WO2012014228A1

WO2012014228A1 - A method to by-pass allosteric domain activity of an enzyme so as to alter its feedback or feed-forward inhibition or activation

Info

Publication number: WO2012014228A1
Application number: PCT/IN2011/000496
Authority: WO
Inventors: Abhishek Narain Singh
Original assignee: Abhishek Narain Singh
Priority date: 2010-07-28
Filing date: 2011-07-27
Publication date: 2012-02-02

Abstract

A method to bypass allosteric domain activity of an enzyme so as to alter the feedback/ feed-forward inhibition/ activation, comprises of (i) a method of increasing the downstream metabolic product such in fermentation or for genetically modified crop, where the wild type organism is feedback or feed-forward inhibited at one of its enzymatic reaction by introducing transgene to be expressed in a common cellular compartment such as cytosol and (ii) a method of decreasing the down stream metabolic product such as for therapeutic purpose, such as controlling the uncontrolled growth of tumor, where the wild type organism is feedback or feed-forward activated at one of its enzymatic reaction, by introducing transgene to be expressed in a common cellular compartment cytosol for that enzyme.

Description

A Method to By-pass Allosteric Domain Activity of an Enzyme so as to Alter its Feedback or Feed-Forward Inhibition or Activation

FIELD OF INVENTION

This invention relates to a method to bypass the allosteric effect of an enzyme such as feedback or feed-forward or a combination of feedback and feed-forward inhibition or activation, in prokaryote or a eukaryote to increase levels of downstream products such as vitamin, or decrease levels of downstream undesired product such as tumor and also to a method for •increasing the levels of vitamin B5 along with additional novel metabolic pathway modification step for the pathway to further increase the level of the vitamin.

BACKGROUND OF INVENTION

Pantothenate (vitamin B5) is the precursor of 4'-phosphopantetheine moiety of coenzyme-A & ACP which acts as a co-factor for a number of essential and non-essential reactions. Commercial methods for its production involve expensive techniques for separating racemic intermediates (Shimizu and Yamada 1992¹). The biosynthetic pathway in bacteria, comprising four enzymatic reactions, is well established, and the enzymes have been fully characterized including the over expression & purification of recombinant enzymes and the determination of their X-ray crystal structures. Biotransformation can potentially increase the yield of pantothenate in plants and algae thereby constituting a less expensive route. There is, therefore, a need to manipulate the pathways in the plants to bypass regulation to increase its level. Pantothenate biosynthesis pathway in microorganisms

In bacteria the first step, ketopantoate hydroxymethyltransferase (KPHMT; ec 2.1.2.1 1) converts a-ketoisovalerate (a-KIVA) into ketopantoate using 5, 10-methylene tetrahydrofolate as a co-factor. A-KIVA is the oxoacid of valine. KPHMT is a class II aldolase that utilises CH2-THF to transfer a hydroxymethyl group to a-KIVA. In purified E. coli KPHMT, Mg²⁺ has been shown to activate the enzyme (Powell and Snell, 1976²) and in the absence of added Mg²⁺, the activity of the enzyme is reduced by more than 10-fold. Subsequently, ketopantoate is reduced by ketopantoate reductase to form pantoate using NADPH as the hydrogen donor. In another branch, L- aspartate is converted to β-alanine by L-aspartate-a-decarboxylase (ADC; EC 4.1.1.15).

Pantoate and β-alanine condense to form pantothenate in the final step catalysed by pantothenate synthetase (PS; EC 6.3.2.1) which is ATP- dependent. This condensation is thought to proceed via a pantoyl adenylate intermediate. Genes for all four enzymes panB, panE, panD, panC have been cloned and characterized in E. coli and the structures of the recombinant enzymes have been solved (Lobley et.al. 2003³). Figure 1 illustrates the committed steps in D-pantothenate biosynthesis pathways in E. coli.

Pantothenate biosynthesis pathway in plants

Plant pantothenate synthetases have a subunit Mr of approximately 34kDa and are 65% similar to the E. coli PS counter part at the amino acid level. Interestingly, pantoate inhibits the plant PS enzyme at concentration greater than 0.5mM (Genschel et.al., 1999⁴), whereas the E. coli PS has normal Michaelis kinetics with this substrate. No N-terminal extension sequence on PS indicated that it was cytosolic which was confirmed by GFP- targeting experiments (Ottenholf et.al., 2004⁵). Southern analysis of the genomic DNA indicated the presence of only a single copy of the panC gene which was confirmed once the genome sequence was available.

KPR in plants has not yet been identified due to various difficulties such as the sequence similarity amongst the bacterial KPRs itself being very low (Matak-Vinkovic et al., 2001⁶). In E. coli, acetohydroxy acid reductoisomerase (AHIR) which reduces acetohydroxy acids can catalyse the reduction of ketopantoate but with much lower efficiency. This enzyme has been shown to be the only enzyme with KPR activity in Corynebactenum glutamicum (Merkamm et al. 2003⁷). A situation like this might prevail in plants. A single gene of AHIR has been found in A. thaliana and spinach. There is also a possibility of an alternate pathway by keto-pantoyl-lactone as substrate can exist. However since the enzyme PS does not use pantoyl lactone as substrate (Genshel et al. 1999), this is a less likely possibility.

The route to β-alanine production for pantothenate in plants is uncertain. In bacteria, β-alanine is produced by the decarboxylation of L- aspartate catalysed by ADC. No genes homologous to ADC could be identified by BLAST searching of plant genome or by using structure based search method known as reverse-FUGUE, inspite of the fact that parxD genes encoding ADC are similar between different bacterial species. Interestingly, no ADC homologue could be identified in yeast S. cerevisiae, suggesting that ADC has not made the transition across the prokaryote-eukaryote border. Instead, β-alanine can be made by two other routes from uracil and spermidine degradation, enzymes for both of which have been found in plants.

Compartmentalization of plant pantothenate biosynthesis pathway makes it more complex than its bacterial counterpart. KPHMT is mitochondrial, whereas PS is in the cytosol. There is similar distribution in yeast cell with KPR found also in cytosol. There must be a transporter protein in plants that is involved in transporting either keto-pantoate or pantoate across the mitochondrial membrane into cytosol in case of plants. It is to be noted that compartmentalization facilitates regulation. Feedforward substrate inhibition of PS by pantoate is clearly another potential site of regulation. Figure 2 summarizes our knowledge in A. thaliana for vitamin B5 pathway.

The method of bypassing allosteric regulation as detailed above was tested by studying the vitamin B5 (pantothenate) pathway in prokaryote Escherichia coli and eukaryote Arabidopsis thaliana.

WO 2010/018196 shows some recent work on the vitamin B5 pathway to modify the enzymes in order to create an insensitive feedback inhibition enzyme. The recent work as indicated above uses manual artificial mutation techniques such as site-directed mutagenesis, saturation mutagenesis, random mutagenesis /directed evolution, chemical or UV mutagenesis of entire cells/ organisms, to introduce mutation at the allosteric domain. The disadvantage of introducing mutation artificially is that by doing so one is not sure how the catalytic activity gets affected after the mutant enzymatic protein has folded. This is because protein folding problem is an unsolved problem. This implies that the technique of artificial introduction of mutation is less reliable than compared to the method described in this invention where we locate for a naturally selected enzyme for the purpose which has acquired loss of allosteric activity by means of selection pressure during evolution. Apart from this artificial introduction of mutation for such purpose to create insensitive enzyme is extremely labor and cost expensive. The method used in the current invention, differs greatly as in this invention an enzyme from another compartment of a eukaryote, such as from a plant mitochondria, which is naturally selected for optimal performance by means of absence of allosteric domain, is expressed into the cytosol of the bacteria or eukaryote such as plant, or into a common compartment other than cytosol. The expression can be made into the cytosol of any other prokaryote or eukaryote (such as plants or algae) to over-express downstream product by means of by-passing feedback inhibition.

WO 2001/000852 shows a method where a plant gene Phosphoenolpyruvate (PEP) carboxylase (EC 4.1. 1.31) is first mutated at certain amino acid residues before transforming it into the host of interest. The DNA fragment is derived from an alfalfa plant, and most preferably, it is derived from a Medicago sativa strain. The plant-derived DNA fragment is modified by one or more nucleotide substitutions, deletions and/or insertions. Most preferably, the modification comprises deleting the nucleotides encoding the amino acid sequence: Met-Ala-Ser-Ile-Asp-Ala-Gln- Leu-Arg. It is then combined with the other sequence from a microbe to form a chimeric functional enzyme insesitive to feed-forward activation by acetyl- CoA and feedback inhibition by aspartic acid. The current invention differs from the above patent as, firstly, the enzyme to be transformed is mutated/ deleted/ substituted at certain amino acids. Secondly, the enzyme is a chimeric molecule having its other half of the sequence obtained from microorganism. Thirdly, there is no mention of specifically looking for a gene in a eukaryote such as plant from within a cellular compartment, as the enzyme PEP is expressed in the cytosol of plant such as in Medicago sativa as used in the invention.

WO 2006/034501 specifies a transgenic plant where a transgene is introduced to the plant which is known to have no feedback inhibition. The current method in the invention differs in the sense that the inventor specifically focuses on introducing a transgene which is natively present in another cellular compartment of a cell. This is not the case with this patent as the enzymes being introduced into the cytosol of the plant was initially present in cytosol of E. coli bacteria or mammal itself.

WO 2005/ 111202 shows a method of introducing a recombinant enzyme mutated homoserine transsuccinylase with reduced sensitivity for the feedback inhibitors S-adenosylmethionine and methionine. Here again the mutations introduced are via standard laboratory techniques such as site directed mutagenesis.

WO 2007/083100 shows a method of introducing an E. coli modified enzyme to human for therapeutic reasons such as by modification to the E. coli enzyme and replacing it with the native enzyme. Similar concept can be applied to the current invention where by introduction of an enzyme by the above method activation can be controlled to suppress tumor. If required the method can also be applied for inhibition control.

WO 2005/003357 again details technique of creating an insensitive feedback regulation by means of introducing recombinant enzymes which have been artificially mutated using standard lab techniques such as site directed mutagenesis, etc.

WO 2005/087940 also again details method of randomly mutating sections of the gene by artificial lab techniques in order to increase downstream flux.

WO 2004/ 111214 again mentions feedback control of an enzyme by standard mutagenesis techniques such as site directed mutation, etc.

Strategy to increase levels of vitamin B5 in microbes, plants, algae and other suitable host. Applying the above detailed method for the panB gene of a reaction pathway for the KPHMT enzyme such as by transforming a panB gene from another compartment like panBl from A. thaliana's mitochondria into the cytosol of the organism of interest leads to by-passing of feedback inhibition. As plant PS enzyme coded by panC gene is feedforward inhibited by pantoate substrate, the strategy would be to introduce bacterial panC gene such as from E. coli into the plant of interest in which inventors aim to increase the levels of vitamin B5. As there is still ambiguity as to whether the enzyme KPR coded by panE gene is expressed in cytosol of the plant or not, transforming a bacterial KPR such as that of E. coli into the plant of interest would also lead to increase in levels of vitamin B5 especially when it is used in combination with expressing panB gene into the cytosol as well, as that ensures enrichment of the cytosol with ketopantoate which is the substrate for the KPR enzyme. As introduction of C. glutamicum 's panD gene coding for ADC enzyme into the host E. coli had shown increased levels of beta-alanine and thus pantothenate, transformation of the same gene into other hosts of interest such as plants would lead to increase in levels of vitamin B5.

OBJECTS OF INVENTION

The main objective of the present invention is to develop a process to increase levels of vitamin B5 in microbes, plant, algae and other suitable host.

Another objective of the present invention is to by-pass allosteric domain activity of an enzyme so as to alter the feed back or feed forward inhibition or activation in eukaryote or prokaryote.

Yet another objective of the invention is to develop a process wherein it has been applied to vitamin B5 pathway at pan B gene expression. A further objection of the invention is to develop a process to introduce pan D gene of C. glutamicum in eukaryote, pan E gene of E. coli in eukaryote, pan C gene of E. coli in eukaryote.

The foregoing has outlined some of the pertinent objectives of the invention. These objectives should be construed to be merely illustrative of some of the more prominent features and applications of the intended invention. Many other beneficial results can be obtained by applying the disclosed invention in a different manner or modifying the invention within the scope of disclosure. Accordingly, other objectives and a full understanding of the invention and the detailed description of the preferred embodiment in addition to the scope of invention are to be defined by the claims.

STATEMENT OF INVENTION

Accordingly the present invention provides a method to bypass allosteric domain activity of an enzyme so as to alter the feedback and feedforward inhibition or activation, comprises of (i) a method of increasing the downstream metabolic product such in fermentation or for genetically modified crop, where the wild type organism is feed back or feed forward inhibited at one of its enzymatic reaction by introducing transgene to be expressed in a common cellular compartment such as cytosol and (ii) a method of decreasing the down stream metabolic product such as for therapeutic purpose, such as obstructing the uncontrolled growth of tumor, where the wild type organism is feedback or feed-forward activated at one of its enzymatic reaction, by introducing transgene to be expressed in a common cellular compartment cytosol for that enzyme.

BRIEF DESCRIPTION OF INVENTION Further objects and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawings wherin:

Figure 1: Shows allosteric and Catalytic domains of Enzyme

Phosphofructokinase

Figure 2: Shows typical feedback regulation by downstream metabolites

Figure 3: Shows a semi-permeable allows selective transportation across its walls.

Figure4: Shows Loss of allosteric domain after removing the compartment barrier after significant time period.

Figure 5: Pantothenate biosynthesis pathway in Escherichia coli. Committed steps in D-pantothenate synthesis comprises of four enzymes from a-KIVA and L-aspartate viz., KPHMT, KPR, ADC and PS.

These enzymes are coded by the genes panB, pane, panD and panC respectively. Enzyme names are given in red. The enzyme structures are KPHMT (lm3u), KPR (1KS9), ADC (1AW8) and PS

(1HLO). Source: Lobley et al., Biochem. Soc. Trans. (2003) 31,

(563-571)

Figure 6: Shows our knowledge of pantothenate biosynthesis in model plant

A. thaliana. The enzymes have been colored red while the substrates and products are in black.

Figure 7: Demonstrates pBluescript vector for transformation in E. coli

Figure 8: Demonstrates pGreen35S vector used for transformation in plants.

Over here panE gene has been cloned into the multiple cloning sites. Figure 9: Shows test of the workability of the bioassay method exposing different levels in microgram of pantothenate with two-fold dilution

Figure 10: Shows transgenic E. coli with A.thaliana panBl gene

Figure 11: Shows transgenic A. thaliana with panE gene from E. coli

Figure 12: Demonstrates transgenic A. thaliana with panD gene from C.

glutamicum

Figure 13: Shows transgenic A. thaliana with panC gene from E. coli

While the invention is described in conjunction with the illustrated embodiment, it is not intended to limit the invention to such embodiment. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the spirit and scope of the inventions disclosure as defined by the claims.

DETAIL DESCRIPTION OF INVENTION

For the purpose of promoting an understanding of the principles of the invention, reference is now to be made to the embodiment illustres in the drawings and specific language is used to describe the same. It is nevertheless to be understood that no limitations of the scope of invention is hereby intended, such alterations and further modifications in the illustrated bag and such further applications of the principals of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

The current invention illustrates a novel method which can be applied to a range of metabolic pathway to cause a de-regulation at the allosteric control of enzyme. The invention method has been applied to vitamin B5 pathway at panB gene expression as an example of its workability. Other strategies to increase vitamin B5 content in microbes, plants and algae have also been demonstrated such as introduction of panD gene of C. glutamicum in eukaryote, panE gene of E. coli in eukaryote, panC gene of E. coli in eukaryote.

The current invention proposes a concept drawn from Charles Darwin's theory of evolution to be applied to metabolic pathway engineering (MPE) in order to change the downstream flux of the desired metabolite for our advantage. An allosteric domain of an enzyme is that region of an enzyme where the binding of an upstream or downstream metabolite can effectively result in change of the structure of the catalytic domain resulting in either up-regulation of the enzyme activity or a down-regulation also known as inhibition of its activity. An enzyme can have one or more of such allosteric domain where the binding of a metabolite to it indirectly affects the structure and thus function of the catalytic domain. Figure 1 highlights the structure of an enzyme called phosphofructokinase with catalytic and allosteric domains being pointed out.

"Inhibition" includes both the reduction of activity of the polypeptide and the complete lack of activity as well. "Host microorganism" means the microorganism that is transformed with the introduced genetic material.

"DNA fragment" refers to a fraction of a deoxyribonucleic acid molecule.

"Expression," as used herein, is intended to mean the production of the protein product encoded by a gene.

"Activator," as used herein, includes both a substance necessary for the polypeptide to become active in the first place, as well as a substance which merely accentuates activity. "Transformation" herein refers to the transfer of a foreign gene into a host cell either as part of the host cell genomic DNA or as an independent molecule, and its genetically stable inheritance.

Figure 2 shows is a generalized feedback inhibition /activation pathway reaction, where protein 'P' along with the cofactors if any is the enzyme having one or many allosteric domain being inhibited by several downstream products. A similar situation can exist for a feed-forward inhibition /activation. Needless to mention, each reaction shown has an enzyme catalyzing the step on its own which may have similar allosteric domains.

A straightforward solution to avoid this feedback effect would be to compartmentalize the reaction which enzyme P is involved such that the downstream products cannot cross the compartment barrier and bind to P's allosteric domain. However, this technique introduces a new bottleneck of compartment entry transportation of the reacting substrates of the enzyme P which over here are A and B, and compartment exit transportation of C. Figure 3. shows the situation.

With the introduction of a semi-permeable membrane barrier, though we have been able to control the feedback influence such as inhibition, however this will not result in an increase in downstream product, at least not substantially, since now even though the previous bottleneck of feedback inhibition is removed, there are new bottlenecks such as the transportation of 'C outside the compartment and transportation of A and/or B inside the compartment.

For the sake of economic energy cellular metabolism and substance metabolism, we know that the production of downstream products is strictly controlled by means of such allosteric feedback inhibition. Now that the functionality of flux control which was by means of the allosteric domain has been substituted by a new bottleneck as of compartment entry and exit of metabolites, the allosteric domain of the enzyme inside the compartment is redundant. Though the introduction of a compartment is not serving our purpose of improving the downstream products yield, let us imagine that we leave this reaction in this state in-vivo for say 10⁶ years or longer or theoretically speaking for an infinite time. In that situation, there would be three kinds of mutations that would develop over the period:

1. The catalytic domain of P will get mutated. All these mutations will not be selected as the catalytic functionality of the enzyme is vital to the survival of the organism.

2. The catalytic, allosteric and other structural domains of P will get mutated. As these mutations would likely also decrease the catalytic function of the enzyme, they would also face strong selection pressure for not getting accepted and propagated.

3. The allosteric domains of P will get mutated. Only those mutations will survive which do not have an adverse effect on the structure of the catalytic site of the enzyme.

As an outcome of the these possibilities, only those mutational changes will survive which do not decrease the catalytic function of the active site of the enzyme such as by retaining its domain structure. As the functionality of allosteric domain of the enzyme is lost due to introduction of a more serious compartment transportation bottleneck, any mutational changes on that domain which do not indirectly influence the structure of catalytic domain will be accepted and retained due to loss of selection pressure. This selection pressure of evolution is derived straight from the Darwin's theory of survival of the fittest. This implies that over a long period of time such as 10⁶ years, the enzyme P would change to P' where the series of mutations on the allosteric domain of P would have made it no more good for exhibiting the allosteric functionality, while retaining its catalytic domain functionality.

At this point if the compartment barrier is removed that had been created 10⁶ years earlier, then in effect the bottleneck of compartment entry and exit is removed and also the new enzyme P' which is now exposed for interaction directly to the downstream products is also not in a state to exhibit any feedback inhibition! Figure 4 illustrates the situation.

Thus the inventor is able to by-pass the allosteric effect (such as the feedback inhibition in the above example) and thereby increases flux of the downstream product. Similar strategy can be adopted for a reverse purpose, which is if the allosteric domain acts as an activator or enhancer of increasing the downstream product, then applying the above technique will allow one to get rid of the allosteric domain and the reaction would not proceed as fast as it was earlier.

The concern now is the practical implementation of the concept as it is not feasible to wait for 10⁶ years or so for obtaining such an enzyme which looses its allosteric characteristic. The approach lies in going backward 10⁶ years or longer rather than forward, and see where the evolution introduced a compartment such as to transform to a eukaryote (yeast, animal, plant, etc.) where multiple compartment in the form of organelles exist differentiating it from its ancestral prokaryote having a single compartment called the cytosol. Thus, for the metabolic pathways which exist in common to a prokaryote and a eukaryote, if there are intermediate reactions where a part of the reaction which shows feedback inhibition controlled in prokaryote is actually being occurring inside an organelle compartment for the eukaryote, this would exactly depict the situation as described in the approach above. For such intermediate reaction(s) if the enzyme of the eukaryote which is originally functional inside a prokaryote is later expressed inside a prokaryotic or eukaryotic cytosol, such that now all the reactions occur in a single compartment, this would lead to bypassing of allosteric control and also bypassing of compartmental control leading to removal of the bottlenecks.

SOLUTIONS. BUFFERS AND MEDIA

LB Media, LB Agar, Sodium Acetate solution, Amino Acid solutions: L- arginine 2.53% (w/v),L-histidine 0.31% (w/v), L-proline 4.6% (w/v), L- Adenine 1.35% (w/v), Nutrient Media: A 100 ml of nutrient media was prepared which comprised of 1M Glucose, 50mM MgS04, 50μΜ FeS04, and 741μΜ Thiamine, GB1 buffer, ½ MS Media prep: ½ MS media is the minimal media for seed selection which comprises of 2.3 gm MS / litre.

Bacterial culture Inoculums preparation

Suspended a vitamin B5 auxotroph of E. coli bacteria in some amount of L.B. media in a steriline and kept it in 37°C overnight. Did streaking on agar plates on separate plate and left it overnight to grow which was then suspended the pellet in 500μ1 GB1 lx buffer.

Pantothenate Extraction

Sonicated bacterial culture which had the transformed vector containing A. thaliana panB gene inserted. The media in itself was containing some vitamin B5. The supernatant was then removed using a syringe and the content was poured via filter to eppendorf tube. Ligation of the cut A. thaliana panB gene insert to the cut pGreen0029:35S vector

The ligation reaction was carried out such that the insert and the vector total comprises of 4 μΐ and in the ratio such that they compensate for the intensity of the band in order to make approximate concentration 1 : 1. total volume was 10 μΐ.

Sterile D.I. water

T4 DNA 10X buffer

pGreen0029:35S cut vector

A. thaliana panB gene cut insert

T4 DNA ligase

MICROBIAL BIOASSAY TO QUANTIFY THE AMOUNT OF PANTOTHENATE

Take 100 mg of D-pantothenate and top it up to 1 ml with sterile water in an eppendorf tube. Use this to prepare lOmg, lmg, 100μg, 10μg and ^g sample in eppendorf tube by successive addition of 100 μΐ of previous sample and 900 μΐ of sterile water. Make 2 Jars with 100 ml of GB1 media each. A 100ml of GB1 media comprises of 10ml GB1 buffer lOx, 500 μΐ Adenine soltution, 500 μΐ L-Arginine solution, 500 μΐ L-histidine solution, 500 μΐ L- proline solution and 2 ml of Nutrient media. To one of the jar add 50 μΐ of the vitaminB5 auxotroph bacterial suspension that was preserved and mix it. Put 200 μΐ of standard pantothenate sample followed by the plant extract pantothenate sample in the first colum of the 96-well plate. Add ΙΟΟμΙ of GB1 media in the rest of the wells. With multi-channel micropipette take out 100 μΐ from the 1^st column and mix it to the next column. Repeat this for the next columns until you reach the last column where you would take out ΙΟΟμΙ and throw it in waste. Now top up all the wells with 100 μΐ of GB1 Media containing the auxotroph bacteria. Transgenic Organism

Since KPHMT (E.colij has also been shown to undergo allosteric inhibition by pantoate (50μΜ), pantothenate (500μΜ) & CoA (ImM), thought to be important in the regulation of the pantothenate pathway, we decided to apply the method detailed in this invention to the pantothenate pathway at this enzyme. Transgenic organism for panBl can be made by cloning the gene of interest such as panBl of Arabidopsis thaliana in this case in to the transformation vector such as pBluescript for E. coli K12. Figure 7 is the diagram for the vector.

Similarly, if one desires to express the A. thaliana panBl gene into the cytosol then an appropriate vector such as pGreen35S can be deployed, such as after cutting the N-terminal sequence which will prevent the gene to be expressed into the mitochondria resulting in its expression in cytosol.

All documents cited in the description are incorporated herein by reference. The present invention is not intended to be limited in scope by the specific embodiments and examples which are intended as illustration of a number of aspects of the scope of this invention. Those skilled in art will know or to be able to ascertain using no more than routine experimentations many equivalents to the specific embodiments of the invention described herein.

It is to be further noted that present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant embodiments employing the concepts and features of this invention are intended to be within the scope of the present invention which is further set forth under the following claims: Abbreviations used: a-KTVA, a-ketoisovaleric acid, ACP, acyl carrier protein, ADC, L-aspartate-a- decarboxylase (EC 4.1.1.15), BSA, bovine serum albumin, CoA, coenzyme A, 0Ή2Ο, distilled water, EDTA, ethylene diamine tetraacetic acid, KPHMT, ketopantoate hydroxymethyltransferase (EC 2.1.2.1 1), KPR, ketopantoate reductase (EC 1.1.1.169), MS, Murashige and Skoog, PCR, polymerase chain reaction, PS, pantothenate synthetase (EC 6.3.2.1), TAE, Tris-acetate

References

1. Shimizu S, Kataoka M, Shimizu K, Hirakata M, Sakamoto K, Yamada H.(1992). "Purification and characterization of a novel lactonohydrolase, catalyzing the hydrolysis of aldonate lactones and aromatic lactones from Fusarium oxysporum". Eur J Biochem. 209:383-390.

2. Powers SG, Snell EE. (1976). "Ketopantoate hydroxymethylltransf erase II Physical, catalytic and regulatory properties" J Biol Chem. 251(12); 3786- 3793.

3. Lobley et.al. Biochem Soc. Trans. (2003) 31, 563-571.

4. Genschel U, Powell CA, Abell C, Smith AG. (1999) "The final step of pantothenate biosynthesis in higher plants: cloning and characterization of pantothenate synthetase from Lotus japonicas and oryza sativum (rice). Biochem J. 341 (Pt 3); 669-678.

5. Ottenhof HH, Ashurst JL, Whitney HM, Saldanha SA, Schmitzberger F, Gweon HS, Blundell TL, Abell C. Smith AG (2004) "Organisation of the pentothenate (vitamin B5) biosynthesis pathways in higher plants" Plant J. 37(1): 61-72. Matak-Vinkovic D, Vinkovic M, Saldanha SA, Ashurst JL, Von Delft F, Inouse T, MiguelRN, Smith AG, Blundell TL, Abell C. (2001) "Crystal structure of Escherichia coli ketopentoate reductase at 1.7 A resolution and insight into the enzyme mechanism." Biochemistry 40(48); 14493- 500. Merkamm M, Chassagnole C, Lindley ND, Guyonvarch A. (2003). "Ketopantoate reductase activity is only encoded by ilvc in corynebacterium glutamicum" J Biotechnol. 104(1-3): 253-260

Claims

We Claim

1. A method to bypass allosteric domain activity of an enzyme so as to alter the feedback / feed-forward inhibition / activation, comprises of : i) a method of increasing the downstream metabolic product such in fermentation or for genetically modified crop, where the wild type organism is feedback or feed-forward inhibited at one of its enzymatic reaction by introducing transgene to be expressed in a common cellular compartment such as cytosol and ii) a method of decreasing the down stream metabolic product such as for therapeutic purpose, such as controlling the uncontrolled growth of tumor, where the wild type organism is feedback or feedforward activated at one of its enzymatic reaction, by introducing transgene to be expressed in a common cellular compartment cytosol for that enzyme.

2. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1, wherein the common cellular compartment can be some other compartment as well, and the transgene being natively found to have been expressed in another compartment of the same eukaryotic cell or be taken from another eukaryotic cellular compartment to be expressed in the eukaryote or prokaryote of interest.

3. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1, wherein the transgene which is native to the cellular compartment need not necessarily be artificially mutated.

4. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1 and 2, wherein the transformation will lead to total or partial removal of allosteric effect such as feed back or feed-forward inhibition or activation.

5. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1 , wherein the region of the eukaryotic gene to be considered would be exon sequence.

6. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1 and 2, wherein expressing plant KPHMT1 enzyme expressed by panBl gene which is originally expressed in mitochondria, into the cytosol of the same plant such as A. thaliana or different plant, another eukaryote, or in prokaryote such as Escherichia coli or any other bacteria will lead to application of the method, leading to increase in levels of vitamin B5, the enzyme can be KPHMT2 of A. thaliana or any other plant or eukaryote having the enzyme expressed in a compartment other than cytosol.

7. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1 and 2, wherein the transgene is either able to integrate into the host organism's chromosome or can be present in the cell as plasmid that replicates along with the cell reproduction.

8. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1 and 2, wherein it can also be done in vitro in a common chemical reaction unit.

9. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 6, wherein the increase in vitamin B5 levels is at least 10% more than originally present in wild type E.coli.

10. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 6, wherein the expression of an enzyme KPR from E.coli in a single or multiple copies in to the cytosol of plant such as A. thaliana would lead to increase in levels of vitamin B5 by at least 7%.

1 1. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 6, wherein expression of enzyme ADC by inserting panD gene from C. glutamicum in a single or multiple copies into the cytosol of E. coli bacteria or A. thaliana plant or any other eukaryote or prokaryote will lead to increase in levels of Vitamin B5 by at least 3%.

12. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 6, wherein expression of enzyme pantothenate synthetase PS from E. coli in a single or multiple copies into the cytosol of plants such as A. thaliana or any other eukaryote of plant kingdom such as algae will lead to increase in levels of vitamin B5 by at least 4% as PS of plants are known to be feed forward inhibited by pantoate.

13. A method to bypass allosteric domain activity of an enzyme, as claimed in Claim 1, 6, 10, 1 1, and 12, which when combined together independently or as an operon would result in increase of vitamin B5 level.