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 melanin biosynthetic pathway and methods for the producing the same. More particularly, the invention is directed to biological synthesis of melanin pigment for multiple applications and methods for obtaining improved melanin yield in Saccharomyces cereviciae by strain engineering.
Background of the Invention

Melanin pigments are dark brown to black color polymeric molecules derived from phenolic or indolic compounds and often found in all living organisms. It has multiple applications in medicine (antimicrobial, antiviral, antitumor, antioxidant, and anti-inflammation), pharmaceutical, cosmetic applications (capacity to absorb broad spectrum electromagnetic radiations and dissipates as heat), and environmental application (bioremediation of contaminated sites due to their binding capacity to heavy metals and radionuclides). In humans, melanin is the prominent pigment responsible for the colour of skin, hair and eyes. Few microorganisms capable of producing these valuable coloring metabolites and used them as survival mechanism to withstand the extreme environmental conditions. Due to their distinctive properties and biocompatible, there is an increasing demand for melanin production. Based on their chemical structure’s melanin pigments are categorized, namely, eumelanin, pheomelanin, neuromelanin and allomelanin. Tyrosinase is responsible for melanin formation. Eumelanin is the black-to-brown subgroup of melanin formed by oxidative polymerization of tyrosine derivatives such as L-3,4-dihydroxyphenylalanine (L-Dopa), and it is the most common melanin found in animals, including humans. Eumelanin is, therefore, by far the most relevant source from a biological and technological perspective and has been widely studied and used as a model for synthetic melanin. Pheomelanin is another type of animal melanin, found in red hair, freckles or feathers, which differs from eumelanin by the presence of sulfur in the composition since its precursor is 5-cysteinyl-Dopa. Neuromelanin is explicitly produced within human neurons by the oxidation of dopamine and other catecholamine precursors. In plants, fungi and bacteria, the identified melanin is called allomelanin. This group encompasses a variety of non-nitrogenous subgroups of melanin derived from different catecholic and dihydroxynaphtalene precursors, which are usually mentioned as catechol melanin (in plants), DHN-melanin and pyomelanin (in bacteria and fungi). The advantages of microbial melanin production in comparison with plant extraction and chemical synthesis is no seasonal growth constrains, cost-effectiveness, and eco-friendliness. Generally, most microbial melanins are formed through the transformation of either tyrosine (DOPA-pathway) or malonyl-coenzyme A (DHN-pathway), facilitated by different sets of enzymes. The DOPA pathway is very similar to mammalian melanin synthesis. In this pathway, the melanin precursor, tyrosine, is converted to L-Dopa, then to dopaquinone by tyrosinase. Dopaquinones are highly active and spontaneously oxidized and autopolymerized to form melanin. Synthesis of melanin via the DOPA-pathway is referred to as DOPA-melanin or eumelanin. Microbial melanin is a valuable source of natural melanin. This study used S. cerevisiae as a chassis for the reconstruction of melanin biosynthetic pathway for cost effective cum sustainable production of melanin 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 present invention relates to nucleic acid encoding melanin producing enzyme operably fused to promoter and terminator and the process for obtaining a high yield of the melanin.
In one aspect, the invention provides a recombinant vector containing nucleic acid encoding melanin producing enzyme.
In another aspect, the invention provides a recombinant host cell which can optimally produce melanin.
In a further aspect, the invention provides an improved process of melanin production
Brief Summary of the Drawings

The features of the present disclosure will become fully apparent from the following description taken in conjunction with the accompanying figures. With the understanding that the figures depict only several embodiments in accordance with the disclosure and are not to be considered limiting of its scope, the disclosure will be described further through use of the accompanying figures.
Figure 1 illlustrates melanin biosynthetic pathway,
Figure 2 illlustrates a recombinant vectors carrying melanin synthesis gene pESCLEU_Tyr1 and their regulatory elements,
Figure 3 illlustrates engineered yeast culture producing melanin pigment.

Detailed Description of the Invention

The present invention discloses nucleic acid encoding melanin producing enzyme which are optimally expressed in a heterologous host.
The invention contemplates that nucleic acid would have better expression in a heterologous host leading to better yield of melanin.
The invention contemplates a multidimensional approach for achieving a high rate of melanin synthesis in a heterologous host. This approach coupled with engineering the nucleotide sequence of the native gene encoding Tyrosinase enzyme to match the preferred codon system of the host cell gives a greater efficiency in protein expression.
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 acid encoding Tyrosinase enzyme 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 acid encoding the Tyrosinase enzyme, 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.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term "comprises" or "comprising" is generally used in the sense to include, that is to say permitting the presence of one or more features or components.
As used herein, the term “disclosure” or “present disclosure” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular disclosure but encompasses all possible embodiments as described in the specification and the claims.
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 gene encoding Tyrosinase enzyme has been operably fused with promoter and terminator of Saccharomyces cerevisiae.
Although disclosure and exemplification has been provided by way of illustrations and examples for the purpose of clarity and understanding, it is apparent to a person skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting the scope of the present disclosure.
The present invention discloses a nucleic acid encoding Tyrosinase enzyme in which the gene encoding Tyrosinase enzyme 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 acid is represented by SEQ ID Numbers: 1.
The present disclosure also relates to a polypeptide encoded by the nucleic acid sequence as in SEQ ID Numbers: 2 or any variant thereof.
In another aspect, the present disclosure discloses suitable vector comprising the nucleic acid for optimal expression of Tyrosinase enzyme 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 sequence encoding the Tyrosinase enzyme is 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 Pact1, Ppgk1, and Psptdh3 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 Tyrosinase enzyme 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, BJ5464. Commercially available Saccharomyces cereviciae, BJ5464, was used in the preferred embodiment of the invention.
In another embodiment, the process for production of melaninis 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 a vectors comprising the nucleic acids in selection media without Leucine and comprises glucose, yeast nitrogen base without amino acids.
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 Tyrosinase (Tyr1) gene in Saccharomyces cereviciae
Gene encoding for Tyrosinase enzyme is cloned in pESCLEU. The resulting plasmid is pESCLEU_Tyr1 (Melanin). 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 Tyrosinase enzyme is represented by SEQ ID Numbers: 1.
The open reading frame is codon optimized for Saccharomyces cereviciae and are artificially synthesized and cloned in pESCLEU.
The recombinant plasmids contain promoters, open reading frames and terminators. The recombinant vectors are represented in Figure 2.
Example 2: Polynucleotide sequence for expression of Tyrosinase enzyme and corresponding polypeptide sequence
The polynucleotide sequences and corresponding translated proteins sequences represented by SEQ ID Numbers: 1 and 2.
Example 3: Development of recombinant host cell by transformation with recombinant plasmid
Recombinant plasmid pESCLEU_Melanin (Constitutive or Inducible expression of Tyrosinase) as described in foregoing example carrying the gene for Tyrosinase enzyme SEQ ID Numbers: 1 was 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 streaked on selection plate and observed for color change indicating melanin synthesis.
, Claims:5. CLAIMS
I/We Claim:
1. A nucleic acid sequence encoding a Tyrosinase enzyme, wherein the sequence is optimized for expression in Saccharomyces cerevisiae and comprises the nucleotide sequence of SEQ ID NO: 1.
2. An expression vector comprising the nucleic acid sequence of claim 1, wherein the nucleic acid sequence is operably linked to:
a strong constitutive or inducible promoter for expression in Saccharomyces cerevisiae; and
a terminator sequence.
3. A process for producing a recombinant Saccharomyces cerevisiae host cell capable of expressing Tyrosinase and producing melanin, comprising the steps of:
synthesizing a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1;
constructing a recombinant pESCLEU vector harboring the nucleic acid of SEQ ID NO: 1, wherein the nucleic acid is operably linked to a constitutive or inducible promoter and a terminator; and
transforming a Saccharomyces cerevisiae host cell with the recombinant pESCLEU_Tyr1 vector.
4. The process as claimed in Claim 3, wherein the Saccharomyces cerevisiae host cell is strain BJ5464.
5. A method for producing melanin, comprising the steps of:
culturing the recombinant Saccharomyces cerevisiae host cell of claim 5 in a suitable culture medium;
inducing Tyrosinase expression (if using an inducible promoter) by adding an appropriate inducer to the culture medium;
maintaining the culture under conditions that promote melanin production; and
harvesting the melanin from the culture medium or the recombinant yeast cells