa; Ishida et al . , 1997) that bound tightly to the Ni⁺ column suggesting that it was modified with a hisε-tag. The purified enzyme (Fig. 3, lane 5) had a specific activity of 0.834 μmol mg protein^~1h^~1 with D-tagatose as substrate and also catalysed the conversion of D-psicose to D-fructose and L-sorbose to L-tagatose. It is concluded that the gene cloned from A. tumefaciens is an active tagatose epimerase with the capacity to convert L- sorbose to L-tagatose. Cloning and Functional Expression of Fucose Isomerase from E. coli K12

The gene encoding fucose isomerase was cloned from E. coli strain K12, and the gene sequenced. Sequence data corresponding to the first 500 bases from the 5' and 3' ends suggested that the gene cloned corresponded to fucose isomerase as shown in Fig. 4. This sequence was cloned into pDESTl7 and the plasmid was then introduced into E. coli BL21. Soluble proteins were extracted and applied to a Ni⁺ column and sequentially eluted with 125 mM, 250 mM, 375 mM and 500 mM imidazole in 20 mM NaPO₄, 0.5 M NaCl pH 7.4. Protein fractions were run on SDS-PAGE gels and stained with coomassie brilliant blue (Fig. 5) . A strongly expressed protein band was present that eluted in all fractions and had a molecular weight corresponding to that of E. coli fucose isomerase (~ 65 kDa; Seeman and Schulz, 1997). The fractions eluting in 250-500 mM imidazole (lanes 5-7) were combined and observed to catalyse the conversion of D-ribulose to D-arabinose with a specific activity of 265.5 μmol mg protein^"1 h^"1. The enzyme also catalysed the conversion of L- galactose to L-tagatose. These data suggest the gene cloned from E. coli encoded a functional fucose isomerase.

Synthesis of L-galactose from L-sorbose by recombinant enzymes 1 A combination of purified tagatose epimerase and

2 fucose isomerase demonstrated the capacity to

3 synthesise L-galactose when added to a 20 % solution

4 of L-sorbose in 50 mM Tris pH 7.5 buffer (Fig. 6) .

5 After 3 days incubation, the ratio of L-sorbose :L-

6 tagatose: L-galactose was 5.9:1.0:2.0. Similarly,

7 the enzymes were able to catalyse the reaction when

8 suspended in a dialysis sack in yeast growth medium

9 (medium 3) . 10

11 Synthesis of AsA from L-sorbose by recombinant

12 enzymes and yeast 1.3

14 Recombinant FI and TE were added to a solution of

15 20% L-sorbose in 50 mM tris pH 7.5 and incubated at

16 37°C for 3 days to allow the conversion of L-sorbose

17 to L-galactose. The enzyme mixture was then diluted

18 1:10 with sugar-free yeast medium to give a final

19 sugar (L-sorbose + L-tagatose + L-galactose)

20 content of 2%. S. cerevisiae INVScI cells were

21 grown in a flask containing yeast medium (medium 3)

22 supplemented with 2% D-glucose. Cells were harvested

23 by centrifugation, washed once in fresh sugar-free

24 yeast medium and added to the diluted, enzyme

25 mixture. The culture was incubated at 3O⁰C and

26 aliquots were taken after 24 hr intervals. Yeast 7 cells were extracted in 5% metaphosphoric acid

28 containing 5 mM TCEP and analysed for AsA content. 9 Fig. 7 demonstrates that yeast cells cultured in

30 this fashion contained AsA after 24 h incubation. 31 A similar experiment was set up where the TE and FI enzymes were suspended in a dialysis membrane and pre-incubated in yeast medium containing 2% L- sorbose. The flask was incubated for 3 days before adding S. cerevisiae INVScI. Aliquots of the culture were taken at 24 hr intervals and HPLC analysis of cell extracts demonstrated the presence of AsA.

Conclusions

We have demonstrated that a gene cloned from Agrobacterium tumefaciens strain C58 (accession number NC003063) encodes a sugar epimerase with broad substrate specificity that is capable of the conversion of L-sorbose to L-tagatose. Similarly, we have demonstrated that fucose isomerase from E. col± K12 (NC 000913) is able to catalyse the conversion of L-tagatose to L-galactose. When both enzymes were combined either in a buffer consisting of 50 mM tris pH 7.5 or in a dialysis sack in a medium suitable for the culture of yeast cells, they catalysed the conversion of L-sorbose to L- galactose. We further demonstrate that when yeast cells were added to such a reaction mixture they become competent for the synthesis of AsA. We contend that this constitutes a novel method for the production of AsA from the cheap starting material L-sorbose. In addition to yeast cells, the method would be suitable for the production of AsA using any other biological system that contain enzymes and their appropriate co-factors for the synthesis of AsA from L-galactose, such as plant cell cultures, filamentous fungi and microalgae.

References

Berry A, Running JA, Severson DK, Burlingame RP. 2002. Vitamin C production in microorganisms and plants. US Patent 2002012979.

Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

Business Communications Company, Inc. 2004. World markets for citric, ascorbic, isoascorbic acids: highlighting antioxidants in food. Report GA-115.

Hancock RD, Galpin JR, Viola R. 2000. Biosynthesis of L-ascorbic acid (vitamin C) by Saccharomyces cerevisiae. FEMS Microbiol. Lett. 186:245-250.

Hancock RD and Viola R. 2002. Biotechnological approaches for L-ascorbic acid production. Trends Biotechnol. 20:299-305.

Ishida Y, Kamiya T, Itoh H, Kimura Y, Izumori K. 1997. Cloning and characterization of the D-tagatose 3-epimerase gene from Pseudomonas cichorii ST-24. J. Ferm. Bioeng. 85:529-534. Okamura M. 1998. Separative determination of ascorbic acid analogues contained in mushrooms by high-performance liquid chromatography. J. Nutr. Sci. Vitamin. 44:25-35.

Running JA, Burlingame RP, Berry A. 2003. The pathway of L-ascorbic acid biosynthesis in the colourless microalga Prototheca moriformis . J. Exp. Bot. 54:1841-1849.

Seeman JE, Schulz GE. 1997. Structure and mechanism of L-fucose isomerase from Escherichia coli. J. MoI. Biol. 273:256-268.

Wheeler, G. L., Jones, M.A. and Smirnoff, N. 1998. The biosynthetic pathway of vitamin C in higher plants. Nature 393:365-369.

Claims

1. A method for the production of L-ascorbic acid, said method comprising co-incubating the enzymes tagatose epimerase and fucose isomerase with a micro-organism able to convert L- galactose to L-ascorbic acid.

2. The method as claimed in Claim 1 wherein said micro-organism is a yeast.

3. The method as claimed in Claim 2 wherein said yeast is Saccharomyces cerevisiae.

4. The method as claimed in Claim 1 wherein said micro-organism is a fungus.

5. The method as claimed in Claim 4 wherein said fungus is Aspergillus nidulans.

6. The method as claimed in any one of Claims 1 to 5 wherein said enzyme tagatose epimerase is obtained from Agrobacterium tumefaciens C58.

7. The method as claimed in any one of Claims 1 to 6 wherein said enzyme fucose isomerase is obtained from E coli.

8. The method as claimed in any one of Claims 1 to 7 wherein the enzyme tagatose epimerase is expressed from a recombinant genetic construct.

9. The method as claimed in any one of Claims 1 to 8 wherein the enzyme fucose isomerase is expressed from a recombinant genetic construct.

10. The method as claimed in any one of Claims 1 to 9 wherein said micro-organism expresses tagatose epimerase.

11. The method as claimed in any one of Claims 1 to 10 wherein said micro-organism expresses fucose isomerase.

12. A recombinant polynucleotide comprising a sequence as set out in SEQ ID No 1 (tagatose epimerase) , SEQ ID No 3, (fucose isomerase) , or homologs thereof.

13. A vector comprising the recombinant polynucleotide of Claim 12.

14. A host cell transformed with the vector of Claim 13.

15. The host cell as claimed in Claim 14 able to express the enzyme tagatose epimerase and/or fucose isomerase.

16. A micro-organism having an enhanced L-ascorbic acid content, wherein said micro-organism is able to express tagatose epimerase and fucose isomerase, and to convert L-galactose to L- ascorbic acid.