WO2020121335A1

WO2020121335A1 - A process for producing tirucalladienol for synthesis of limonoids

Info

Publication number: WO2020121335A1
Application number: PCT/IN2019/050915
Authority: WO
Inventors: Venkataramaiah Thulasiram HIREKODATHAKALLU; Avinash PANDREKA; Chaya Shivanagouda PATIL
Original assignee: Council Of Scientific & Industrial Research
Priority date: 2018-12-14
Filing date: 2019-12-13
Publication date: 2020-06-18

Abstract

The invention relates to the construction of recombinant DNA constructs encoding tirucalladienol synthase; and squalene epoxidase operably linked to a regulatory sequence. The production of tricualldienol in a yeast is increased by coexpression with squalene epoxidase. Further, tirucalladeinol synthase engineering leads to increase in the efficiency of the synthesis process towards tirucalladienol.

Description

A PROCESS FOR PRODUCING TIRUCALLADIENOL FOR SYNTHESIS OF

LIMONOIDS

FIELD OF THE INVENTION

The present invention relates to a process for tirucalladienol production by co expressing enzymes involved in limonoid biosynthesis. More particularly, the present invention relates to co-expressing DNA sequences encoding tirucalladienol synthase and squalene epoxidase involved in limonoid biosynthesis for improving tirucalladienol production. Further, the present invention relates to mutant tirucalladienol synthase having amino acid substitutions at active sites to obtain tirucalladienol.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF PRIOR ART

Plants produce a diverse array of metabolites which play a critical role in plant interactions with the environment such as pathogens and herbivores, abiotic stress and attracting other organisms for pollination and seed dispersal. Apart from their role in plants, secondary metabolites have proved beneficial to humans as medicines, drugs, dyes, biofuels, fragrance and essential oils. Over 75,000 isoprenoids have been identified in different organisms. Triterpenoids are one of the classes of isoprenoids synthesized from isoprene units through C30 squalene intermediate. They have various biological properties like anti-inflammatory, anti-viral, anti-cancer, and insecticidal and have been used for treatment of vascular diseases.

Limonoids are tetranortriterpenoids occurring in Meliaceae family. A total of 300 limonoids were identified, majority of which were accounted to be in Azadirachta indica (Neem) and Melia azedarach (Chinaberry). Limonoids are abundant in neem seeds as compared to other tissues. Based on structural similarities, neem limonoids are divided into basic and C-seco limonoids. Gedunin, azadiradione, nimbin, salannin and azadirachtin are the most important limonoids from neem showing different biological activities. Azadirachtin, azadirone, 6-deacetyl nimbin, gedunin, nimbin, nimbolide and salannin have anti-cancer activity. 17 -epi- 17 -hydroxy azadiradione, 7-acetyl-16, 17- dehydro- 16-hydroxy neotrichilenone, 7-deacetyl gedunin, nimocinol and nimbin exhibit an inhibitory effect on 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation. Limonoids such as meldenin, isomeldenin and nimocinol isolated from fresh neem leaves have been found to demonstrate anti-malarial activity against chloroquine-resistant Plasmodium falciparum. Other limonoids such as azadirachtin A, nimbolide and 6-deacetyl nimbin have been reported to interfere with transmissible Plasmodium stages.

Neem limonoids have been extensively studied in the past 30 years and are demonstrated to have insecticidal activity against 413 species in 16 different insect orders. The biological properties of limonoids against insects include repellence, feeding and oviposition deterrence, growth disruption, reduced fitness and sterility.

The mevalonate pathway (MVA) and non-mevalonate pathway (MEP) pathways lead to biosynthesis of 2, 3-oxidosqualene, which serves as a precursor for limonoid biosynthesis. Further, 2, 3-oxidosqualene is cyclized by triterpene cyclases. Based on oxygenated C30 compounds isolated from Meliaceae, the precursor cyclic molecule (proto-limonoid skeleton) for limonoid biosynthesis has been predicted to be a euphol or a tirucallol derivative. When tritium labelled euphol, tirucallol, A⁷-tirucallol and butyrospermol are fed to leaves of neem, all are incorporated into nimbolide. However, euphol is more effectively incorporated into nimbolide as compared to others. Hence, the predicted protolimonoid skeleton for limonoid biosynthesis in neem is A⁷-isomer of euphane (butyrospermol) or tirucallane. This cyclic product undergoes modification like hydroxylation, dehydrogenation, epoxidation, acetylation and tigloylation to form diverse limonoids in neem ( Scheme for limonoid production is illustrated in Fig.l). Therefore, there is a long lasting need in the art to identify genes involved in limonoid biosynthesis and metabolic engineering for large-scale production. Sequencing and functional annotation of the transcriptome are primary tools for discovery of novel genes, especially in non-model plants for which full genome sequencing is not economically feasible. Integration of transcriptomics and metabolic fingerprinting helps understanding limonoid biosynthesis.

It is established that plants are major sources of isoprenoids which have a great impact on humans. However, plant-based supply of isoprenoids is very low and techniques employed to isolate them are tedious and lead to consumption of numerous natural sources. Many of these metabolites are very complex, and chemical synthesis often requires several steps and difficult reactions, resulting in low yield or incorrect stereochemistry and high cost. In light of the drawbacks posed by chemical procedures for isoprenoid isolation, there is an emergent need to focus on engineering of metabolic pathways for large-scale and cost-effective metabolite production in heterologous hosts.

Prior art literature has focussed on metabolic engineering of isoprenoids. Saccharomyces cerevisiae has been employed as a preferred host over E.coli as it contains a native MVA pathway and is considered as a better system for cytochrome P450 enzymes (Hong, K.-K. & Nielsen, J. Metabolic engineering of Saccharomyces cerevisiae : a key cell factory platform for future biorefineries. Cellular and Molecular Life Sciences 69, 2671-2690 (2012)).

Through metabolic engineering, increased artemisinic acid production is observed up to 7.7 g L ¹ (Paddon, C.J. et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496, 528 (2013)). Studies on triterpenoid production through metabolic engineering are in the initial stages b-amyrin production in yeast is improved to 6 mg L ¹ (50%) by expressing b-amyrin synthase (. Artemisia annua) and 3- hydroxyl-3-methylglutaryl-CoA reductase (HMGR) under GAL promoter while down regulating lanosterol synthase [Kirby, J., Romanini, D.W., Paradise, E.M. & Keasling, J.D. Engineering triterpene production in Saccharomyces cerevisiae- b-amyrin synthase from Artemisia annua. The FEBS journal 275, 1852-1859 (2008)]. Overexpressing truncated HMGR, farnesyl pyrophosphate synthase, squalene synthase and 2,3- oxidosqualene synthase genes, together with increasing protopanaxadiol synthase activity through codon optimization and two-phase extractive fermentation results in protopanaxadiol (1.2 gm L ¹) and dammarenediol-II (1.5 gm L ¹) production [Dai, Z. et al. Metabolic engineering of Saccharomyces cerevisiae for production of ginsenosides. Metabolic engineering 20, 146-156 (2013).]. Yeast has been used for the production of a wide variety of products like biofuels, protein drugs and non-ribosomal peptides.

Even though metabolic engineering has been employed in prior art literature to arrive at the expression of enzymes involved in isoprenoid synthesis, said prior art disclosures are completely silent on expression of precursors and enzymes involved in limonoids synthesis in Azadirachta indica.

The metabolite extract of A. indica which is rich in limonoids (tetranortriterpenoids) has been indicated to be of utmost importance as a bioinsecticide, hence, there is an urgent need to produce formulations containing limonoids of A. indica origin, since;

(i) limonoids are present in minimal concentrations in natural extracts of A. indica,

(ii) extraction processes are tedious to isolate tetranortriterpenoids as well as precursors of limonoids, and

(iii) chemical synthesis procedures are difficult due to complex structures of limonoids.

Therefore, there is a need in the art for a method for producing tirucalladienol for prudetion of limonoids of A. indica origin.

OBJECTS OF THE INVENTION

An object of the present invention is to provide a process for producing tirucalladienol in increased concentrations, for use as a precursor in the synthesis of limonoid.

Another object of the present invention is to provide polynucleotide sequences encoding squalene epoxidase (SQO) and ti ucal la-7, 24-4ίoh-3b-o1 synthase (TTS) involved in synthesis of limonoid.

Yet another object of the present invention is to provide for increased tirucalladienol production by co-expressing a first vector comprising a polynucleotide sequence encoding timcalla-7,24-dien-3P-ol synthase (TTS) along with a second vector comprising a polyncleotide sequence encoding squalene epoxidase under strong promoter GAL.

Still another object of the present invention is to provide a recombinant yeast as an expression system for production of tirucalladienol. SUMMARY OF THE INVENTION

An aspect of the present invention provides a process for producing tirucalladienol comprising;

(i) cloning a nucleic acid sequence encoding tirucalladienol synthase having the polynucleotide sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33 into a first vector and expressing said first vector into a host organism;

(ii) cloning a nucleic acid sequence encoding squalene epoxidase having the polynucleotide sequence as set forth in SEQ ID NO: 3 into a second vector;

(iii) co-expressing the second vector of step (ii) into the host organism harbouring the first vector of step (i);

(iv) culturing the host organism of step (iii) at 30 °C for 24 hours in a nutrient medium;

(v) saponifying the host organism of step (iv) and extracting with n-hexane to obtain a crude metabolite extract;

(vi) purifying the crude metabolite extract obtained in step (v) with a column containing silver nitrate impregnated silica gel and dichloromethane as a solvent to obtain tirucalladienol.

Another aspect of the present invention provides a nucleic acid sequence encoding tirucalladienol synthase having atleast 85% sequence identity with SEQ ID NO. 1, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33.

Another aspect of the present invention provides a mutant tirucalladienol synthase having an amino acid substitution at one or more amino acid positions selected from the group consisting of amino acid positions Y125, F260, T413, V484, V534, L553 and V550 according to the amino acid position of the Azadirachta indica tirucalladienol synthase having the amino acid sequence as set forth in SEQ ID NO. 2 encoded by SEQ ID NO: 1. Yet another aspect of the present invention provides a mutant tirucalladienol synthase having an amino acid substitution at one or more amino acid positions selected from the group consisting of amino acid positions Y125, F260, T413, V484, V534, L553 and V550 according to the amino acid position of the Azadirachta indica tirucalladienol synthase having the amino acid sequence as set forth in SEQ ID NO. 2 encoded by SEQ ID NO: 1, wherein said mutant tirucalladienol synthase is having the amino acid sequence selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 16, SEQ ID NO. 20, SEQ ID NO. 24, SEQ ID NO. 28, SEQ ID NO. 32 and SEQ ID NO. 36.

Still another aspect of the present invention provides a first recombinant DNA construct comprising a polynucleotide having the sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33, wherein the polynucleotide encodes tirucalladienol synthase.

Another aspect of the present invention provides a second recombinant DNA construct comprising a polynucleotide having the sequence as set forth in SEQ ID NO: 3 operably linked to a GAL1 promoter, wherein the polynucleotide encodes squalene epoxidase.

Yet another aspect of the present invention provides a first vector comprsing the first recombinant DNA construct, wherein the first vector is selected from the group consisting of pYES2/CT, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

Still another aspect of the present invention provides a second vector comprising the second recombinant DNA construct, wherein the second vector is selected from the group consisting of pESC-LEU, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET- 32, pET28 and pETDuet.

Another aspect of the present invention provides a host organism transformed with the first vector comprsing the first recombinant DNA construct and the second vector comprising the second recombinant DNA construct.

Yet another aspect of the present invention provides a host organism transformed with the first vector comprsing the first recombinant DNA construct and the second vector comprising the second recombinant DNA construct, wherein the host organism is a yeast.

Still another aspect of the present invention provides a host organism transformed with the first vector comprsing the first recombinant DNA construct and the second vector comprising the second recombinant DNA construct, wherein the host organism is a yeast INVScl.

Yet another aspect of the present invention provides an expression of recombinant DNA constructs in a host cell and purification of metabolites related to limonoids.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 depicts limonoid biosynthesis in neem.

Figure 2 depicts statistics of differential gene expression analysis between flower and kernel.

Figure 3 depicts the total ion chromatograms of fractions in tirucalladienol purification by 5% AgN0₃ silica column.

Figure 4 depicts ¹ H NMR spectrum (CDCb, 700 MHz) of tirucalladienol.

Figure 5 depicts ¹³C NMR spectrum (CDCI3, 700 MHz) of tirucalladienol.

Figure 6 depicts DEPT- 135 NMR spectrum (CDCI3, 700 MHz) of tirucalladienol.

Figure 7 depicts HSQC DEPT- 135 NMR spectrum (CDCb, 700 MHz) of tirucalladienol.

Figure 8 depicts HMBC NMR spectrum (CDCb, 700 MHz) of tirucalladienol.

Figure 9 depicts COSY NMR spectrum (CDCb, 700 MHz) of tirucalladienol.

Figure 10 depicts NOESY spectrum (CDCb, 700 MHz) of tirucalladienol.

Figure 11 depicts relative fold change of squalene, ergosterol, lanosterol and tirucalladienol. The graph shows the abundance of lanosterol and tirucalladienol in INVScl host cell by coexpression of TTS and SQO. The tirucalldienol production was increased to 6.6 mg/L (coexpression of TTS and SQO) as compared to TTS expression where the tirucalldienol production was 3.1 mg/L. Figure 12 depicts multiple sequence alignment of tirucalladienol synthase. Amino acid sequences of TTS {A. indica), Z7TTS ( Euphorbia tirucalli, AB206469), /ATTS ( Rhizophora stylosa, BAF80442), /fcTTS ( Kandelia candel, BAF35580) and Tc/TTS ( Ailanthus altissima, DD135972) are used for multiple sequence alignment. The highly conserved DCTAE motif is indicated in orange, and QW motifs are indicated in by line above the alignment blue and active site residues in red colour letters.

Figure 13 depicts total ion chromatograms of mutant tirucalladienol synthase.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The invention can be understood in depth from the detailed description provided below along with the list of sequences which forms a part of the present application.

The sequence descriptions and sequence listing attached hereto obey with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications.

SEQ ID NO.l: Nucleotide sequence of cDNA encoding tirucalladienol synthase (2283 bp)

SEQ ID NO.2: Amino acid sequence of tirucalladienol synthase enoded by cDNA fragment of SEQ ID NO: 1 (760 aa)

SEQ ID NO.3: Nucleotide sequence of cDNA encoding squalene epoxidase (1593 bp)

SEQ ID NO.4: Amino acid sequence of squalene epoxidase encoded by cDNA fragment of SEQ ID NO. 3 (530 aa)

SEQ ID NO.5: Nucleotide sequence of an oligonucleotide primer used to amplify SEQ ID NO. 1 (43 bp)

SEQ ID NO.6: Nucleotide sequence of an oligonucleotide primer used to amplify SEQ ID NO. 1 (34 bp)

SEQ ID NO.7: Nucleotide sequence of an oligonucleotide primer used to amplify SEQ ID NO. 3 (30 bp)

SEQ ID NO.8: Nucleotide sequence of an oligonucleotide primer used to amplify SEQ ID NO. 3 (51 bp) SEQ ID NO.9: Nucleotide sequence of cDNA encoding mutant tirucalladienol synthase Y125F (2283 bp)

SEQ ID NO.10: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at Y125F in SEQ ID NO.l to generate SEQ ID NO.9 (27 bp)

SEQ ID NO.11: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at Y125F in SEQ ID NO.l to generate SEQ ID NO.9 (27bp)

SEQ ID NO.12: Polypeptide sequence (encoded by SEQ ID NO: 9) having a mutation at the 125^th position according to the numbering of SEQ ID NO.2, wherein, Y (tyrosine) is replaced by F (phenylalanine) (760 aa)

SEQ ID NO.13: Nucleotide sequence of cDNA encoding mutant tirucalladienol synthase F260Y (2283 bp)

SEQ ID NO.14: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at F260Y in SEQ ID NO.l to generate SEQ ID NO.13 (27 bp)

SEQ ID NO.15: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at F260Y in SEQ ID NO.l to generate SEQ ID NO.13 (27 bp)

SEQ ID NO.16: Polypeptide sequence (encoded by SEQ ID NO: 13) having a mutation at the 260^th position according to the numbering of SEQ ID NO.2, wherein F (phenylalanine) is replaced by Y (tyrosine) (760 aa)

SEQ ID NO.17: Nucleotide sequence of cDNA encoding mutant tirucalladienol synthase T413S (2283 bp)

SEQ ID NO.18: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at T413S in SEQ ID NO.l to generate SEQ ID NO.17 (31 bp)

SEQ ID NO.19: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at T413S in SEQ ID NO.l to generate SEQ ID NO.17 (31 bp)

SEQ ID NO.20: Polypeptide sequence (encoded by SEQ ID NO 17) having a mutation at the 413^th position according to numbering of SEQ ID NO.2, wherein T (threonine) is replaced by S (serine) (760 aa)

SEQ ID N0.21: Nucleotide sequence of cDNA encoding mutant tirucalladienol synthase V484L (2283 bp) SEQ ID N0.22: Nucleotide sequence of an oligonucleotide primer to insert a mutation at V484L in SEQ ID NO. 1 to generate SEQ ID N0.21 (34 bp)

SEQ ID N0.23: Nucleotide sequence of an oligonucleotide primer to insert a mutation at V484L in SEQ ID NO. 1 to generate SEQ ID NO. 21 (34 bp)

SEQ ID N0.24: Polypeptide sequence (encoded by SEQ ID NO: 21) having a mutation at the 484^th position according to the numbering of SEQ ID NO.2, wherein V (valine) is replaced by L (leucine) (760 aa)

SEQ ID NO.25: Nucleotide sequence of cDNA encoding mutated tirucalladienol synthase V534A (2283 bp)

SEQ ID N0.26: Nucleotide sequence of an oligonucleotide primer to insert a mutation at V534A in SEQ ID NO.l to generate SEQ ID N0.25.

SEQ ID NO.27: is a nucleotide sequence of an oligonucleotide primer to insert a mutation at V534A in SEQ ID NO.l to generate SEQ ID N0.25.

SEQ ID NO.28: is a polypeptide sequence (encoded by SEQ ID NO. 25) having a mutation at the 534^th position according to the numbering of SEQ ID NO.2, wherein V (valine) is replaced by A (alanine) (760 aa)

SEQ ID NO.29: Nucleotide sequence of cDNA encoding mutant tirucalladienol synthase L553F (2283 bp)

SEQ ID NO.30: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at L553F in SEQ ID NO. 1 to generate SEQ ID NO.29 (27 bp)

SEQ ID N0.31: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at L553F in SEQ ID NO.l to generate SEQ ID N0.29 (27 bp)

SEQ ID NO.32: Polypeptide sequence (encoded by SEQ ID NO. 29) having a mutation at the 553^rd position according to numbering of SEQ ID NO.2, wherein L (leucine) is replaced by F (phenylalanine) (760 aa)

SEQ ID NO.33: Nucleotide sequence of cDNA encoding mutant tirucalladienol synthase V550T (2283 bp)

SEQ ID NO.34: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at V550T in SEQ ID NO.l to generate SEQ ID NO.33 (27 bp) SEQ ID NO.35: Nucleotide sequence of an oligonucleotide primer used to insert a mutation at V550T in SEQ ID NO.l to generate SEQ ID NO.33 (27 bp)

SEQ ID NO.36: is a polypeptide sequence (encoded by SEQ ID NO. 33) having a mutation at the 550^th position according to the numbering of SEQ ID NO.2, wherein V (valine) is replaced by T (threonine) (760 aa)

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

In this context of the present invention, a number of terms are utilized. The terms “polynucleotide/nucleic acid” are used interchangeably herein. These terms indicate nucleotide sequences that may be a polymer of RNA or DNA that is single- or double- stranded, that optionally contains synthetic or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.

The invention uses high-throughput techniques to identify limonoid biosynthetic genes. It is necessary to select tissues where limonoid production is high as well low. It was observed that production of limonoids is very high in seeds, kernel and pericarp as compared to other tissues. The application of differential gene expression analysis across these tissues results in rapid identification of genes involved in limonoid biosynthesis.

The methods of the present invention use differential gene expression approach for isolation of candidate genes and functional identification of these genes by heterologous expression in yeast. For example, corresponding cDNA sequences may be identified by differential gene expression analysis of high-throughput data between neem seed (limonoid content is high) and flower (limonoid content is low).

The present invention employs nucleic acid amplification for isolation of limonoid biosynthesis genes. The nucleic acid to be used as a template for amplification can be isolated from tissues with standard methodologies. The nucleic acid can be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it is desired to convert RNA to a cDNA.

“PCR” or“polymerase chain reaction” is a technique for synthesis of large quantities of specific DNA segments.

A pairs of primers selectively hybridize to nucleic acids in specific conditions that permit selective hybridization. The term“primer,” is defined as any nucleic acid that is capable of priming synthesis of a nascent nucleic acid in a template-dependent process. Primers are single or double-stranded oligonucleotides forming 20 to 25 base pairs in length and in some cases, longer sequences can be employed. Once primer and template are hybridized, polymerase enzyme facilitates template-dependent nucleic acid synthesis. Multiple rounds of amplification are conducted until sufficient amount of amplification product is produced.

The term“recombinant DNA construct” means, a recombinant nucleic acid sequence is made by an artificial combination of two separated nucleotide segments, e.g. by ligation or chemical synthesis or by the manipulation of isolated segments of nucleic acids by molecular biology techniques.

The term“vector” means a DNA molecule which can self-replicate in a host cell and/or to which another DNA segment can be operatively linked to bringing about replication of the attached segment.

The term“transformation” means the transfer of nucleic acid fragments or recombinant DNA constructs into a host organism which may include either a yeast cell or a bacterial cell. For example, transformation of a plasmid into a microorganism by heat shock or electroporation.

The term“expression”, as used herein refers to transcription and stable accumulation of sense mRNA derived from a polynucleotide in a host organism which includes a yeast cell and/or a bacterial cell. The expression may also refer to translation of mRNA into a polypeptide.

The present invention employs metabolite extraction from the host organism and its purification.“Metabolite extraction” refers to the isolation of metabolites from the expressed host by partition coefficient principle and further purification of invention product which is an intermediate of limonoid biosynthesis.

The term “characterization” means confirmation of metabolite which is produced through limonoid biosynthetic pathway genes by using different techniques like GC- MS (Gas Chromatography and Mass Spectrum) and NMR (Nuclear Magnetic Resonance). More than 150 limonoids and their derivatives were isolated and characterized from neem. Limonoid abundance varies across neem tissue. The mature seed kernel and pericarp were found to contain highest amount of triterpenoids. The C- seco limonoids were observed to be high in kernel as compared to other tissues, whereas pericarp, flower and leaf contain ring-intact limonoids.

The present invention provides polynucleotides encoding enzymes involved in limonoid biosynthesis. Accordingly, the present invention provides a cDNA sequence having SEQ ID NO. 1 encoding timcalla-7,24-dien-3P-ol synthase (TTS) having the amino acid sequence as set forth in SEQ ID NO. 2, and a cDNA sequence having SEQ ID NO. 3 encoding squalene epoxidase having the amino acid sequence as set forth in SEQ ID NO. 4.

The present invention provides a process for production of timcalladienol involving nucleotide sequences having atleast 85% sequence identity with SEQ ID NO. 1 encoding timcalldienol synthase and nucleotide sequences having atleast 85% sequence identity with SEQ ID NO. 3 encoding squalene epoxidase. These sequences are co expressed in a host organism cultured in a nutrient medium to yield timcalladienol in concentrations ranging from 5mg/L to 8 mg/L.

The present invention also provides a yeast expression system comprising recombinant DNA constructs of the present invention. The altered expression levels of the cDNA sequences in the recombinant yeast expression system results in an increased production of timcalladienol.

The present invention further provides an expression of recombinant DNA constmcts in a host oragism and purification of metabolites related to limonoids.

An embodiment of the present invention provides a process for producing timcalladienol comprising; (i) cloning a nucleic acid sequence encoding timcalladienol synthase having the polynucleotide sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33 into a first vector and expressing said first vector into a host organism;

(vi) purifying the crude metabolite extract obtained in step (v) with a column containing silver nitrate impregnated silica gel and dichloromethane as a solvent to obtain timcalladienol.

In yet another embodiment of the present invention, there is provided a method of producing timcalladienol, wherein the first vector is selected from the group consisting of pYES2/CT, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

In still another embodiment of the present invention, there is provided a method of producing timcalladienol, wherein the second vector is selected from the group consisting of pESC-LEU, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

In another embodiment of the present invention, there is provided a method of producing timcalladienol, wherein the host organism is a yeast INVScl.

In yet another embodiment of the present invention, there is provided a method of producing timcalladienol, wherein the nutrient medium comprises 2% galactose containing complete supplement mixture (CSM) without the amino acid selected markers uracil and leucine. In still another embodiment of the present invention, there is provided a method of producing tirucalladienol, wherein the tirucalladienol obtained is in a concentration ranging from 5mg/L to 8mg/L.

An embodiment of the present invention provides a nucleic acid sequence encoding tirucalladienol synthase having atleast 85% sequence identity with SEQ ID NO. 1, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33.

Another embodiment of the present invention provides a mutant tirucalladienol synthase having an amino acid substitution at one or more amino acid positions selected from the group consisting of amino acid positions Y125, F260, T413, V484, V534, L553 and V550 according to the amino acid position of the Azadirachta indica tirucalladienol synthase having the amino acid sequence as set forth in SEQ ID NO. 2 encoded by SEQ ID NO: 1.

Yet another embodiment of the present invention provides a mutant tirucalladienol synthase derived from wild type tirucalladienol synthase of Azadirachta indica, wherein the mutant is having an amino acid substitution at one or more amino acid positions selected from the group consisting of amino acid positions Y125, F260, T413, V484, V534, L553 and V550 according to the amino acid numbering of the Azadirachta indica tirucalladienol synthase having the amino acid sequence as set forth in SEQ ID NO. 2 encoded by SEQ ID NO: 1.

Accordingly, the amino acid substitutions in the active site residues of the amino acid sequence of tirucalladienol synthase (SEQ ID NO. 2) is selected from the group comprising Y125F (SEQ ID NO. 12), F260Y (SEQ ID NO. 16), T413S (SEQ ID NO. 20) V484L (SEQ ID NO. 24), V534A (SEQ ID NO. 28), L553F (SEQ ID NO. 32), and V550T (SEQ ID NO. 36) to obtain an increased production of tirucalladienol.

Still another embodiment of the present invention provides a mutant tirucalladienol synthase, wherein said mutant tirucalladienol synthase is having the amino acid sequence selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 16, SEQ ID NO. 20, SEQ ID NO. 24, SEQ ID NO. 28, SEQ ID NO. 32 and SEQ ID NO. 36. Another embodiment of the present invention provides timcalladienol produced by the process to be used as a precursor for the synthesis of limonoids.

Yet another embodiment of the present invention provides a first recombinant DNA construct comprising a polynucleotide having the sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33, wherein the polynucleotide encodes timcalladienol synthase.

Still another embodiment of the present invention provides a second recombinant DNA construct comprising a polynucleotide having the sequence as set forth in SEQ ID NO: 3 operably linked to a GAL1 promoter, wherein the polynucleotide encodes squalene epoxidase.

Another embodiment of the present invention provides a first vector comprising the first recombinant DNA construct having a polynucleotide encoding timcalladienol synthase, wherein the first vector is selected from the group consisting of pYES2/CT, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

Yet another embodiment of the present invention provides a second vector comprising the second recombinant DNA construct having a polynucleotide encoding squalene epoxidase, wherein the second vector is selected from the group consisting of pESC- LEU, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

Still another embodiment of the present invention provides a host organism transformed with the first vector comprising the first recombinant DNA construct and the second vector comprising the second recombinant DNA constmct.

In another embodiment of the present invention, there is provided a host organism transformed with the first vector comprising the first recombinant DNA constmct and the second vector comprising the second recombinant DNA constmct, wherein the host organism is a yeast.

In yet another embodiment of the present invention, there is provided a host organism transformed with the first vector comprising the first recombinant DNA constmct and the second vector comprising the second recombinant DNA constmct, wherein the host organism is a yeast INVScl. The present invention provides cDNA sequences encoding enzymes involved in limonoid biosynthesis. Specifically provided herein is a cDNA sequence encoding timcalladienol synthase represented by SEQ ID NO: 1 and cDNA sequence encoding squalene epoxidase represented by SEQ ID NO: 3. These cDNA sequences have 100% sequence identity with the nucleotide sequences isolated from Azadirachta indica.

In the present invention, to identify genes involved in limonoids biosynthesis in neem, tissue-specific transcriptome analysis was done in the pericarp, kernel, flower and leaf. Total of 127,815 transcripts was generated and functional annotation was done by KEGG and Pfam analysis. Based on these analyses, genes for terpenoid metabolic pathways in neem were predicted. To identify the genes involved in limonoid biosynthesis in A. indica, differential gene expression was carried out. 3-hydroxy-3- methyl-glutaryl-coenzyme A synthase [HMGS], 3-hydroxy-3-methyl-glutaryl- coenzyme A reductase [HMGR] (key genes from the MVA pathway), farnesyl diphosphate synthase [FDS], squalene synthase [SQS], squalene epoxidase [SQO] and timcalladienol synthase [TTS] were highly expressed in the kernel as compared to flowers. Downstream enzymes of limonoid biosynthesis were also predicted.

The first committed step for limonoid biosynthesis is the cyclization of 2,3- oxidosqualene by triterpene cyclases. In plants, 2,3-oxidosqualene is involved in production of steroids and triterpenoids. In steroid biosynthesis, cycloartenol is synthesized from 2,3-oxidosqualene by the action of cycloartenol synthase and further modified to phytosterol, while other triterpene cyclases convert 2,3-oxidosqualene into triterpene cyclic product, which is further modified by CYP450 enzymes into triterpenoids. The process for limonoid biosynthesis in neem involves euphol or timcallol or butyrospermol or timcalladienol as triterpene cyclic product.

In another embodiment of the present invention, there is provided a yeast expression vector comprising cDNA sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33 encoding timcalladienol synthase.

The yeast expression vector is pYES2/CT. The cDNA sequences encoding timcalladienol synthase are cloned into a pYES2/CT vector and expressed in yeast INVScl host cells. Saponification and n-hexane extraction of yeast cells is performed to isolate the metabolites. Silver nitrate column is used to purify tirucalladienol synthase metabolite. Based on GC-MS and NMR analyses, the metabolite is identified as tirucalladienol.

Further, the polynucleotide sequence encoding squalene epoxidase is selected and cloned into pESC-LEU vector. Functional characterization of SQO is done by co expressing with tirucalladienol synthase. A two-fold increase in production of tirucalladienol is observed.

Another embodiment of the present invention provides variants of polynucleotide encoding tirucalladienol synthase. In a preferred embodiment, the polynucleotide sequence encoding tirucalladienol synthase is having at least 90% sequence identity with SEQ ID NO: 1. Accordingly, the polypeptide sequences encoded by these variants of SEQ ID NO. 1 are having at least 90% sequence identity with the amino acid sequence as provided in SEQ ID NO. 2.

Another embodiment of the present invention provides variants of polynucleotide encoding squalene epoxide. In a preferred embodiment, the polynucleotide sequence endocing squalene epoxide is having at least 90% sequence identity with SEQ ID NO:

3.

In another preferred embodiment of the present invention, there is provided polynucleotide sequences having at least 85% sequence identity with SEQ ID NO: 1 and SEQ ID NO. 3, encoding tirucalladienol synthase and squalene epoxide, respectively. Further, polypeptide sequences encoded by variants of SEQ ID NO: 1 and SEQ ID NO: 3 have at least 85% sequence identity with SEQ ID NO. 2 and SEQ ID NO. 4, respectively.

The nucleic acid sequences encoding tirucalladienol synthase having atleast 85% sequence identity with SEQ ID NO. 1 cloned from Azadirachta indica are selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33.

In another embodiment, the present invention provides amino acid residues identified at the active site of tirucalladienol synthase (SEQ ID NO.2) selected from the group comprising N120, P123, Y125, F126, W220, W258, F260, C261, V264, 1369, G370, C371, V372, T413, W419, F475, V484, D486, C487, V534, W535, V550, L553, L556, E564, C565, W613, Y619, V728, F729, M730, L735 and Y737.

Accordingly, the amino acid variants having substitutions at the active site of the amino acid sequence of tirucalladienol synthase (SEQ ID NO.2) is selected from the group comprising Y125F (SEQ ID N0.12), F260Y (SEQ ID NO.16), T413S (SEQ ID NO.20), V484L (SEQ ID NO: 24), V534A (SEQ ID NO: 28), L553F (SEQ ID NO: 32) and V550T (SEQ ID NO: 36).

More preferably, amino acid variants having substitutions at the active site residues of the amino acid sequence of tirucalladienol synthase (SEQ ID NO. 2) is selected from the group comprising Y125F (SEQ ID NO. 12), F260Y (SEQ ID NO. 16) and T413S (SEQ ID NO.20) to result in an increased production of tirucalladienol.

The mutants of TTS with substitutions at positions Y125F (SEQ ID NO. 12), F260Y (SEQ ID NO.16) and T413S (SEQ ID NO.20) result in an increase in production of tirucalladienol by 20% as compared to tirucalladienol production by native tirucalladienol synthase.

In addition, the present invention relates to the identification of domains active site residues (FIG.12) which stabilize the carbocation in tirucalladienol synthase (TTS) by using coordinates from human lanosterol synthase (PDB ID: 1W6K).

Further, eight active site residues were selected for mutagenesis to enhance the efficiency of TTS for the production of tirucalladienol. Y125F (SEQ ID NO. 12), F260Y (SEQ ID NO. 16) and T413S (SEQ ID NO.20) mutations have stabilized the carbocation and increased the production of tirucalladienol by 20% as compared to wild tirucalladienol synthase in neem (Fig.13).

In an embodiment, the present invention provides tirucalladienol produced by the present process as a precursor for the synthesis of limonoids. Tirucalladienol is employed as a precursor involved in limonoids synthesis in Azadirachta indica.

EXAMPLES

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. Example 1: Identification of genes involved in limonoid biosynthesis

Limonoids in neem are diverse in skeletal architecture and their abundance is highly tissue-specific. The mature seed kernel and pericarp of initial stages were found to contain the highest amount of total limonoids. Fruit pericarp, flower and leaf contained majorly the basic limonoids (azadirone, azadiradione, epoxyazadiradione, nimocinol, gedunin). C-seco limonoids (nimbin, nimbanal, nimbinene, 6-deacetylnimbinene, salannin, salannol acetate, 6-deacetylnimbin, 3-deacetylsalannin, azadirachtin A and B) were high in the kernel as compared to the other tissues. This observation shows that genes which are involved in limonoid biosynthesis are highly expressed in fruit kernel and pericarp.

RNA was isolated from tissues where the limonoid content is high, such as kernel, pericarp, flower and leaves. Paired-end reads were generated by using Illumina Nextseq500. Low-quality reads were removed and assembled by Trinity with hash length 25. Transcripts from all the tissues were clustered by using CD-HIT.

For functional annotation, tissue- specific transcriptomes were submitted to Blastx with NCBI non-redundant database and an E-value cutoff of 10⁵. Total of 36546 (78.95%), 33947 (77.04%), 37163 (79.99%) and 37477 (78.38%) transcripts were annotated from kernel, pericarp, leaves and flower respectively. The clustered 127518 transcripts were submitted to KAAS analysis by taking the plant reference database. 17823 (14%) transcripts were assigned 4037 unique KO numbers, which covered 250 pathways. Peptide sequences of transcripts with length more than 20 amino acids were submitted to Pfam analysis. 53168 transcripts were assigned unique Pfam IDs. A total of 4930 different Pfam IDs were assigned to the transcripts. From this analysis, MVA and MEP pathway genes were identified.

RNA-Seq can be used for both discovery and quantification of transcripts in a single experiment. The expression level of each transcript is measured by the number of reads that map to it, which is expected to correlate directly with its abundance level. One of the applications of the expression level of the transcriptome is to identify differentially expressed genes in two or more conditions/tissues. Neem limonoids production is low in flower and highest in kernel hence differentially expression analysis was done between these tissues (FIG.2). DESeq2 was used to predict the genes involved in limonoids biosynthesis. 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase [Az^’HMGS], 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase [T/HMGRJ (key genes from the MVA pathway), famesyl diphosphate synthase [AzFDS], squalene synthase [Az^'SQS], squalene epoxidase [Az^'SQO] and tirucalladienol synthase [Az^'TTS l] were highly expressed in kernel as compared to flowers. Downstream enzymes of limonoid biosynthesis such as CYP 450, methyltransferases, acyltransferases, desaturase and glycosyltransferases were also predicted.

Example 2: Cloning and functional characterization of tirucalladienol synthase

Triterpene synthase catalyzes the first committed step in triterpenoid biosynthesis by cyclizing 2,3-oxidosqualene. Functional expression of tirucalladienol synthase (TTS) of neem (A. indica ) in yeast (INVScl) was done by cloning of coding sequence of TTS into expression vector pYES2/CT. The ORF of TTS (SEQ ID NO. 1) was 2,283 bp and codes for a protein having 760 amino acids (SEQ ID NO. 2). TTS showed a maximum identity with several characterized triterpene synthases such as 87% sequence identity to tirucalladienol synthase from Ailanthus altissima [NCBI Accession No. - DD135972], 71 % identity with b-amyrin synthase from Betula platyphylla [UniProt: Q8W3Z1], Glycyrrhiza glabra [UniProt: Q9MB42]. TTS consisted of DCTAE motif and six copies of QW [(K/R)(G/A)XX (F/Y/W)(L/I/V) XXXQXXXGXW] motifs. The aspartate residue in DCTAE motif involves in the protonation of epoxide ring of 2,3- oxidosqualene in order to start a cascade cyclization. QW motifs are the structural elements present in all triterpene synthase.

The open reading frame of TTS (SEQ ID NO.l) was amplified from neem fruit cDNA by introducing EcoRI and Notl sites using forward primer (SEQ ID NO.5) and reverse primer (SEQ ID NO. 6), respectively. Full-length PCR product was cloned into expression vector pYES2/CT using T4 DNA ligase. The expression of TTS was carried out in S. cerevisiae yeast strain INVScl. Yeast cells harbouring the expression construct were inoculated into 2% glucose containing CSM-URA and incubated at 30°C. The overnight grown culture was induced by transferring it into 2 % galactose containing CSM-URA and incubated at 30°C for 24 h. After culturing, the cell pellet was saponified with 10% KOH in 80% ethanol at 70°C for 2 h and then extracted thrice with equal volumes of n-hexane. The crude metabolite extract obtained were passed through anhydrous sodium sulphate and concentrated to 50pl. The extracted samples were analyzed by using GC-MS.

To characterize the TTS metabolite, 25 L of yeast culture was grown in 2% galactose containing CSM-URA medium. Crude metabolite extract was obtained from an n- hexane extract of saponified yeast pellet as described above. 120 mg of crude metabolite extract was obtained from the saponification of 130 g of yeast cell pellet. The crude metabolite extract was loaded onto the column containing 5 % silver nitrate impregnated silica gel (230 - 400 mesh size) and dichloromethane (DCM) was used as a solvent. The column was passed through 100 % DCM in which lanosterol and TTS metabolic product were eluted. Squalene and ergosterol were eluted when the polarity was increased to 5% by methanol. All the eluted fractions were analyzed by GC-MS (FIG 3). The TTS metabolite mass, structure and stereochemistry were analyzed using different techniques such as by GC-MS, ^ NMR, ¹³C-NMR, DEPT, HMBC, HSQC, COSY and NOSY (FIG 4 - 10). A. indica TTS has been functionally expressed in yeast, and shown to produce Timcalla-7,24-dien-3P-ol (tirucalladienol) as an individual product.

*H NMR (700 MHz) d: 5.26 (1H, dt, J=3.41 Hz, 2.95 Hz, H-7), 5.11 (1H, t, J=6.47 Hz, H-24), 3.25 (1H, dd, J=11.41, 3.92 Hz, H-3a), 2.18-2.24 (1H, m, H-9), 2.12-2.17 (1H, m, H-6a), 2.01-2.08 (1H, m, H-23R), 1.91-2.00 (2H, m, H-6b and H-16b), 1.83-1.90 (1H, m, H-23S), 1.79 (1H, dd, J=13.62, 9.88 Hz, H-12b), 1.69 (3H, s, H-26), 1.68 (1H, br. s., H-1b), 1.66 (1H, d, J=3.75 Hz, H-2a), 1.62-1.64 (1H, m, H-12a), 1.61 (3H, s, H- 27), 1.59 (1H, d, J=3.75 Hz, H-2b), 1.53 (2H, m, J=8.86 Hz, H-I Ib and H-15a), 1.49 (1H, d, H-17b), 1.47 (1H, m, H-15b), 1.45 (2H, m, H-l la and H-22S), 1.39 (1H, m, H- 20a), 1.32 (1H, dd, J=12.09, 5.62 Hz, H-5a), 1.26 (1H, br. s, H-16a), 1.12-1.18 (lH,m, H-la), 1.01-1.08 (1H, m, H-22R), 0.98 (6H, s, H-28 and H-30), 0.89 (3H, d, J=6.47 Hz, H-21), 0.87 (3H, s, H-29), 0.82 (3H, s, H-18), 0.75 (3H, s, H-19).

¹³C NMR (700 MHz) d: 145.88 (C-8), 130.91 (C-25), 125.2 (C-24), 117.78 (C-7), 79.25 (C-3), 52.93 (C-17), 51.13 (C-14), 50.6 (C-5), 48.93 (C-9), 43.5 (C-13), 38.94 (C-4), 37.17 (C-l), 36.17 (C-22), 35.95 (C-20), 34.92 (C-10), 34.01 (C-15), 33.77 (C- 12), 28.2 (C-16), 27.67 (C-2), 27.59 (C-28), 27.25 (C-30), 25.71 (C-26), 25 (C-23), 23.92 (C-6), 21.89 (C-18), 18.31 (C-21), 18.11 (C-l l), 17.62 (C-27), 14.71 (C-29), 13.1 (C-19).

Example 3: Cloning and functional characterization of squalene epoxidase

Squalene epoxidase (SQO) oxidizes squalene into 2,3-oxidosqualene, which act as the substrate for synthesis of triterpenoids. The SQO was cloned into an expression vector pESC-LEU. The expression vector pESC-LEU was coexpressed in yeast strain INVScl harbouring TTS expression construct. The production of tirucalladienol was increased by two folds.

The coding sequence of SQO (SEQ ID NO. 3) was 1,593 bp and codes for a protein of 530 amino acids (SEQ ID NO. 4). SQO showed a maximum identity with several characterized SQOs such as 78 % identity to SQO from Arabidopsis thaliana [UniProt: 081000], 46 % identity to SQO from Homo sapiens [UniProt: Q14534] and 37 % identity to SQO from Candida albicans SC5314 [UniProt: Q92206]. SQO consists of Rossman fold GXGXXG motif, DG and GD motifs, as present in fungus and vertebrates. The GXGXXG motif binds with dinucleotide of a FAD, DG motif interacts with diphosphate group of FAD and GD motif may interact with ribityl moiety of a FAD.

The open reading frame of SQO (SEQ ID NO. 3) was amplified from neem fruit cDNA by forward primer (SEQ ID NO. 7) and reverse primer (SEQ ID NO. 8). The PCR product with introduced BamHI and Hindlll sites was cloned into a pESC-LEU vector under a strong GAL1 promoter. SQO expression construct was transformed into INVScl cells containing TTS-pYES2/CT vector. A single colony was inoculated into 2% glucose containing complete supplement mixture (CSM) without the respective amino acid selection marker (URA and LEU) and incubated at 30°C. An overnight grown culture was induced by transferring it into 2 % galactose containing (CSM, 500 ml) without the respective amino acid selection marker and incubated at 30°C for 24 h. Cholesterol was used as an internal standard for the quantification of change in metabolite concentration in yeast extracts. After culturing, the yeast cells were saponified and extracted with n-hexane. Crude Metabolite extracts were passed through anhydrous sodium sulphate and concentrated to 500 pL. Extracted samples were analyzed by using GC-FID. The relative abundance of sterol was estimated by comparing with a standard graph of cholesterol. When SQO and TTS were co expressed in a yeast, the production of tirucalldienol was increased to 6.6 mg/L as compared to tirucalldienol production with only TTS expression, which is 3.1 mg/L. There was a two-fold increase in production of tirucalladienol. Production of squalene was decreased to 1.2 mg/L in coexpression of SQO and TTS as compared to only SQO expression where squalene was around 2.4 mg/L. Similar results were observed with ergosterol. These results indicate that SQO converts squalene into 2,3-oxidosqualene (FIG. 11).

Example 4: Tirucalladeinol synthase engineering

The production of secondary metabolites in heterologous systems involves multiple modifications such as expression of polynucleotides which encodes for the enzymes involved in the biosynthetic pathway, change their expression levels to optimize the metabolic flux and modifying the primary metabolic pathway in yeast such that the metabolic flux is high towards precursor for production of secondary metabolites. Also by employing mutagenesis, an increase in efficiency of enzymes towards the substrate and product can be achieved.

In the present invention, the polynucleotide sequence encoding for tirucalladienol synthase was mutated to increase the efficiency towards the production of tirucalladienol. First, the homology model of tirucalladienol synthase was generated by considering the coordinates of human lanosterol synthase (1W6K). A total of 33 active site residues which were involved in cyclization of 2,3-oxidosqualene were identified (Fig. 12). These active residues identified are N120, P123, Y125, F126, W220, W258, F260, C261, V264, 1369, G370, C371, V372, T413, W419, F475, V484, D486, C487, V534, W535, V550, L553, L556, E564, C565, W613, Y619, V728, F729, M730, L735 and Y737. Out of these, eight active site residues were selected for mutagenesis: Y125, F260, T413, V484, V534, V550, L553 and L556.

F260 and T413 play a key role for p interaction with dammarenyl cation and when compared to most of the pentacyclic triterpene synthase, F260, T413 were mutated to tyrosine and serine respectively. Y125, V550, L553 and L556 were involved in stabilizing the side chain of timcalladienol. In most of the pentacyclic triterpene synthase, the hydrophobic residue at position L553 is replaced by aromatic amino acid like phenylalanine. The hydrophobic residue at position 550 was replaced with a hydroxy amino acid like serine or threonine. V484 and V534 are most likely to be involved in the stabilization of B-ring confirmation in the dammarenyl cation.

Primers (SEQ ID NOs. 10-11, 14-15, 18-19, 22-23, 26-27, 30-31, 34-35) were designed based on the mutation to be carried out in a polynucleotide sequence of timcalladienol synthase. The polymerase chain reaction was carried out with a plasmid having timcalladienol synthase (TTS-pYES2/CT) as a template and the amplified plasmid was restriction digested with Dpnl. All the methylated template plasmids were digested by Dpnl and only mutated plasmids were present in the reaction. Insertion of mutations was further confirmed through sequencing. Further the mutated plasmids were expressed in INVScl cells. Metabolites were extracted from saponified yeast cell pellet and the samples were analysed using GC-FID.

Mutants (variants) of TTS at V550T (SEQ ID NO: 36), L553F (SEQ ID NO:32), V534A (SEQ ID NO:28) and V484L (SEQ ID NO: 24) resulted in drastic reduction of timcalladienol production, which indicates that these amino acids play a key role in dammarenyl cation stabilization. Y125F (SEQ ID NO: 12), F260Y (SEQ ID NO: 16) and T413S (SEQ ID NO:20) mutants (variants) of TTS resulted in an increase in the production of timcalladienol by 20% as compared to timcalladienol synthase (Table 1 and FIG.12).

Table 1

ADVANTAGES OF THE PRESENT INVENTION

• The present invention provides details about the identification of polynucleotides encoding genes involved in the limonoid biosynthesis from Azadirachta indica.

• The invention is significant as limonoids produced in neem are known to possess medicinal and insecticidal properties. The production of limonoids can further be increased by metabolic engineering of polynucleotides involved in this pathway.

Claims

We claim,

1. A process for producing tirucalladienol comprising;

(i) cloning a nucleic acid sequence encoding tirucalladienol synthase having the polynueotide sequence selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33 into a first vector and expressing said first vector in a host organism;

(iii) co-expressing the second vector of step (ii) into the host organism harboring the first vector of step (i);

(vi) purifying the crude metabolite extract obtained in step (v) with a column containing silver nitrate impregnated silica gel and dichloromethane as a solvent to obtain tirucalldienol.

2. The process as claimed in claim 1, wherein the first vector is selected from the group consisting of pYES2/CT, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

3. The process as claimed in claim 1, wherein the second vector is selected from the group consisting of pESC-LEU, pESC-URA, pESC-TRP, pESC-HIS, pRS- 315, pET-32, pET28 and pETDuet.

4. The process as claimed in claim 1, wherein the host organism is a yeast InvScl.

5. The process as claimed in claim 1, wherein the nutrient medium comprising 2% galactose solution containing complete supplement mixture (CSM) without the amino acid selection markers uracil and leucine.

6. The method as claimed in claim 1, wherein the timcalladienol obtained is in a concentration ranging from 5mg/L to 8mg/L.

7. A nucleic acid sequence encoding timcalladienol synthase having atleast 85% sequence identity with SEQ ID NO. 1, wherein said nucleic acid sequence is selected from the group consisting of SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33.

8. A mutant timcalladienol synthase having an amino acid substitution at one or more amino acid positions selected from the group consisting of amino acid positions Y125, F260, T413, V484, V534, L553 and V550 according to the amino acid position of the Azadirachta indica timcalladienol synthase having the amino acid sequence as set forth in SEQ ID NO. 2 encoded by SEQ ID NO: 1.

9. The mutant timcalladienol synthase as claimed in claim 8, wherein said mutant timcalladienol synthase is having the amino acid sequence selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 16, SEQ ID NO. 20, SEQ ID NO. 24, SEQ ID NO. 28, SEQ ID NO. 32 and SEQ ID NO. 36.

10. A first recombinant DNA constmct comprising a polynucleotide having the sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 9, SEQ ID NO. 13, SEQ ID NO. 17, SEQ ID NO. 21, SEQ ID NO. 25, SEQ ID NO. 29 and SEQ ID NO. 33, wherein the polynucleotide encodes timcalladienol synthase.

11. A second recombinant DNA constmct comprising a polynucleotide having the sequence as set forth in SEQ ID NO: 3 operably linked to a GAL1 promoter, wherein the polynucleotide encodes squalene epoxidase.

12. A first vector comprsing the first recombinant DNA constmct as claimed in 10, wherein the first vector is selected from the group consisting of pYES2/CT, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

13. A second vector comprising the second recombinant DNA constmct as claimed in claim 11, wherein the second vector is selected from the group consisting of pESC-LEU, pESC-URA, pESC-TRP, pESC-HIS, pRS-315, pET-32, pET28 and pETDuet.

14. A host organism transformed with a first vector as claimed in claim 12 and a second vector as claimed in claim 13.

15. The host organism as claimed in claim 14, wherein the host organism is a yeast INVScl.