Description:4. DESCRIPTION
Technical Field of the Invention

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

Background of the Invention

Betanins are group of natural plant pigments derived from tyrosine in Beta vulgaris (beet). Betanin, red-violet pigment, found in beet root plant and sold as beet root extract. Betanin has wide variety of applications in foodstuffs, cosmetics, and pharmaceuticals as natural red coloring agent. It also has strong antioxidant activity that can extend the shelf-life of dairy and meat products. It has attractive features such as high-water solubility, robust stability and color intensity over a broad range of neutral and acidic conditions (pH 3–7), lack of intrinsic flavor, high extinction coefficient compared to most artificial red dyes.
Although, red beetroot extract used as source of betanin, it also carries undesired earthy flavors due to the presence of geosmin and various pyrazines. From Tyrosine, Betanin is formed via three enzymatic reactions. L-tyrosine is initially 3-hydroxylated to form 3, 4-dihydroxy-L-phenylalanine (L-DOPA), and L-DOPA is subsequently oxidized and cyclized to cyclo-DOPA. The CYP76AD1 subfamily of cytochrome P450 enzymes (P450s) takes part in this conversion process. Alternatively, L-DOPA is converted to betalamic acid by DOPA-4, 5-dioxygenase enzyme (DODA). And the cyclo-DOPA 5-O-glucosyltransferase (cDOPA5GT) catalyzes glycosylation at the 5-O position of cyclo-DOPA, allowing subsequent spontaneous condensation of the cyclo-DOPA-5-O-glucoside with betalamic acid to form betanin.
Due to the low enzymatic activity of CYP76AD1-a, biosynthesis of betanin is very limited in microbes. Majority of artificial red colorants available in market are produced through chemical synthesis which is not sustainable.
Extraction of natural dyes from plants is not ideal as plant cultivation requires a lot of land, water and also product yield depend on climate change. Plant-cell culture systems are possible option but it leaves difficulties in scale up and genetic manipulations. Alternative ideal production platform is engineered microbes for production of plant pigments. Saccharomyces cerevisiae (baker's yeast) is generally regarded as safe (GRAS) microorganism can be engineered with heterologous biochemical pathways for production of molecules of interest.
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 Betanin enzymes has been operably fused with promoters and terminators of Saccharomyces cerevisiae
A few patents related to the production of betalain and nucleic acid coding enzymes have been discussed below:
The patent JP2016182044A (Method for manufacturing betacyanins) relates to a method for manufacturing betacyanins that can be stably supplied and also can be industrialized. The method comprises of converting a material into betacyanins in aqueous medium (conversion step) under the presence of a microorganism having enzymatic activity of hydroxylating 3-position of a phenol ring of tyrosine, DOPA4,5-dioxygenase activity, L-DOPA oxidase activity, and enzymatic activity of adding sugar to a phenolic hydroxyl group or a processed product thereof.
The patent US9909130B2 (Production of carotenoids in oleaginous yeast and fungi) relates to systems for producing engineered oleaginous yeast or fungi that express carotenoids. In this invention, strains that both (i) accumulate lipid, often in the form of cytoplasmic oil bodies and typically to at least about 20% of their dry cell weight; and (ii) produce carotenoid(s) at a level at least about 1%, and in some embodiments at least about 3-20%, of their dry cell weight, are generated through manipulation of host cells (i.e., strains, including, e.g., naturally-occurring strains, strains which have been previously modified, etc.). These manipulated host cells are then used to produce carotenoids, so that carotenoids that partition into the lipid bodies can readily be isolated.
In our present invention, S. cerevisiae is employed as a chassis for the reconstruction of betanin biosynthetic pathway for cost effective cum sustainable production of betanin with high yield.

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 sustainable method of synthesis of beanin with a high yield using with engineering the nucleotide sequence of the native genes encoding betanin enzymes to match the preferred codon system of the host cell gives a greater efficiency in protein expression..
Another objective of our present invention is to provide a method using nucleic acid encoding betanin enzymes in which the genes encoding betanin enzymes has been operably fused to promoters and terminators of Saccharomyces cerevisiae and having optimal expression levels in heterologous hosts
The present invention relates to nucleic acids encoding betanin producing enzymes operably fused to promoters and terminators and the process for obtaining a high yield of the betanin.
In one aspect, the invention provides a recombinant vector containing nucleic acids encoding betanin producing enzymes.
In another aspect, the invention provides a recombinant host cell which can optimally produce betanin.
In a further aspect, the invention provides an improved process for production for Betanin

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: Diagram showing the betanin biosynthetic pathway, Figure 2 is a recombinant vector carrying betanin synthesis genes pESCLEU_CYP76AD1W13L – DOD – cDOPA5GT and their regulatory elements, 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.

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.
Furthermore, the methods, preparation and use of the nucleic acids encoding betanin enzymes employ, unless otherwise indicated, conventional techniques in recombinant DNA technology, fermentation technology and related fields. These techniques, their principles, and requirements are explained in the literature and known to a person skilled in the art.
Before the method of generating the nucleic acids encoding the betanin enzymes, vectors, recombinant hosts, methods of downstream processing and other embodiments of the present disclosure are disclosed and described, it is to be understood that the terminologies used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.
The present invention discloses nucleic acids encoding betanin producing enzymes which are optimally expressed in a heterologous host leading to better yield of betanin.
The invention contemplates a multidimensional approach for achieving a high rate of betanin synthesis in a heterologous host. This approach coupled with engineering the nucleotide sequence of the native genes encoding betanin enzymes to match the preferred codon system of the host cell gives a greater efficiency in protein expression.
The present invention discloses a nucleic acid encoding betanin enzymes in which the genes encoding betanin enzymes has been operably fused to promoters and terminators of Saccharomyces cerevisiae and having optimal expression levels in heterologous hosts. In a preferred embodiment, the nucleic acids are represented by SEQ ID Numbers: 1, 3, 5.
The present disclosure also relates to a polypeptides encoded by the nucleic acid sequences as in SEQ ID Numbers: 2, 4, 6 or any variant thereof.
In another aspect, the present disclosure discloses suitable vectors comprising the nucleic acid for optimal expression of betanin enzymes in a heterologous host. In yet another aspect, 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 aspect, the vector is preferably an expression vector in which the DNA sequences encoding the betanin 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 vector is a pESCLEU.
Any suitable promoter can be used. In a preferred embodiment, the strong constitutive promoters Ptdh3, Pgpd, and Ptef2 are 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 betanin 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 betanin is provided. In a preferred embodiment, the process of production includes the steps of culturing host cells transformed with a vector comprising nucleic acids of SEQ ID Numbers: 1, 3, 5 in a suitable culture medium.
In another embodiment, the process of culturing host cells transformed with vectors comprising the nucleic acids in selection media without histidine, Methionine, uracil and comprises glucose, yeast nitrogen base without amino acids.
A few examples pertaining to different embodiments of our present invention have been discussed 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 betanin genes of in Saccharomyces cereviciae
Genes encoding for betanin enzymes CYP76AD1W13L are cloned in pESCLEU. The resulting plasmid is pESCLEU_CYP76AD1W13L – DOD – cDOPA5GT. 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 betanin enzymes are represented by SEQ ID Numbers: 1, 3, 5.
These open reading frames are codon optimized for Saccharomyces cereviciae and are artificially synthesized and cloned in pESCLEU by Genscript.
The recombinant plasmids contain promoters, open reading frames and terminators. The recombinant vector is represented in Figure 2.
Example 2: Polynucleotide sequences for expression of betanin enzymes and corresponding polypeptide sequence
The polynucleotide sequences and corresponding translated proteins sequences represented by SEQ ID Numbers: 1, 2, 3, 4, 5, 6.
Example 3: Development of recombinant host cell by transformation with recombinant plasmid
Recombinant pESCLEU_CYP76AD1W13L – DOD – cDOPA5GT plasmid as described in foregoing example carrying the genes for betanin enzymes SEQ ID Numbers: 1, 3, 5 were used.
Host cells were electroporated with the plasmid 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 betanin synthesis.
The nucleotide sequence of our present invention are given below:
CYP76AD1W13L (Mutation W13L, Beta vulgaris cultivar C869 Accession No: HQ656024.1)
DNA (Seq ID No: 1)
ATGGATCATGCTACTTTGGCAATGATCTTGGCTATCTTGTTTATTTCATTCCATTTCATCAAATTGTTATTTTCT CAACAAACTACAAAATTGTTACCACCAGGTCCAAAGCCATTGCCAATCATCGGTAACATCTTGGAAGTTGGTAAA AAGCCACATAGATCATTCGCTAATTTGGCAAAGATTCATGGTCCATTGATTTCTTTGAGATTAGGTTCAGTTACT ACAATTGTTGTTTCTTCAGCTGATGTTGCAAAGGAAATGTTCTTGAAGAAAGATCATCCATTGTCAAACAGAACA ATCCCAAACTCTGTTACTGCTGGTGACCATCATAAATTAACTATGTCTTGGTTGCCAGTTTCACCAAAGTGGAGA AACTTCAGAAAGATCACTGCTGTTCATTTGTTATCTCCACAAAGATTAGATGCTTGTCAAACTTTTAGACATGCA AAGGTTCAACAATTGTACGAATACGTTCAAGAATGTGCTCAAAAAGGTCAAGCAGTTGATATTGGTAAAGCTGCT TTTACTACATCTTTAAATTTGTTGTCAAAGTTGTTTTTCTCTGTTGAATTGGCTCATCATAAGTCTCATACATCA CAAGAATTCAAAGAATTGATCTGGAACATCATGGAAGATATCGGTAAACCAAACTACGCAGATTACTTCCCAATT TTGGGTTGTGTTGATCCATCAGGTATTAGAAGAAGATTGGCTTGTTCTTTCGATAAGTTGATCGCAGTTTTTCAA GGTATCATCTGTGAAAGATTAGCTCCAGATTCTTCAACTACAACTACAACTACAACTGATGATGTTTTGGATGTT TTGTTACAATTGTTTAAACAAAACGAATTGACTATGGGTGAAATTAATCATTTGTTGGTTGATATCTTCGATGCT GGTACAGATACAACTTCTTCAACTTTCGAATGGGTTATGACAGAATTGATCAGAAACCCAGAAATGATGGAAAAG GCACAAGAAGAAATTAAACAAGTTTTGGGTAAAGATAAGCAAATCCAAGAATCTGATATTATTAATTTGCCATAT TTGCAAGCTATTATTAAGGAAACTTTGAGATTACATCCACCAACAGTTTTCTTGTTACCAAGAAAAGCAGATACA GATGTTGAATTGTATGGTTACATCGTTCCAAAGGATGCTCAAATCTTGGTTAATTTGTGGGCAATCGGTAGAGAT CCAAATGCTTGGCAAAATGCAGATATTTTCTCTCCAGAAAGATTCATTGGTTGTGAAATCGATGTTAAGGGTAGA GATTTCGGTTTGTTACCATTTGGTGCTGGTAGAAGAATTTGTCCTGGTATGAATTTGGCTATCAGAATGTTGACA TTGATGTTGGCAACTTTGTTACAATTTTTCAACTGGAAGTTGGAAGGTGACATTTCTCCAAAGGATTTGGATATG GATGAAAAGTTCGGTATCGCTTTGCAAAAGACTAAGCCATTGAAGTTGATCCCAATCCCAAGATACTAA
Protein (Seq ID No: 2)

MDHATLAMILAILFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNILEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNSVTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECAQKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADYFPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFKQNELTMGEINHLLVDIFDAGTDTTSSTFEWVMTELIRNPEMMEKAQEEIKQVLGKDKQIQESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGRDPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQFFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRY
DOPA 4, 5-dioxygenase (DOD, Mirabilis jalapa, Accession No: AB435372.1)
DNA (Seq ID No: 3)
ATGAAGGGTACTTACTACATCAACCATGGTGACCCATTGATGTATTTGAAGAAACATATCAAGTTGAGACAATTT TTAGAAGGTTGGCAAGAAAATGTTGTTATTGAAAAGCCAAAGTCTATCTTGATCATTTCAGCTCATTGGGATACT AACGTTCCAACAGTTAACTTCGTTGAACATTGTGATACTATTCATGATTTTGATGATTACCCAGATCCATTGTAT CAAATTCAATACAGAGCTCCAGGTGCACCAAATTTGGCTAAGAAAGTTGAAGAATTGTTGAAGGAATCTGGTATG GAATGTGAAATCGATACAAAAAGAGGTTTGGATCATGCTGCATGGTTTCCATTGATGTTCATGTACCCAGAAGCT AACATCCCAATCTGTGAATTGTCTGTTCAACCATCAAAGGATGGTATCCATCATTACAACGTTGGTAAAGCATTG TCACCATTGTTACAACAAGGTGTTTTGATCATCGGTTCTGGTGGTACTGTTCATCCATCAGATGATACACCACAT TGTCCAAATGGTGTTGCTCCATGGGCAATTGAATTTGATAATTGGTTGGAAGATGCTTTGTTATCTGGTAGATAC GAAGATGTTAACAACTTCAAGAAATTAGCACCAAATTGGGAAATTTCACATCCAGGTCAAGAACATTTGTACCCA TTACATGTTGCTTTGGGTGCTGCTGGTAAAAATCCAAAAACTCAATTGATCCATAGATCTTGGGCTGCAAATGGT GTTTTTGGTTACTCAACTTACAACTTCACACCAACTACACAAAAGACTGATTAA
Protein (Seq ID No: 4)
MKGTYYINHGDPLMYLKKHIKLRQFLEGWQENVVIEKPKSILIISAHWDTNVPTVNFVEHCDTIHDFDDYPDPLYQIQYRAPGAPNLAKKVEELLKESGMECEIDTKRGLDHAAWFPLMFMYPEANIPICELSVQPSKDGIHHYNVGKALSPLLQQGVLIIGSGGTVHPSDDTPHCPNGVAPWAIEFDNWLEDALLSGRYEDVNNFKKLAPNWEISHPGQEHLYPLHVALGAAGKNPKTQLIHRSWAANGVFGYSTYNFTPTTQKTD
Cyclo-DOPA 5-O-glucosyltransferase (Mirabilis jalapa, Accession No: AB182643.1)
DNA (Seq ID No: 5)
ATGACTGCTATTAAAATGAACACAAACGGTGAAGGTGAAACTCAACATATCTTGATGATCCCTTTTATGGCTCAA GGTCACTTAAGACCATTTTTAGAATTGGCAATGTTCTTGTACAAAAGATCTCATGTTATTATTACATTGTTAACT ACACCATTAAATGCAGGTTTCTTGAGACATTTGTTGCATCATCATTCTTACTCTTCATCTGGTATCAGAATCGTT GAATTGCCTTTTAATTCAACAAATCATGGTTTGCCACCAGGTATCGAAAACACTGATAAGTTGACATTGCCATTG GTTGTTTCTTTGTTCCATTCTACTATCTCATTGGATCCACATTTGAGAGATTACATCTCTAGACATTTCTCACCA GCTAGACCACCATTATGTGTTATCCATGATGTTTTCTTGGGTTGGGTTGATCAAGTTGCAAAAGATGTTGGTTCA ACAGGTGTTGTTTTTACTACAGGTGGTGCTTATGGTACTTCTGCATACGTTTCAATCTGGAACGATTTGCCACAT CAAAACTACTCTGATGATCAAGAATTCCCATTACCAGGTTTCCCAGAAAACCATAAGTTTAGAAGATCACAATTG CATAGATTTTTGAGATACGCTGATGGTTCTGATGATTGGTCAAAATACTTTCAACCACAATTGAGACAATCTATG AAGTCATTCGGTTGGTTGTGTAACTCTGTTGAAGAAATTGAAACATTAGGTTTTTCAATCTTGAGAAACTACACT AAGTTGCCAATCTGGGGTATTGGTCCATTGATTGCTTCTCCAGTTCAACATTCATCTTCAGATAACAACTCAACA GGTGCAGAATTCGTTCAATGGTTGTCTTTGAAGGAACCAGATTCAGTTTTGTACATCTCTTTCGGTTCACAAAAC ACTATCTCTCCAACACAAATGATGGAATTAGCTGCTGGTTTGGAATCTTCAGAAAAACCATTTTTGTGGGTTATT AGAGCTCCATTCGGTTTCGATATCAACGAAGAAATGAGGCCAGAATGGTTACCAGAAGGTTTCGAAGAAAGAATG AAGGTTAAGAAACAGGGTAAATTGGTTTACAAGTTGGGTCCACAATTGGAAATCTTGAACCATGAATCTATCGGT GGTTTCTTGACTCATTGTGGTTGGAACTCTATCTTGGAATCATTGAGAGAAGGTGTTCCAATGTTAGGTTGGCCA TTGGCTGCTGAACAAGCTTACAATTTGAAGTACTTGGAAGATGAAATGGGTGTTGCTGTTGAATTAGCAAGAGGT TTGGAAGGTGAAATCTCTAAGGAAAAGGTTAAGAGAATTGTTGAAATGATCTTGGAAAGAAACGAAGGTTCAAAA GGTTGGGAAATGAAAAATAGAGCTGTTGAAATGGGTAAAAAGTTGAAGGATGCAGTTAACGAAGAAAAGGAATTG AAGGGTTCTTCAGTTAAGGCAATCGATGATTTCTTGGATGCTGTTATGCAAGCAAAATTAGAACCATCTTTGCAA TAA
Protein (Seq ID No: 6)
MTAIKMNTNGEGETQHILMIPFMAQGHLRPFLELAMFLYKRSHVIITLLTTPLNAGFLRHLLHHHSYSSSGIRIVELPFNSTNHGLPPGIENTDKLTLPLVVSLFHSTISLDPHLRDYISRHFSPARPPLCVIHDVFLGWVDQVAKDVGSTGVVFTTGGAYGTSAYVSIWNDLPHQNYSDDQEFPLPGFPENHKFRRSQLHRFLRYADGSDDWSKYFQPQLRQSMKSFGWLCNSVEEIETLGFSILRNYTKLPIWGIGPLIASPVQHSSSDNNSTGAEFVQWLSLKEPDSVLYISFGSQNTISPTQMMELAAGLESSEKPFLWVIRAPFGFDINEEMRPEWLPEGFEERMKVKKQGKLVYKLGPQLEILNHESIGGFLTHCGWNSILESLREGVPMLGWPLAAEQAYNLKYLEDEMGVAVELARGLEGEISKEKVKRIVEMILERNEGSKGWEMKNRAVEMGKKLKDAVNEEKELKGSSVKAIDDFLDAVMQAKLEPSLQ
, C , Claims:5. CLAIMS
I/We Claim:
1. A method for producing a recombinant host cell capable of expressing betanin enzymes comprising the steps of:
a. synthesizing nucleic acids comprising the nucleotide sequence of SEQ ID Numbers: 1, 3, 5;
b. constructing a recombinant pESCLEU vector harbouring the nucleic acids of SEQ ID NO: 1, 3, 5 wherein the nucleic acids are operably linked to constitutive promoters; and
c. transforming a Saccharomyces cereviciae, BY4741, host cell with the recombinant pESCLEU_CYP76AD1W13L – DOD – cDOPA5GT vector to obtain a recombinant host cell.
wherein the host cell is Saccharomyces cereviciae, BY4741, wherein the vectors are pESCLEU and the constitutive promoters are Ptdh3, Pgpd, and Ptef2, and , wherein the nucleic acids are operably linked to strong constitutive promoter.
2. The method for producing a recombinant host cell capable of expressing betanin enzymes, as claimed in claim 1, wherein the nucleic acids comprise the nucleotide sequences of SEQ ID Numbers: 1, 3, 5 and the nucleic acid encodes betanin enzymes in a host cell, wherein the nucleotide sequence are given by:
CYP76AD1W13L (Mutation W13L, Beta vulgaris cultivar C869 Accession No: HQ656024.1)
DNA (Seq ID No: 1)
ATGGATCATGCTACTTTGGCAATGATCTTGGCTATCTTGTTTATTTCATTCCATTTCATCAAATTGTTATTTTCT CAACAAACTACAAAATTGTTACCACCAGGTCCAAAGCCATTGCCAATCATCGGTAACATCTTGGAAGTTGGTAAA AAGCCACATAGATCATTCGCTAATTTGGCAAAGATTCATGGTCCATTGATTTCTTTGAGATTAGGTTCAGTTACT ACAATTGTTGTTTCTTCAGCTGATGTTGCAAAGGAAATGTTCTTGAAGAAAGATCATCCATTGTCAAACAGAACA ATCCCAAACTCTGTTACTGCTGGTGACCATCATAAATTAACTATGTCTTGGTTGCCAGTTTCACCAAAGTGGAGA AACTTCAGAAAGATCACTGCTGTTCATTTGTTATCTCCACAAAGATTAGATGCTTGTCAAACTTTTAGACATGCA AAGGTTCAACAATTGTACGAATACGTTCAAGAATGTGCTCAAAAAGGTCAAGCAGTTGATATTGGTAAAGCTGCT TTTACTACATCTTTAAATTTGTTGTCAAAGTTGTTTTTCTCTGTTGAATTGGCTCATCATAAGTCTCATACATCA CAAGAATTCAAAGAATTGATCTGGAACATCATGGAAGATATCGGTAAACCAAACTACGCAGATTACTTCCCAATT TTGGGTTGTGTTGATCCATCAGGTATTAGAAGAAGATTGGCTTGTTCTTTCGATAAGTTGATCGCAGTTTTTCAA GGTATCATCTGTGAAAGATTAGCTCCAGATTCTTCAACTACAACTACAACTACAACTGATGATGTTTTGGATGTT TTGTTACAATTGTTTAAACAAAACGAATTGACTATGGGTGAAATTAATCATTTGTTGGTTGATATCTTCGATGCT GGTACAGATACAACTTCTTCAACTTTCGAATGGGTTATGACAGAATTGATCAGAAACCCAGAAATGATGGAAAAG GCACAAGAAGAAATTAAACAAGTTTTGGGTAAAGATAAGCAAATCCAAGAATCTGATATTATTAATTTGCCATAT TTGCAAGCTATTATTAAGGAAACTTTGAGATTACATCCACCAACAGTTTTCTTGTTACCAAGAAAAGCAGATACA GATGTTGAATTGTATGGTTACATCGTTCCAAAGGATGCTCAAATCTTGGTTAATTTGTGGGCAATCGGTAGAGAT CCAAATGCTTGGCAAAATGCAGATATTTTCTCTCCAGAAAGATTCATTGGTTGTGAAATCGATGTTAAGGGTAGA GATTTCGGTTTGTTACCATTTGGTGCTGGTAGAAGAATTTGTCCTGGTATGAATTTGGCTATCAGAATGTTGACA TTGATGTTGGCAACTTTGTTACAATTTTTCAACTGGAAGTTGGAAGGTGACATTTCTCCAAAGGATTTGGATATG GATGAAAAGTTCGGTATCGCTTTGCAAAAGACTAAGCCATTGAAGTTGATCCCAATCCCAAGATACTAA
Protein (Seq ID No: 2)
MDHATLAMILAILFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNILEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNSVTAGDHHKLTMSWLPVSPKWRNFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECAQKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADYFPILGCVDPSGIRRRLACSFDKLIAVFQGIICERLAPDSSTTTTTTTDDVLDVLLQLFKQNELTMGEINHLLVDIFDAGTDTTSSTFEWVMTELIRNPEMMEKAQEEIKQVLGKDKQIQESDIINLPYLQAIIKETLRLHPPTVFLLPRKADTDVELYGYIVPKDAQILVNLWAIGRDPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQFFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRY
DOPA 4, 5-dioxygenase (DOD, Mirabilis jalapa, Accession No: AB435372.1)
DNA (Seq ID No: 3)
ATGAAGGGTACTTACTACATCAACCATGGTGACCCATTGATGTATTTGAAGAAACATATCAAGTTGAGACAATTT TTAGAAGGTTGGCAAGAAAATGTTGTTATTGAAAAGCCAAAGTCTATCTTGATCATTTCAGCTCATTGGGATACT AACGTTCCAACAGTTAACTTCGTTGAACATTGTGATACTATTCATGATTTTGATGATTACCCAGATCCATTGTAT CAAATTCAATACAGAGCTCCAGGTGCACCAAATTTGGCTAAGAAAGTTGAAGAATTGTTGAAGGAATCTGGTATG GAATGTGAAATCGATACAAAAAGAGGTTTGGATCATGCTGCATGGTTTCCATTGATGTTCATGTACCCAGAAGCT AACATCCCAATCTGTGAATTGTCTGTTCAACCATCAAAGGATGGTATCCATCATTACAACGTTGGTAAAGCATTG TCACCATTGTTACAACAAGGTGTTTTGATCATCGGTTCTGGTGGTACTGTTCATCCATCAGATGATACACCACAT TGTCCAAATGGTGTTGCTCCATGGGCAATTGAATTTGATAATTGGTTGGAAGATGCTTTGTTATCTGGTAGATAC GAAGATGTTAACAACTTCAAGAAATTAGCACCAAATTGGGAAATTTCACATCCAGGTCAAGAACATTTGTACCCA TTACATGTTGCTTTGGGTGCTGCTGGTAAAAATCCAAAAACTCAATTGATCCATAGATCTTGGGCTGCAAATGGT GTTTTTGGTTACTCAACTTACAACTTCACACCAACTACACAAAAGACTGATTAA
Protein (Seq ID No: 4)
MKGTYYINHGDPLMYLKKHIKLRQFLEGWQENVVIEKPKSILIISAHWDTNVPTVNFVEHCDTIHDFDDYPDPLYQIQYRAPGAPNLAKKVEELLKESGMECEIDTKRGLDHAAWFPLMFMYPEANIPICELSVQPSKDGIHHYNVGKALSPLLQQGVLIIGSGGTVHPSDDTPHCPNGVAPWAIEFDNWLEDALLSGRYEDVNNFKKLAPNWEISHPGQEHLYPLHVALGAAGKNPKTQLIHRSWAANGVFGYSTYNFTPTTQKTD
Cyclo-DOPA 5-O-glucosyltransferase (Mirabilis jalapa, Accession No: AB182643.1)
DNA (Seq ID No: 5)
ATGACTGCTATTAAAATGAACACAAACGGTGAAGGTGAAACTCAACATATCTTGATGATCCCTTTTATGGCTCAA GGTCACTTAAGACCATTTTTAGAATTGGCAATGTTCTTGTACAAAAGATCTCATGTTATTATTACATTGTTAACT ACACCATTAAATGCAGGTTTCTTGAGACATTTGTTGCATCATCATTCTTACTCTTCATCTGGTATCAGAATCGTT GAATTGCCTTTTAATTCAACAAATCATGGTTTGCCACCAGGTATCGAAAACACTGATAAGTTGACATTGCCATTG GTTGTTTCTTTGTTCCATTCTACTATCTCATTGGATCCACATTTGAGAGATTACATCTCTAGACATTTCTCACCA GCTAGACCACCATTATGTGTTATCCATGATGTTTTCTTGGGTTGGGTTGATCAAGTTGCAAAAGATGTTGGTTCA ACAGGTGTTGTTTTTACTACAGGTGGTGCTTATGGTACTTCTGCATACGTTTCAATCTGGAACGATTTGCCACAT CAAAACTACTCTGATGATCAAGAATTCCCATTACCAGGTTTCCCAGAAAACCATAAGTTTAGAAGATCACAATTG CATAGATTTTTGAGATACGCTGATGGTTCTGATGATTGGTCAAAATACTTTCAACCACAATTGAGACAATCTATG AAGTCATTCGGTTGGTTGTGTAACTCTGTTGAAGAAATTGAAACATTAGGTTTTTCAATCTTGAGAAACTACACT AAGTTGCCAATCTGGGGTATTGGTCCATTGATTGCTTCTCCAGTTCAACATTCATCTTCAGATAACAACTCAACA GGTGCAGAATTCGTTCAATGGTTGTCTTTGAAGGAACCAGATTCAGTTTTGTACATCTCTTTCGGTTCACAAAAC ACTATCTCTCCAACACAAATGATGGAATTAGCTGCTGGTTTGGAATCTTCAGAAAAACCATTTTTGTGGGTTATT AGAGCTCCATTCGGTTTCGATATCAACGAAGAAATGAGGCCAGAATGGTTACCAGAAGGTTTCGAAGAAAGAATG AAGGTTAAGAAACAGGGTAAATTGGTTTACAAGTTGGGTCCACAATTGGAAATCTTGAACCATGAATCTATCGGT GGTTTCTTGACTCATTGTGGTTGGAACTCTATCTTGGAATCATTGAGAGAAGGTGTTCCAATGTTAGGTTGGCCA TTGGCTGCTGAACAAGCTTACAATTTGAAGTACTTGGAAGATGAAATGGGTGTTGCTGTTGAATTAGCAAGAGGT TTGGAAGGTGAAATCTCTAAGGAAAAGGTTAAGAGAATTGTTGAAATGATCTTGGAAAGAAACGAAGGTTCAAAA GGTTGGGAAATGAAAAATAGAGCTGTTGAAATGGGTAAAAAGTTGAAGGATGCAGTTAACGAAGAAAAGGAATTG AAGGGTTCTTCAGTTAAGGCAATCGATGATTTCTTGGATGCTGTTATGCAAGCAAAATTAGAACCATCTTTGCAA TAA
Protein (Seq ID No: 6)
MTAIKMNTNGEGETQHILMIPFMAQGHLRPFLELAMFLYKRSHVIITLLTTPLNAGFLRHLLHHHSYSSSGIRIVELPFNSTNHGLPPGIENTDKLTLPLVVSLFHSTISLDPHLRDYISRHFSPARPPLCVIHDVFLGWVDQVAKDVGSTGVVFTTGGAYGTSAYVSIWNDLPHQNYSDDQEFPLPGFPENHKFRRSQLHRFLRYADGSDDWSKYFQPQLRQSMKSFGWLCNSVEEIETLGFSILRNYTKLPIWGIGPLIASPVQHSSSDNNSTGAEFVQWLSLKEPDSVLYISFGSQNTISPTQMMELAAGLESSEKPFLWVIRAPFGFDINEEMRPEWLPEGFEERMKVKKQGKLVYKLGPQLEILNHESIGGFLTHCGWNSILESLREGVPMLGWPLAAEQAYNLKYLEDEMGVAVELARGLEGEISKEKVKRIVEMILERNEGSKGWEMKNRAVEMGKKLKDAVNEEKELKGSSVKAIDDFLDAVMQAKLEPSLQ