Description:4. DESCRIPTION
Technical Field of the Invention

The present invention is directed to the field of synthetic biology. Particularly, the invention relates to construction of biosynthetic pathway for violacein synthesis and methods for the producing the same. More particularly, the invention is directed to a biological synthesis of violacein pigment for various applications and methods for obtaining improved yield of violacein in Saccharomyces cereviciae by strain engineering.

Background of the Invention

Violacein is a deep purple color pigment with wide range of application in medicine (strong antibacterial, anticancer, antiviral, trypanocidal, and antiprotozoal), Textile, cosmetics, and pharmaceutical industries.
As used herein, the term “gene” refers to a nucleic acid fragment corresponding to specific amino acid sequence that expresses a specific protein with regulatory sequences. “Native gene” or “wild type gene” refers to a gene as found in nature with its own regulatory sequences.
As used herein, the term “promoter” refers to a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA capable of controlling the expression of a coding sequence or functional RNA which can be native, derived or synthetic. Some promoters are called constitutive as they are active in all circumstances in the cell, while others are called inducible as they are regulated and become active in response to specific stimuli.
As used herein, the term “gene expression”, refers to the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
As used herein, the term “transformation” as used herein, refers to the transfer of a nucleic acid fragment into a host organism either in the form of plasmid or integrated stably to the chromosome of the host organisms resulting in genetically stable inheritance. A cloning vector is a small piece of DNA, mostly a plasmid, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning or transformation purposes.
The term “host cell” includes an individual cell or cell culture which can be, or has been, a recipient for the subject of expression constructs. Host cells include progeny of a single host cell. Host cell can be any expression host including prokaryotic cell such as but not limited to Bacillus subtilis, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum or eukaryotic system, such as, but not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha.
The term “recombinant strain” refers to a host cell which has been transfected or transformed with the expression constructs or vectors of this invention.
The term “expression cassette” denotes a gene sequence used for cloning in expression vectors or in to integration vectors or integrated in to coding or noncoding regions of chromosome of the host cell in a single or multiple copy numbers, where the expression cassette directs the host cell's machinery to make RNA and protein encoded by the expression cassette.
The term “expression construct” is used here to refer to a functional unit that is built in a vector for the purpose of expressing recombinant proteins/peptides, when introduced into an appropriate host cell, can be transcribed and translated into a fusion protein which is displayed on the cell wall.
The term “nucleic acid” or “nucleotide sequence” is used to refer to an artificially synthesized nucleic acid in which the genes encoding violacein genes has been operably fused to promoters and terminators of Saccharomyces cerevisiae.
Violacein is derived from tryptophan biosynthetic pathway of marine pathogenic bacteria, such as Janthinobacterium lividum, Chromobacterium violaceum, and Pseudoalteromonas luteoviolacea. From L-Tryptophan, the violacein biosynthetic pathway involves five enzymatic reactions operated by vioA, vioB, vioC, vioD, and vioE. Two molecules of L-tryptophan are oxidatively condensed by vioA and vioB to form indole-3-pyruvic acid (IPA). Then, IPA is decarboxylated via vioE to form protodeoxyviolaceinic acid. Subsequently, protodeoxyviolaceinate is oxidized to violacein by vioD and vioC. Expression of VioA, VioB, and VioE results in prodeoxyviolacein. Combinatorial expression of VioABE, with VioC or VioD results in Deoxyviolacein and proviolacein respectively.
Majority of the reported violacein-producing microorganisms are pathogenic Gram-negative bacteria, for example C. violaceum and J. lividum, known to cause serious skin infection in immune-compromised people. This limits their industrial application. Chemical synthesis of violacein resulted in production of hazardous byproducts that causes environmental pollution.
To overcome this problem, an alternative appreciated method is biological production of textile dyes/pigments in engineered microorganisms by using rDNA technology. Therefore, reconstruction of violacein biosynthetic pathway in industrially acceptable hosts for stable and high production of violacein has been attracting strategy.
Saccharomyces cereviciae has been widely used and engineered for production of chemicals interest as it is nonpathogenic and classified as ‘generally regarded as safe’ (GRAS) by the US Food and Drug Administration (FDA).
A few patents related to the topic have been discussed below:
The patent US20110172826A1 (Device including altered microorganisms, and methods and systems of use) relates to a method for administration to at least one biological tissue of at least one device including at least one altered microorganism, and includes at least one nucleic acid construct encoding at least one therapeutic agent using an auxotrophic microorganism, at least one nucleic acid construct encoding at least one therapeutic agent, and at least one metabolite. This is different from our present invention which involves no therapeutic agents and involves recombinant eukaryotic host cell, and atleast one vector/s and promoter/s.
The patent US20060205017A1 (Microorganism genomics, compositions and methods related thereto) relates to a cell engineered with a replicable vector including heterologous genomic DNA isolated from a source of uncultivated microorganisms, which host cell produces a compound in a manner dependent on expression of at least one opening reading frame of the genomic DNA.
Our present invention is linked to cost effective sustainable production of violacein in ecofriendly manner by using engineered microorganisms.

Brief Summary of the Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

The primary objective of this invention is to provide a method of nucleic acids encoding violacein producing enzymes (VioA, VioB, VioC, VioD and VioE) operably fused to Saccharomyces cereviciae promoters and terminators and the process for obtaining a high yield of the violacein.
Another objective of our present invention is the cost-effective sustainable production of violacein in ecofriendly manner by using engineered microorganisms.

The present invention relates to a method of nucleic acids encoding violacein producing enzymes (VioA, VioB, VioC, VioD and VioE) operably fused to Saccharomyces cereviciae promoters and terminators and the process for obtaining a high yield of the violacein.
In one aspect, the invention provides a recombinant vectors containing nucleic acids encoding violacein producing enzymes.
In another aspect, the invention provides a recombinant host cell which can optimally produce violacein.
In a further aspect, the invention provides an improved process for production for violacein in a sustainable manner.

Brief Summary of the Drawings

The invention will be further understood from the following detailed description of a preferred embodiment taken in conjunction with an appended drawing, in which:

Fig 1 illustrates the diagram showing the genes circuit of recombinant plasmids pESCHIS_violacein 1 and pESCURA_violacein 2 which shows the gene construction for expression of violacein enzymes., in accordance with our present invention.

Detailed Description of the Invention

The present disclosure emphasises that its application is not restricted to specific details of construction and component arrangement, as illustrated in the drawings. It is adaptable to various embodiments and implementations. The phraseology and terminology used should be regarded for descriptive purposes, not as limitations.

The terms "including," "comprising," or "having" and variations thereof are meant to encompass listed items and their equivalents, as well as additional items. The terms "a" and "an" do not denote quantity limitations but signify the presence of at least one of the referenced items. Terms like "first," "second," and "third" are used to distinguish elements without implying order, quantity, or importance.

The present invention discloses nucleic acids encoding violacein producing enzymes which are optimally expressed in a heterologous host. The nucleic acids would have better expression in a heterologous host leading to better yield of violacein in a multidimensional approach for achieving a high rate of violacein synthesis in a heterologous host. This approach coupled with engineering the nucleotide sequence of the native genes encoding violacein enzymes to match the preferred codon system of the host cell gives a greater efficiency in protein expression.
The present invention discloses nucleic acids encoding violacein enzymes in which the genes encoding violacein enzymes has been operably fused to promoters and terminators of Saccharomyces cerevisiae and having optimal expression levels in heterologous hosts and have been presented below:
VioA (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 1)
ATGAAGCATTCTTCAGATATCTGTATCGTTGGTGCAGGTATCTCTGGTTTATCATGTGCTACTTATTTGTTGGAATCTCCAGCATGTAGAGGTTTATCATTGAGAATCTTCGATATGCAAACAGAAGCTGGTGGTAGAATCAGATCTAAAAATTTGGATGGTAAAGCTGCAATTGAATTGGGTGCTGGTAGATACTCTCCACAATTGCATCCACAATTCCAATCAGTTATGCAATCTTACTCACAAAGATCTGAAAGATACCCTTTTACTCAATTGAAGTTTAAAAACAGAGTTCAACAAACATTGAAGTCTGCTATGAGAGAATTATCACCAAGATTGAAAGAACATGGTAAAGAATCATTTTTGAACTTTGTTTCAAGATACCAGGGTATGGATTCTGCTATCGGTATGATCAAATCAATGGGTTACGATGCATTATTTTTGCCAAACATCTCTGCAGAAATGGCTTACGATATCGTTGGTAAACATCCAGAAATCCAATCTTTTACTGAAAACGATGCAAACCAATGGTTTTCTGCTGTTGATGGTTTTGATGGTTTAATCGCTAGAATGAAGGATAAGGTTAAAGCAGGTGGTGCTAAATTTTCTTTGGGTTTTAGATTGGCTTCAGTTGAAAGAGATCAAGATGGTTATAGATTAGCATTGGTTGGTGACGATGGTTGGCAATTGACACATCATGCAAGACATTTGATTTTGGCTATTCCACCATCAGCAATGGCTGGTTTAAATTTGGATTTTCCAGCTGCATGGGGTCATTCTAGATACGGTTCATTACCATTGTTTAAAGGTTTCTTGTCTTACGATGAACCATGGTGGAGAGATTACAAGTTGGAAGATCAAGTTTTGATCGTTGATAACCCATTGAGAAAGGTTTACTTCAAGGGTGACAAGTACGTTTTCTTTTATACTGATTCTGAAATGGCTGCATATTGGAGAGCATGTGTTGCTGATGGTGAAGATGGTTACTTGGAACAAGTTAGAGTTCATTTGGCTGCAGCTTTGGGTATTGGTGCAGCTTCTATTCCACAACCATCACAACATGTTCATAAATATTGGGCTCATGGTGTTGAATTTTGTCAAGATGCAGCTGCAGCTGATAGACCACCAGCATTGTTGAACAGAGATTCTGGTATCATCGCATGTTCAGATGCTTACACAGAACATTGTGGTTGGATGGAAGGTGGTTTGTTATCTGCAAGAGCAGCTTCAGCTTTGTTATTGCAAAGAATGGCTGGTTAA
Protein (Seq ID No:2)
MKHSSDICIVGAGISGLSCATYLLESPACRGLSLRIFDMQTEAGGRIRSKNLDGKAAIELGAGRYSPQLHPQFQSVMQSYSQRSERYPFTQLKFKNRVQQTLKSAMRELSPRLKEHGKESFLNFVSRYQGMDSAIGMIKSMGYDALFLPNISAEMAYDIVGKHPEIQSFTENDANQWFSAVDGFDGLIARMKDKVKAGGAKFSLGFRLASVERDQDGYRLALVGDDGWQLTHHARHLILAIPPSAMAGLNLDFPAAWGHSRYGSLPLFKGFLSYDEPWWRDYKLEDQVLIVDNPLRKVYFKGDKYVFFYTDSEMAAYWRACVADGEDGYLEQVRVHLAAALGIGAASIPQPSQHVHKYWAHGVEFCQDAAAADRPPALLNRDSGIIACSDAYTEHCGWMEGGLLSARAASALLLQRMAG
VioB (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 3)

ATGTCCATTTTGGACTTCCCAAGATTCCATTTCAGAGGTTGTGCTCGTGTTAACGCTCCTACTGCTAACAGAGATCCACATGGTCACATTGACATGGCTTCTAACACTGTTTCTGCTGGTGGCCAACCAGTCGACTTGGCTAGACCACCAGCTGAATTCCACCAATATTTGAGACAATTAGGTCCACACTTTGACTCTGAAGGCAAGCCAGCGGATGATGGTGTCTTTTCCTTGGCTCTGGGTCACAACGCTAACGGTAACAATCATTTCTCTTGGGAAGGTGCTGTTGTCACCGCTGTCGAACCAAGAGCTGGTGCTGCTGAAGCTGATGATTCTTTGATCGGTGCTAAACTCGCCTTATGGGGTCATTACAACGAATATCTTAGAACTTCTTTCAACAGAGCTAGATGGGTTGACAACGATCCAACTAGACCAGACGCTGTCCAAATTTACGCCGGTCAATTCGTCATCTCTCCACCAGGTGCTGCCGCTCATGCCCCTTCTCTGTTCACTGCTGACATTGACCAAGCACACGGTGCCAGATGGGTTGGTGGTGGTCACGTTTGTGAACGTAGCGGGCACTTTTTGGACGACGATTTCGGTCGGGTCAGAGTTTTCCAATTCTCATTACCAAAGTCTGATCCATACTTCTTATTCAATTTAGGTGATGACGGTTCTGCTACCTTGCAAGCTTTGCAAAGAGCCCTAGAGGACGATGACGCTCTAGGTTTGACGGTTCAATACACCTTATTCAACATGTCCACCCCAAGACAACCAGACACTCCAGTTTTCTACGACATGGTCGGTACTGTCGGTGTCTGGAGAAGAGATGAATTGGCTTCTTACCCAGCTGGTAGATTGTTGCAACCAGCTGGTGCTGGATTAGGTCCATTGACTTTGCAAATCAGAGATGACAGAGTTAGCCTGAACTTGTGTGCCGCTTTGCCATTCACCACCAGAGCACCTAGAATCGACGAACCAGGTAGATTGACTCATGCTTTGGGTCCAAAACTTGCTTTGGGTGACTTGCTTTTGAAGGACAGAGGTGGTGCTTTGGTCGCTAGAATCCCACAACAAGCTTGTTTGGATTACTGGAGAAACCACGGTATCGTCGATGTTCCATTGTTGAACCCACCACAAGGTTCTTTGTCTTTGTCCTCTGAACTAGCTAGGTGGGACGAAGTTGACTGGGTTTCTCAAACGGATGCCAACAACTTGTACTTGGAAGCTCCAGATGCTGCTAGAGGTTTGTCCTTCCCAGAAAGAGTAACTGTTAGATCCTACTACAGAGGTGAGCGTAGACCAAGACCAGCTATTCCTTTCAAGGTTGTTGGTGATGGTCAAGCTGCAGTTGAAGCTAGACAAAGAGATGGTGATGCTGAATGGTTGATTACCGGTTTGAAGCCAGGTGTTGCTCGTATCGAATTGGATGGTGGTGTCCAAAGAGTCAACTTGAGAATCTTGCCAGACGACTGGCAATTGGCTGAAGTTCCAGCCGAACAAGTTGACTACGCCTTCTTGTACAGACACGTTATGTCATACTACGAATTAGTTTACCCATTCATGTCTGACAAGGTTTTCTCTTTGGCTGATAGATGCAAGTGTGAAACTTACGCTCGGTTGATGTGGCAAATGTGTGATCCACAAAACCGTGACAAATCTTATTACATGCCATCCACCAGAGAACTGTCCAGACCAAAGGCTGGTTTGTTCTTGAAGTACCTCGCTAACGTTGAAAGGGCTGCCCCATCCGCTGCTCCCGACGGTGGAGCCCCAGCTAGAATCGAAAACAGAGCCCAACTGGTGGAGGCCTTAAAGACCGCTGTTGATTTGGAATTGTCTATCATGTTGCAATACGTCTACGCTGCTTACTCCATTCCAAACTACGCTCAAGGTGAACAATTGGCTCAATCCGGAGCTTGGTCCCAAGAACAATTGGAATTGGCTTGTGGTGGGGGTGACAGACGTAGAGATGGTGGTTTGAGAGGTGCCATTTTGGAAATTGCTCACGAAGAAATGATTCACTACTTGGTCGTTAACAACTTACTGATGGCTTTGGGTGAACCATTCCACCCAGGTGCTGCTCGTGTTGGTGATGAAGCCAGAAGAGCTTTCGGTTTGGACACTGAATTTTCTTTCGAACCATTCTCCGAATACGTTTTGGCTAGATTCATCAAGTTGGAATGGCCAGCTTTCATTCCATCTCCAGCCAAGTCCATTGCTGCTTTCTACGCTTCCATCAGACAAGCTTTCGAAGAATTGCCAGACTTGTTCGATGGTGCTGCTGGTAAGAGAGGTGGTGAACACCACTTGTTCCTGAACGAATTGACCAACAGAGCTTTCCCAGGTTACCAACTAGAAGTTTTCGACAGAGATTCCGCCTTGTTCGGTATCAGATTCGTTACCGAACAGGGTGAAGGTGGTGCTTTGGACAGTCCACACTTCGAACACTCACACTTCCAAAGATTGAGATCTTTGGCCGGTAGATTGATGGCCCAACCAGCCCCATTTGAACCTGCTTTGCCAGCTTTAAAGAACGCCGTATTGGCTCCGAGAGAAGGTTGTAACCTCGTTACTGAACCACAAGCCAGAGCCTTGATGAGATTATACCAAGGTGGCTACGAATTGATGTTCGCTATGATGGCTCAACACTTTGCTGCTCAACCGGCTGGTTCCTTGAGAAGATCTCGTCTTATGAATGCTGCGATCGACTTGATGACTGGTCTGTTAAGACCACTCTCCACTGCTCTAATGAACTTGCCATCTGGTGTTCCAGGTAGAAATGCTGCCCCACCTGTCCCAGCCGCAGCTGACTGTAAAGTCTTTGATGATTATTCTTTGGGGTGCCAAATGTTGGCTAAGAGATGTCAAGCCTTGGCTGACGAAGCAGCCGGTTTAGAACCAGGTTGGGTTGCTGACGCGCCTAAGGAATTGTTGGCTTTTTACTGTCGTCAATTGATGGACCTCGCCTGTGGTAAGTTGACCCGTGAAGCTTGA
Protein (Seq ID No: 4)
MSILDFPRFHFRGCARVNAPTANRDPHGHIDMASNTVSAGGQPVDLARPPAEFHQYLRQLGPHFDSEGKPADDGVFSLALGHNANGNNHFSWEGAVVTAVEPRAGAAEADDSLIGAKLALWGHYNEYLRTSFNRARWVDNDPTRPDAVQIYAGQFVISPPGAAAHAPSLFTADIDQAHGARWVGGGHVCERSGHFLDDDFGRVRVFQFSLPKSDPYFLFNLGDDGSATLQALQRALEDDDALGLTVQYTLFNMSTPRQPDTPVFYDMVGTVGVWRRDELASYPAGRLLQPAGAGLGPLTLQIRDDRVSLNLCAALPFTTRAPRIDEPGRLTHALGPKLALGDLLLKDRGGALVARIPQQACLDYWRNHGIVDVPLLNPPQGSLSLSSELARWDEVDWVSQTDANNLYLEAPDAARGLSFPERVTVRSYYRGERRPRPAIPFKVVGDGQAAVEARQRDGDAEWLITGLKPGVARIELDGGVQRVNLRILPDDWQLAEVPAEQVDYAFLYRHVMSYYELVYPFMSDKVFSLADRCKCETYARLMWQMCDPQNRDKSYYMPSTRELSRPKAGLFLKYLANVERAAPSAAPDGGAPARIENRAQLVEALKTAVDLELSIMLQYVYAAYSIPNYAQGEQLAQSGAWSQEQLELACGGGDRRRDGGLRGAILEIAHEEMIHYLVVNNLLMALGEPFHPGAARVGDEARRAFGLDTEFSFEPFSEYVLARFIKLEWPAFIPSPAKSIAAFYASIRQAFEELPDLFDGAAGKRGGEHHLFLNELTNRAFPGYQLEVFDRDSALFGIRFVTEQGEGGALDSPHFEHSHFQRLRSLAGRLMAQPAPFEPALPALKNAVLAPREGCNLVTEPQARALMRLYQGGYELMFAMMAQHFAAQPAGSLRRSRLMNAAIDLMTGLLRPLSTALMNLPSGVPGRNAAPPVPAAADCKVFDDYSLGCQMLAKRCQALADEAAGLEPGWVADAPKELLAFYCRQLMDLACGKLTREA
VioC (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 5)
ATGCAAAAAGCAATTATTGTTGGTGGTGGTTTGGCTGGTGGTTTAACTGCAATATATTTGGCTAGAAGAGGTTACGAAGTTCATGTTGTTGAAAAGAGAGGTGACCCATTACAAGATTTGTCATCTTATGTTGATGCAGTTTCTTCAAGAGCTATTGGTGTTTCTATGACAGTTAGAGGTATTAAAGCAGTTTTGGCTGCAGGTATTCCAAGAGCTGAATTAGATCAATGTGGTGAACCAATTGTTGGTATGGCATTTTCAGTTGGTGGTAAATTCAGAATCAGAGAATTACAACCATTGGAAGGTTTATCTCCATTATCATTGAACAGAGCTGCATTCCAAAGATTGTTAAACAGACATGCTAACAGAAACGGTGTTAGATACCATTTCGAACATAAGTGTTTGGATGTTGATTTGGAGGGTAAATCTGTTTTGATCCAATCAAAAGATGGTCAATTGCAAAGATTACAAGGTGACATGGTTATTGGTGCTGATGGTGCACATTCTGCTGTTAGACAAGCTATGCAATCTGGTATGAGAAGATTCGAATTCCAACAAACTTTCTTTAGACATGGTTACAAGACATTGGTTTTGCCAGATGCTGAAGCATTGGGTTACAGAAAGGATTTGTTGTATTTCTTTGGTATGGATTCTGGTGGTTTATTTGCTGGTAGAGCTGCAACTATTCCAGATGGTTCTGTTTCAATTGCAGTTTGTTTGCCATATGAAGGTACTCCATCATTAGCTACACAAGATAGACAAGCAATGAAAGCTTTCTTTCAAAGATACTTTGGTTCTTTGTCAGATGCTGTTAGAGAAGAAATGTTGGAACAATTTTTAGTTAAACCATCTAATGATTTGATTAATGTTAGATCTTCAACTTTCCATTACAAGGGTAACGTTTTGTTGTTGGGTGACGCTGCACATGCTACAGCACCATTTTTGGGTCAGGGTATGAATATGGCATTGGAAGATGCTCATTCATTCGTTACTTTGTTGGATCAACATTGTCATGATCAAGAATTGGCTTTTGCAGAATTCACTGCTCAAAGAAAAGTTCAAGCTGATGCAATGCAAGATATGGCTAGAGCAAACTACGATGTTTTGTCTTGTTCAAACCCAATTTTCTTTTTGAGAGCAAGATACACAAGATACATGCATGGTAAATTTCCAGAATTGTACCCACCAGATATGGCTCAAAAGTTGTACTTCACTTCTGAACCATACGATAAGTTGCAAACAATCCAAAAGAAACAAAACGTTTGGTACAAGTTGGGTAGAGTTAATTAA
Protein (Seq ID No: 6)
MQKAIIVGGGLAGGLTAIYLARRGYEVHVVEKRGDPLQDLSSYVDAVSSRAIGVSMTVRGIKAVLAAGIPRAELDQCGEPIVGMAFSVGGKFRIRELQPLEGLSPLSLNRAAFQRLLNRHANRNGVRYHFEHKCLDVDLEGKSVLIQSKDGQLQRLQGDMVIGADGAHSAVRQAMQSGMRRFEFQQTFFRHGYKTLVLPDAEALGYRKDLLYFFGMDSGGLFAGRAATIPDGSVSIAVCLPYEGTPSLATQDRQAMKAFFQRYFGSLSDAVREEMLEQFLVKPSNDLINVRSSTFHYKGNVLLLGDAAHATAPFLGQGMNMALEDAHSFVTLLDQHCHDQELAFAEFTAQRKVQADAMQDMARANYDVLSCSNPIFFLRARYTRYMHGKFPELYPPDMAQKLYFTSEPYDKLQTIQKKQNVWYKLGRVN
VioD (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 7)
ATGAAGAAAATTTTGGTTATTGGTGCTGGTCCAGCAGGTTTGGTTTTTGCTTCTCAAATGAAGTTGGCAAAGCCA GATTGGCAAATTTCAATTGCTGAAAAGAATGATCCAGAAGAAGTTGCAGGTTGGGGTGTTGTTTTGCCAGGTAGA CCAGGTCAACATCCAGCTAATCCATTGTCTTATTTGGAACATCCAGAAAGATTAGATCCACAATTCTTGGAAGAT TTCAAGTTGATCCATCATAATGAACCAAATTTGATGTCAACTGGTGTTTTGTTATGTGGTGTTGAAAGAAGAGGT TTGGTTCAAGCTTTAAGAGAAAGATGTCAATCTTTGGGTATCGCTATCCATTACGAATCACCATTGTTAGCAAGA GAACAATTGCCATTAGATGATTACGATTTGATTGTTGCTGCAAACGGTGTTAACCATAAGACTTCTCATTTTACA GAAGCTTTGGCACCAAAGTTGGAATACGGTGGTAATAAGTACATTTGGTTTGGTACTTCACAATTGTTCGATCAA ATGAATTTGGTTTTTAGAACACATGGTAAAGATATTTTCATTGCTCATGCATACAAGTACTCTTCAAGAATGTCT ACTTTCGTTGTTGAATGTTCAGAAGAAACATTCGAAAGAGCTAGATTGGGTGAAATGTCTGATGAAGCTTCAGCA GAATATGTTGCTGGTGTTTTTAGAGCAGAATTGGGTGGTCATGGTTTAGTTGCTCAACCAGGTTTGGGTTGGAGA AACTTCATGACTTTGTCTCATGATAAGTCATACGATGGTAAATTGGTTTTGATTGGTGACGCTTTACAATCTGGT CATTTTTCAATTGGTCATGGTACTACAATGGCTGTTGTTGCTGCACAATTGTTAGTTAAAGCATTGTGTGCTGAA GCATCTGTTCCAGCTGCATTAGCTTCATTTGATGCTAGAGCAATGCCATTGGTTAAGTTATTTTCTGAACATGCA AACTCTTCAAGAGATTGGTTTGAAACAGTTGATGATAGAATGCATTTGTCTAATGCTGAATTCATGCAATCATTT GATGCTAGAAGAAAAGCATTGCCACCATTACCAGAAGCTTTGGCAAGAAATTTGGGTTACGCATTGGATAGATAA

Protein (Seq ID No: 8)
MKKILVIGAGPAGLVFASQMKLAKPDWQISIAEKNDPEEVAGWGVVLPGRPGQHPANPLSYLEHPERLDPQFLEDFKLIHHNEPNLMSTGVLLCGVERRGLVQALRERCQSLGIAIHYESPLLAREQLPLDDYDLIVAANGVNHKTSHFTEALAPKLEYGGNKYIWFGTSQLFDQMNLVFRTHGKDIFIAHAYKYSSRMSTFVVECSEETFERARLGEMSDEASAEYVAGVFRAELGGHGLVAQPGLGWRNFMTLSHDKSYDGKLVLIGDALQSGHFSIGHGTTMAVVAAQLLVKALCAEASVPAALASFDARAMPLVKLFSEHANSSRDWFETVDDRMHLSNAEFMQSFDARRKALPPLPEALARNLGYALDR
VioE (Accession no: KX461963.1)
DNA (Seq ID No: 9)
ATGGGTTTGGATTCTTCTTCTGGTGGTGGTGGTTCAGGTGGTTCTGGTGGTGGTGGTAGTGGTGAAAATTTGTAC TTCCAGGGTATGGCTTCTGATTACAAGGATGATGATGATAAGCATATGGAAAATAGAGAACCACCATTGTTACCA GCTAGATGGTCATCTGCATATGTTTCATACTGGTCACCAATGTTGCCAGATGATCAATTGACTTCAGGTTACTGT TGGTTCGATTACGAAAGAGATATCTGTAGAATTGATGGTTTGTTTAATCCATGGTCTGAAAGAGATACAGGTTAT AGATTATGGATGTCAGAAGTTGGTAATGCTGCATCTGGTAGAACTTGGAAACAAAAAGTTGCTTACGGTAGAGAA AGAACAGCATTGGGTGAACAATTATGTGAAAGACCATTGGATGATGAAACTGGTCCATTTGCTGAATTGTTTTTA CCAAGAGATGTTTTGAGAAGATTAGGTGCTAGACATATTGGTAGAAGAGTTGTTTTAGGTAGAGAAGCAGATGGT TGGAGATATCAAAGACCAGGTAAAGGTCCATCAACATTGTACTTAGATGCTGCATCTGGTACTCCATTGAGAATG GTTACAGGTGACGAAGCTTCAAGAGCATCTTTAAGAGATTTCCCAAATGTTTCTGAAGCAGAAATTCCAGTTTTT GCTGCAAAAAGAGAATTTTTGGGTTAA
Protein (Seq ID No: 10)
MGLDSSSGGGGSGGSGGGGSGENLYFQGMASDYKDDDDKHMENREPPLLPARWSSAYVSYWSPMLPDDQLTSGYCWFDYERDICRIDGLFNPWSERDTGYRLWMSEVGNAASGRTWKQKVAYGRERTALGEQLCERPLDDETGPFAELFLPRDVLRRLGARHIGRRVVLGREADGWRYQRPGKGPSTLYLDAASGTPLRMVTGDEASRASLRDFPNVSEAEIPVFAAKREFLG
In a preferred embodiment, the nucleic acids are represented by SEQ ID Numbers: 1, 3, 5, 7, 9.
The current embodiment of our present invention also relates to a polypeptides encoded by the nucleic acid sequences as in SEQ ID numbers: 2, 4, 6, 8, 10 or any variant thereof.
In another embodiment of our present invention, suitable vectors comprising the nucleic acids for optimal expression of violacein enzymes in a heterologous host is utilized. In yet another embodiment of our present invention, the vector of the disclosure is an expression vector which can be conveniently subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector could be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector could be one which, when introduced into a host cell, is integrated into the host cell genome, in part or in its entirety, and replicated together with the chromosomes into which it has been integrated.
In another embodiment of our present invention, the vector is preferably an expression vector in which the DNA sequences encoding the violacein enzymes are operably linked to additional segments required for transcription of the DNA. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in some promoter and proceeds through the DNA sequence coding for the enzyme.
Preferably, the gene can be cloned into any Saccharomyces cerevisiae expression vectors known in the art. In a preferred embodiment, the vectors are pESCHIS and pESCURA.
Any suitable promoter can be used. In a preferred embodiment, the strong constitutive promoters Ptdh3, Ppgk1, Pact1, Psptdh3, and Pfba1are used.
In another embodiment, the host cell into which the DNA construct or the recombinant vector of the disclosure is introduced may be any cell which can produce the violacein enzymes and includes bacteria, yeast, any other microorganism, a mammalian cell, plant cell or any cell culture of said category.
In a preferred embodiment, the host cell is a eukaryotic cell selected from a group comprising Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha or any host known in the art for expression of heterologous pathway enzymes using yeast promoter-based vectors for expression.
In a preferred embodiment, the host-cell is Saccharomyces cereviciae, BY4741. Commercially available Saccharomyces cereviciae, BY4741, was used in the preferred embodiment of the invention.
In another embodiment, the process for production of violacein is provided. In a preferred embodiment, the process of production includes the steps of culturing host cells transformed with a vectors comprising a nucleic acids of SEQ ID Numbers: 1, 3, 5, 7, 9 in a suitable culture medium.
In another embodiment, the process of culturing host cells transformed with a vectors comprising the nucleic acids in selection media without histidine, uracil and comprises glucose, yeast nitrogen base without amino acids.
A few example embodiments of our present invention have been given below:

EXAMPLES
The following examples particularly describe the manner in which the invention is to be performed. But the embodiments disclosed herein do not limit the scope of the invention in any manner.
Example 1: Recombinant plasmid construction for expression of violacein enzymes in Saccharomyces cereviciae
Genes encoding for violacein enzymes VioA-VioB-VioC and VioD-VioE are cloned in pESCHIS and pESCURA respectively. The resulted plasmids are pESCHIS_VioABC and pESCURA_VioDE. The preferred codons for expression in Saccharomyces cereviciae has been used in place of rare codons.
The sequences of the open reading frame encoding for violacein enzymes are represented by SEQ ID Numbers: 1, 3, 5, 7, 9.
These open reading frames are codon optimized for Saccharomyces cereviciae and artificially synthesized and cloned in pESCHIS and pESCURA vectors by Genscript.
The recombinant plasmids contains promoters, open reading frames and terminators. The recombinant vectors are represented in Figure 2.
Example 2: Polynucleotide sequence for expression of violacein enzymes and corresponding polypeptide sequence
The polynucleotide sequences and corresponding translated protein sequences represented by SEQ ID Numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
Example 3: Development of recombinant host cell by transformation with recombinant plasmids
Recombinant pESCHIS_VioABC and pESCURA_VioDE plasmids as described in foregoing example carrying the genes for violacein enzymes SEQ ID Numbers: 1, 3, 5, 7, 9 were used.
Host cells were electroporated with the plasmids as described in foregoing example and recovered in YPD media at 30°C with shaking for 3 hours before plating on selection plate. These plates are incubated for 5 days to get transformants. These colonies are inoculated in selection media and observed for media color change indicating violacein synthesis.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular as is considered appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for the sake of clarity. Generally, nomenclatures used in connection with, and techniques of biotechnology, fermentation technology, genetic engineering and recombinant DNA technology described herein are those well-known and commonly used in the art. Certain references and other documents cited are expressly incorporated herein by reference. In case of conflict, the present specification, including definitions, will control. The materials, methods, figures and examples are illustrative only and not intended to be limiting
, Claims:5. CLAIMS
I/We Claim:
1. A method of biological synthesis of violacein pigment comprising the process of producing a recombinant host cell capable of expressing violacein enzymes, comprising the steps of:
a. synthesizing nucleic acids comprising the nucleotide sequence of SEQ ID Numbers: 1, 3, 5, 7, 9
b. constructing a recombinant vectors pESCHIS and pESCURA harbouring the nucleic acid of SEQ ID Numbers: 1, 3, 5, 7, 9 wherein the nucleic acids are operably linked to constitutive promoters; and
c. transforming a Saccharomyces cereviciae, BY4741, host cell with the recombinant pESCHIS_VioABC and pESCURA_VioDE vectors to obtain a recombinant host cell.
wherein the nucleic acid (which is operably linked to strong constitutive promoter) encodes violacein enzymes in a recombinant eukaryotic host cell, Saccharomyces cereviciae, BY4741 which comprises the vectors, wherein the vectors comprising the nucleic acid are pESCHIS and pESCURA and the constitutive promoters are Ptdh3, Ppgk1, Pact1, Psptdh3, and Pfba1.
2. The nucleotide sequence synthesized as claimed in claim 1, wherein the nucleotide sequence is given by:
VioA (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 1)
ATGAAGCATTCTTCAGATATCTGTATCGTTGGTGCAGGTATCTCTGGTTTATCATGTGCTACTTATTTGTTGGAATCTCCAGCATGTAGAGGTTTATCATTGAGAATCTTCGATATGCAAACAGAAGCTGGTGGTAGAATCAGATCTAAAAATTTGGATGGTAAAGCTGCAATTGAATTGGGTGCTGGTAGATACTCTCCACAATTGCATCCACAATTCCAATCAGTTATGCAATCTTACTCACAAAGATCTGAAAGATACCCTTTTACTCAATTGAAGTTTAAAAACAGAGTTCAACAAACATTGAAGTCTGCTATGAGAGAATTATCACCAAGATTGAAAGAACATGGTAAAGAATCATTTTTGAACTTTGTTTCAAGATACCAGGGTATGGATTCTGCTATCGGTATGATCAAATCAATGGGTTACGATGCATTATTTTTGCCAAACATCTCTGCAGAAATGGCTTACGATATCGTTGGTAAACATCCAGAAATCCAATCTTTTACTGAAAACGATGCAAACCAATGGTTTTCTGCTGTTGATGGTTTTGATGGTTTAATCGCTAGAATGAAGGATAAGGTTAAAGCAGGTGGTGCTAAATTTTCTTTGGGTTTTAGATTGGCTTCAGTTGAAAGAGATCAAGATGGTTATAGATTAGCATTGGTTGGTGACGATGGTTGGCAATTGACACATCATGCAAGACATTTGATTTTGGCTATTCCACCATCAGCAATGGCTGGTTTAAATTTGGATTTTCCAGCTGCATGGGGTCATTCTAGATACGGTTCATTACCATTGTTTAAAGGTTTCTTGTCTTACGATGAACCATGGTGGAGAGATTACAAGTTGGAAGATCAAGTTTTGATCGTTGATAACCCATTGAGAAAGGTTTACTTCAAGGGTGACAAGTACGTTTTCTTTTATACTGATTCTGAAATGGCTGCATATTGGAGAGCATGTGTTGCTGATGGTGAAGATGGTTACTTGGAACAAGTTAGAGTTCATTTGGCTGCAGCTTTGGGTATTGGTGCAGCTTCTATTCCACAACCATCACAACATGTTCATAAATATTGGGCTCATGGTGTTGAATTTTGTCAAGATGCAGCTGCAGCTGATAGACCACCAGCATTGTTGAACAGAGATTCTGGTATCATCGCATGTTCAGATGCTTACACAGAACATTGTGGTTGGATGGAAGGTGGTTTGTTATCTGCAAGAGCAGCTTCAGCTTTGTTATTGCAAAGAATGGCTGGTTAA
Protein (Seq ID No:2)
MKHSSDICIVGAGISGLSCATYLLESPACRGLSLRIFDMQTEAGGRIRSKNLDGKAAIELGAGRYSPQLHPQFQSVMQSYSQRSERYPFTQLKFKNRVQQTLKSAMRELSPRLKEHGKESFLNFVSRYQGMDSAIGMIKSMGYDALFLPNISAEMAYDIVGKHPEIQSFTENDANQWFSAVDGFDGLIARMKDKVKAGGAKFSLGFRLASVERDQDGYRLALVGDDGWQLTHHARHLILAIPPSAMAGLNLDFPAAWGHSRYGSLPLFKGFLSYDEPWWRDYKLEDQVLIVDNPLRKVYFKGDKYVFFYTDSEMAAYWRACVADGEDGYLEQVRVHLAAALGIGAASIPQPSQHVHKYWAHGVEFCQDAAAADRPPALLNRDSGIIACSDAYTEHCGWMEGGLLSARAASALLLQRMAG
VioB (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 3)
ATGTCCATTTTGGACTTCCCAAGATTCCATTTCAGAGGTTGTGCTCGTGTTAACGCTCCTACTGCTAACAGAGATCCACATGGTCACATTGACATGGCTTCTAACACTGTTTCTGCTGGTGGCCAACCAGTCGACTTGGCTAGACCACCAGCTGAATTCCACCAATATTTGAGACAATTAGGTCCACACTTTGACTCTGAAGGCAAGCCAGCGGATGATGGTGTCTTTTCCTTGGCTCTGGGTCACAACGCTAACGGTAACAATCATTTCTCTTGGGAAGGTGCTGTTGTCACCGCTGTCGAACCAAGAGCTGGTGCTGCTGAAGCTGATGATTCTTTGATCGGTGCTAAACTCGCCTTATGGGGTCATTACAACGAATATCTTAGAACTTCTTTCAACAGAGCTAGATGGGTTGACAACGATCCAACTAGACCAGACGCTGTCCAAATTTACGCCGGTCAATTCGTCATCTCTCCACCAGGTGCTGCCGCTCATGCCCCTTCTCTGTTCACTGCTGACATTGACCAAGCACACGGTGCCAGATGGGTTGGTGGTGGTCACGTTTGTGAACGTAGCGGGCACTTTTTGGACGACGATTTCGGTCGGGTCAGAGTTTTCCAATTCTCATTACCAAAGTCTGATCCATACTTCTTATTCAATTTAGGTGATGACGGTTCTGCTACCTTGCAAGCTTTGCAAAGAGCCCTAGAGGACGATGACGCTCTAGGTTTGACGGTTCAATACACCTTATTCAACATGTCCACCCCAAGACAACCAGACACTCCAGTTTTCTACGACATGGTCGGTACTGTCGGTGTCTGGAGAAGAGATGAATTGGCTTCTTACCCAGCTGGTAGATTGTTGCAACCAGCTGGTGCTGGATTAGGTCCATTGACTTTGCAAATCAGAGATGACAGAGTTAGCCTGAACTTGTGTGCCGCTTTGCCATTCACCACCAGAGCACCTAGAATCGACGAACCAGGTAGATTGACTCATGCTTTGGGTCCAAAACTTGCTTTGGGTGACTTGCTTTTGAAGGACAGAGGTGGTGCTTTGGTCGCTAGAATCCCACAACAAGCTTGTTTGGATTACTGGAGAAACCACGGTATCGTCGATGTTCCATTGTTGAACCCACCACAAGGTTCTTTGTCTTTGTCCTCTGAACTAGCTAGGTGGGACGAAGTTGACTGGGTTTCTCAAACGGATGCCAACAACTTGTACTTGGAAGCTCCAGATGCTGCTAGAGGTTTGTCCTTCCCAGAAAGAGTAACTGTTAGATCCTACTACAGAGGTGAGCGTAGACCAAGACCAGCTATTCCTTTCAAGGTTGTTGGTGATGGTCAAGCTGCAGTTGAAGCTAGACAAAGAGATGGTGATGCTGAATGGTTGATTACCGGTTTGAAGCCAGGTGTTGCTCGTATCGAATTGGATGGTGGTGTCCAAAGAGTCAACTTGAGAATCTTGCCAGACGACTGGCAATTGGCTGAAGTTCCAGCCGAACAAGTTGACTACGCCTTCTTGTACAGACACGTTATGTCATACTACGAATTAGTTTACCCATTCATGTCTGACAAGGTTTTCTCTTTGGCTGATAGATGCAAGTGTGAAACTTACGCTCGGTTGATGTGGCAAATGTGTGATCCACAAAACCGTGACAAATCTTATTACATGCCATCCACCAGAGAACTGTCCAGACCAAAGGCTGGTTTGTTCTTGAAGTACCTCGCTAACGTTGAAAGGGCTGCCCCATCCGCTGCTCCCGACGGTGGAGCCCCAGCTAGAATCGAAAACAGAGCCCAACTGGTGGAGGCCTTAAAGACCGCTGTTGATTTGGAATTGTCTATCATGTTGCAATACGTCTACGCTGCTTACTCCATTCCAAACTACGCTCAAGGTGAACAATTGGCTCAATCCGGAGCTTGGTCCCAAGAACAATTGGAATTGGCTTGTGGTGGGGGTGACAGACGTAGAGATGGTGGTTTGAGAGGTGCCATTTTGGAAATTGCTCACGAAGAAATGATTCACTACTTGGTCGTTAACAACTTACTGATGGCTTTGGGTGAACCATTCCACCCAGGTGCTGCTCGTGTTGGTGATGAAGCCAGAAGAGCTTTCGGTTTGGACACTGAATTTTCTTTCGAACCATTCTCCGAATACGTTTTGGCTAGATTCATCAAGTTGGAATGGCCAGCTTTCATTCCATCTCCAGCCAAGTCCATTGCTGCTTTCTACGCTTCCATCAGACAAGCTTTCGAAGAATTGCCAGACTTGTTCGATGGTGCTGCTGGTAAGAGAGGTGGTGAACACCACTTGTTCCTGAACGAATTGACCAACAGAGCTTTCCCAGGTTACCAACTAGAAGTTTTCGACAGAGATTCCGCCTTGTTCGGTATCAGATTCGTTACCGAACAGGGTGAAGGTGGTGCTTTGGACAGTCCACACTTCGAACACTCACACTTCCAAAGATTGAGATCTTTGGCCGGTAGATTGATGGCCCAACCAGCCCCATTTGAACCTGCTTTGCCAGCTTTAAAGAACGCCGTATTGGCTCCGAGAGAAGGTTGTAACCTCGTTACTGAACCACAAGCCAGAGCCTTGATGAGATTATACCAAGGTGGCTACGAATTGATGTTCGCTATGATGGCTCAACACTTTGCTGCTCAACCGGCTGGTTCCTTGAGAAGATCTCGTCTTATGAATGCTGCGATCGACTTGATGACTGGTCTGTTAAGACCACTCTCCACTGCTCTAATGAACTTGCCATCTGGTGTTCCAGGTAGAAATGCTGCCCCACCTGTCCCAGCCGCAGCTGACTGTAAAGTCTTTGATGATTATTCTTTGGGGTGCCAAATGTTGGCTAAGAGATGTCAAGCCTTGGCTGACGAAGCAGCCGGTTTAGAACCAGGTTGGGTTGCTGACGCGCCTAAGGAATTGTTGGCTTTTTACTGTCGTCAATTGATGGACCTCGCCTGTGGTAAGTTGACCCGTGAAGCTTGA
Protein (Seq ID No: 4)
MSILDFPRFHFRGCARVNAPTANRDPHGHIDMASNTVSAGGQPVDLARPPAEFHQYLRQLGPHFDSEGKPADDGVFSLALGHNANGNNHFSWEGAVVTAVEPRAGAAEADDSLIGAKLALWGHYNEYLRTSFNRARWVDNDPTRPDAVQIYAGQFVISPPGAAAHAPSLFTADIDQAHGARWVGGGHVCERSGHFLDDDFGRVRVFQFSLPKSDPYFLFNLGDDGSATLQALQRALEDDDALGLTVQYTLFNMSTPRQPDTPVFYDMVGTVGVWRRDELASYPAGRLLQPAGAGLGPLTLQIRDDRVSLNLCAALPFTTRAPRIDEPGRLTHALGPKLALGDLLLKDRGGALVARIPQQACLDYWRNHGIVDVPLLNPPQGSLSLSSELARWDEVDWVSQTDANNLYLEAPDAARGLSFPERVTVRSYYRGERRPRPAIPFKVVGDGQAAVEARQRDGDAEWLITGLKPGVARIELDGGVQRVNLRILPDDWQLAEVPAEQVDYAFLYRHVMSYYELVYPFMSDKVFSLADRCKCETYARLMWQMCDPQNRDKSYYMPSTRELSRPKAGLFLKYLANVERAAPSAAPDGGAPARIENRAQLVEALKTAVDLELSIMLQYVYAAYSIPNYAQGEQLAQSGAWSQEQLELACGGGDRRRDGGLRGAILEIAHEEMIHYLVVNNLLMALGEPFHPGAARVGDEARRAFGLDTEFSFEPFSEYVLARFIKLEWPAFIPSPAKSIAAFYASIRQAFEELPDLFDGAAGKRGGEHHLFLNELTNRAFPGYQLEVFDRDSALFGIRFVTEQGEGGALDSPHFEHSHFQRLRSLAGRLMAQPAPFEPALPALKNAVLAPREGCNLVTEPQARALMRLYQGGYELMFAMMAQHFAAQPAGSLRRSRLMNAAIDLMTGLLRPLSTALMNLPSGVPGRNAAPPVPAAADCKVFDDYSLGCQMLAKRCQALADEAAGLEPGWVADAPKELLAFYCRQLMDLACGKLTREA
VioC (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 5)
ATGCAAAAAGCAATTATTGTTGGTGGTGGTTTGGCTGGTGGTTTAACTGCAATATATTTGGCTAGAAGAGGTTACGAAGTTCATGTTGTTGAAAAGAGAGGTGACCCATTACAAGATTTGTCATCTTATGTTGATGCAGTTTCTTCAAGAGCTATTGGTGTTTCTATGACAGTTAGAGGTATTAAAGCAGTTTTGGCTGCAGGTATTCCAAGAGCTGAATTAGATCAATGTGGTGAACCAATTGTTGGTATGGCATTTTCAGTTGGTGGTAAATTCAGAATCAGAGAATTACAACCATTGGAAGGTTTATCTCCATTATCATTGAACAGAGCTGCATTCCAAAGATTGTTAAACAGACATGCTAACAGAAACGGTGTTAGATACCATTTCGAACATAAGTGTTTGGATGTTGATTTGGAGGGTAAATCTGTTTTGATCCAATCAAAAGATGGTCAATTGCAAAGATTACAAGGTGACATGGTTATTGGTGCTGATGGTGCACATTCTGCTGTTAGACAAGCTATGCAATCTGGTATGAGAAGATTCGAATTCCAACAAACTTTCTTTAGACATGGTTACAAGACATTGGTTTTGCCAGATGCTGAAGCATTGGGTTACAGAAAGGATTTGTTGTATTTCTTTGGTATGGATTCTGGTGGTTTATTTGCTGGTAGAGCTGCAACTATTCCAGATGGTTCTGTTTCAATTGCAGTTTGTTTGCCATATGAAGGTACTCCATCATTAGCTACACAAGATAGACAAGCAATGAAAGCTTTCTTTCAAAGATACTTTGGTTCTTTGTCAGATGCTGTTAGAGAAGAAATGTTGGAACAATTTTTAGTTAAACCATCTAATGATTTGATTAATGTTAGATCTTCAACTTTCCATTACAAGGGTAACGTTTTGTTGTTGGGTGACGCTGCACATGCTACAGCACCATTTTTGGGTCAGGGTATGAATATGGCATTGGAAGATGCTCATTCATTCGTTACTTTGTTGGATCAACATTGTCATGATCAAGAATTGGCTTTTGCAGAATTCACTGCTCAAAGAAAAGTTCAAGCTGATGCAATGCAAGATATGGCTAGAGCAAACTACGATGTTTTGTCTTGTTCAAACCCAATTTTCTTTTTGAGAGCAAGATACACAAGATACATGCATGGTAAATTTCCAGAATTGTACCCACCAGATATGGCTCAAAAGTTGTACTTCACTTCTGAACCATACGATAAGTTGCAAACAATCCAAAAGAAACAAAACGTTTGGTACAAGTTGGGTAGAGTTAATTAA
Protein (Seq ID No: 6)
MQKAIIVGGGLAGGLTAIYLARRGYEVHVVEKRGDPLQDLSSYVDAVSSRAIGVSMTVRGIKAVLAAGIPRAELDQCGEPIVGMAFSVGGKFRIRELQPLEGLSPLSLNRAAFQRLLNRHANRNGVRYHFEHKCLDVDLEGKSVLIQSKDGQLQRLQGDMVIGADGAHSAVRQAMQSGMRRFEFQQTFFRHGYKTLVLPDAEALGYRKDLLYFFGMDSGGLFAGRAATIPDGSVSIAVCLPYEGTPSLATQDRQAMKAFFQRYFGSLSDAVREEMLEQFLVKPSNDLINVRSSTFHYKGNVLLLGDAAHATAPFLGQGMNMALEDAHSFVTLLDQHCHDQELAFAEFTAQRKVQADAMQDMARANYDVLSCSNPIFFLRARYTRYMHGKFPELYPPDMAQKLYFTSEPYDKLQTIQKKQNVWYKLGRVN
VioD (Chromobacterium violaceum (ATCC 31532), Accession no: LC000628.1)
DNA (Seq ID No: 7)
ATGAAGAAAATTTTGGTTATTGGTGCTGGTCCAGCAGGTTTGGTTTTTGCTTCTCAAATGAAGTTGGCAAAGCCA GATTGGCAAATTTCAATTGCTGAAAAGAATGATCCAGAAGAAGTTGCAGGTTGGGGTGTTGTTTTGCCAGGTAGA CCAGGTCAACATCCAGCTAATCCATTGTCTTATTTGGAACATCCAGAAAGATTAGATCCACAATTCTTGGAAGAT TTCAAGTTGATCCATCATAATGAACCAAATTTGATGTCAACTGGTGTTTTGTTATGTGGTGTTGAAAGAAGAGGT TTGGTTCAAGCTTTAAGAGAAAGATGTCAATCTTTGGGTATCGCTATCCATTACGAATCACCATTGTTAGCAAGA GAACAATTGCCATTAGATGATTACGATTTGATTGTTGCTGCAAACGGTGTTAACCATAAGACTTCTCATTTTACA GAAGCTTTGGCACCAAAGTTGGAATACGGTGGTAATAAGTACATTTGGTTTGGTACTTCACAATTGTTCGATCAA ATGAATTTGGTTTTTAGAACACATGGTAAAGATATTTTCATTGCTCATGCATACAAGTACTCTTCAAGAATGTCT ACTTTCGTTGTTGAATGTTCAGAAGAAACATTCGAAAGAGCTAGATTGGGTGAAATGTCTGATGAAGCTTCAGCA GAATATGTTGCTGGTGTTTTTAGAGCAGAATTGGGTGGTCATGGTTTAGTTGCTCAACCAGGTTTGGGTTGGAGA AACTTCATGACTTTGTCTCATGATAAGTCATACGATGGTAAATTGGTTTTGATTGGTGACGCTTTACAATCTGGT CATTTTTCAATTGGTCATGGTACTACAATGGCTGTTGTTGCTGCACAATTGTTAGTTAAAGCATTGTGTGCTGAA GCATCTGTTCCAGCTGCATTAGCTTCATTTGATGCTAGAGCAATGCCATTGGTTAAGTTATTTTCTGAACATGCA AACTCTTCAAGAGATTGGTTTGAAACAGTTGATGATAGAATGCATTTGTCTAATGCTGAATTCATGCAATCATTT GATGCTAGAAGAAAAGCATTGCCACCATTACCAGAAGCTTTGGCAAGAAATTTGGGTTACGCATTGGATAGATAA
Protein (Seq ID No: 8)
MKKILVIGAGPAGLVFASQMKLAKPDWQISIAEKNDPEEVAGWGVVLPGRPGQHPANPLSYLEHPERLDPQFLEDFKLIHHNEPNLMSTGVLLCGVERRGLVQALRERCQSLGIAIHYESPLLAREQLPLDDYDLIVAANGVNHKTSHFTEALAPKLEYGGNKYIWFGTSQLFDQMNLVFRTHGKDIFIAHAYKYSSRMSTFVVECSEETFERARLGEMSDEASAEYVAGVFRAELGGHGLVAQPGLGWRNFMTLSHDKSYDGKLVLIGDALQSGHFSIGHGTTMAVVAAQLLVKALCAEASVPAALASFDARAMPLVKLFSEHANSSRDWFETVDDRMHLSNAEFMQSFDARRKALPPLPEALARNLGYALDR
VioE (Accession no: KX461963.1)
DNA (Seq ID No: 9)
ATGGGTTTGGATTCTTCTTCTGGTGGTGGTGGTTCAGGTGGTTCTGGTGGTGGTGGTAGTGGTGAAAATTTGTAC TTCCAGGGTATGGCTTCTGATTACAAGGATGATGATGATAAGCATATGGAAAATAGAGAACCACCATTGTTACCA GCTAGATGGTCATCTGCATATGTTTCATACTGGTCACCAATGTTGCCAGATGATCAATTGACTTCAGGTTACTGT TGGTTCGATTACGAAAGAGATATCTGTAGAATTGATGGTTTGTTTAATCCATGGTCTGAAAGAGATACAGGTTAT AGATTATGGATGTCAGAAGTTGGTAATGCTGCATCTGGTAGAACTTGGAAACAAAAAGTTGCTTACGGTAGAGAA AGAACAGCATTGGGTGAACAATTATGTGAAAGACCATTGGATGATGAAACTGGTCCATTTGCTGAATTGTTTTTA CCAAGAGATGTTTTGAGAAGATTAGGTGCTAGACATATTGGTAGAAGAGTTGTTTTAGGTAGAGAAGCAGATGGT TGGAGATATCAAAGACCAGGTAAAGGTCCATCAACATTGTACTTAGATGCTGCATCTGGTACTCCATTGAGAATG GTTACAGGTGACGAAGCTTCAAGAGCATCTTTAAGAGATTTCCCAAATGTTTCTGAAGCAGAAATTCCAGTTTTT GCTGCAAAAAGAGAATTTTTGGGTTAA
Protein (Seq ID No: 10)
MGLDSSSGGGGSGGSGGGGSGENLYFQGMASDYKDDDDKHMENREPPLLPARWSSAYVSYWSPMLPDDQLTSGYCWFDYERDICRIDGLFNPWSERDTGYRLWMSEVGNAASGRTWKQKVAYGRERTALGEQLCERPLDDETGPFAELFLPRDVLRRLGARHIGRRVVLGREADGWRYQRPGKGPSTLYLDAASGTPLRMVTGDEASRASLRDFPNVSEAEIPVFAAKREFLG