DESC:The present disclosure relates to the fields of biotechnology and food technology. Particularly, the present disclosure relates to the field of alternative sweeteners and their production. More particularly, provided herein is means and methods for the production of sweet protein Brazzein and compositions comprising the said protein.

BACKGROUND OF THE DISCLOSURE
Brazzein is a thermostable sweet protein, originally found in a wild African plant Pentadiplandra brazzeana Baillon or Oubli. Brazzein is 2,000 and 500 times sweeter than 2% aqueous solution of sucrose and 10% of sugar, respectively, making it an attractive replacement for sugar. Moreover, Brazzein holds its structure intact irrespective of the heat or pH levels, unlike other sweet proteins that have been reported in the art.
In contrast to artificial sweeteners Brazzein which is a natural product limits calorie intake. While Brazzein holds tremendous potential as an alternative sweetener, there are, however, limitations associated with its production and yield. Particularly, because of technical complexities with the large-scale production, manufacturing of Brazzein was believed to be economically non-viable.
There is therefore a need in the art for means and methods to make the production of Brazzein on a large scale economically feasible. The present disclosure addresses the said need.

SUMMARY OF THE DISCLOSURE
The present disclosure provides an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a constitutive promoter or an inducible promoter, and a terminator; wherein the constitutive promoter is selected from a group comprising GAP, TEF1, and GCW14; and wherein the inducible promoter is selected from a group comprising FLD1, LRA3, AOX1, DAS and ADH3.
In some embodiments, the nucleotide sequence is selected from a group comprising nucleotide sequences encoding SEQ ID Nos. 2-5.
In some embodiments, when the nucleotide sequence encodes SEQ ID No. 1, it is under the control of a constitutive promoter, and a terminator.
In some embodiments, when the nucleotide sequence encodes any of SEQ ID Nos. 2-5, it is under the control of a constitutive promoter or an inducible promoter, and a terminator.
In some embodiments, the terminator is selected from a group comprising alcohol oxidase 1 (Pp_AOX1t), Pp_GAPDHt, Pp_GAP1t and Sc_RPL3t.
In some embodiments, the expression cassette further encodes a secretion signal sequence selected from a group comprising FAK - Alpha-factor, AT – Alpha-factor_T, AA – Alpha-amylase, GA – Glucoamylase, IN – Inulinase, IV – Invertase, KP – Killer protein, LZ – Lysozyme, SA – Serum albumin. EG- Major exo-1,3-beta-glucanase and FAKS-Alpha-factor full.
Further provided herein is an vector comprising the expression cassette as described above.
In some embodiments, the vector is selected from a group comprising is selected from a group comprising pREV11, pREV21, pREV45, pREV46, pREV47, pREV48, pREV49, pREV44, pREV33, pREV34, pREV36, pREV38, pREV54, pREV29, pREV42 and pREV23.
In some embodiments, the vector comprises a single copy of the expression cassette.
In some embodiments, the vector further comprises a selectable marker.
Also envisaged herein is a recombinant yeast expression system comprising the expression cassette or the vector as described above.
In some embodiments, the recombinant protein expression system comprises a single copy of the expression cassette.
In some embodiments, the recombinant protein expression system is a yeast or bacterial expression system. In some embodiments, the yeast expression system is selected from a group comprising K. phaffii, K. lactis and S. cerevisea; and the bacterial expression system is E.coli.
In some embodiments, the expression cassette or the vector are integrated at an integration locus selected from a group comprising Alcohol oxidase 1, Formaldehyde dehydrogenase L-Rhamnonate dehydratase, Dihydroxyacetone phosphate, Alcohol dehydrogenase, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Glycosylphosphatidylinositol and Translation elongation factor-1 alpha.
In some embodiments, the above-described method comprises growing the recombinant protein expression system as described above in a fermentation medium under suitable conditions.
In some embodiments, the growing of the recombinant protein expression system comprises a growth phase and a feeding phase; wherein the growth phase comprises maintaining one or more of DO of at least about 40%, temperature of about 28°C to about 32°C, pH of about 3 to about 7, aeration of about 0.2vvm to about 2.2vvm and/or agitation of about 70rpm to about 150rpm; and the feeding phase comprises maintaining one or more of DO of at least about 40%, temperature of about 23°C to about 27°C, pH of about 2.5 to about 6.5, aeration of about 0.2vvm to about 2.2vvm and/or agitation of about 70rpm to about 150rpm.
In some embodiments, the growth phase comprises polyol feeding to compensate for depletion of carbon source and the feeding phase comprises replacement of polyol feeding with dextrose feeding.
In some embodiments, the growth or fermentation is performed in fed-batch mode and/or wherein the growth or fermentation is performed under aerobic conditions.
In some embodiments, the fermentation medium is a basal salt medium.
In some embodiments, the culture is maintained and fed until a cell density corresponding to a wet (packed) cell volume that is about 35% to about 45% of the total fermentation volume is achieved.
In some embodiments, method further comprises isolation of the Brazzein by centrifugation of the fermentation medium followed by one or more of microfiltration, ultrafiltration and diafiltration.
In some embodiments, method further comprises spray drying or freeze drying the isolated Brazzein.
In some embodiments, the spray drying is performed at an inlet temperature of about 115°C to about 125°C, and outlet temperature of about 76°C to about 78°C.
In some embodiments, the obtained Brazzein or mutant(s) thereof has a purity of at least about 80%.
Also provided herein is a Brazzein mutant represented by sequence(s) selected from a group comprising SEQ ID Nos. 2-5.
Further provided herein is a composition comprising Brazzein and/or its mutants having sequence represented by SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto, in combination with one or more sweeteners and/or bulking agents; wherein the sweeteners are selected from a group comprising low calorie sweeteners and non-calorific sweeteners.
BRIEF DESCRIPTION OF FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:
FIGURE 1 depicts the assembly of wild-type Brazzein (wtBRZ) gene through primer walking.
FIGURE 2 depicts screening and confirmation of pREV21-wtBRZ recombinant plasmid by restriction analysis.
FIGURE 3 depicts screening and confirmation of pREV21-wtBRZ/K. phaffii integrants by molecular screening.
FIGURE 4 depicts screening pREV21-wtBRZ/K. phaffii integrants for the expression of wtBRZ protein by SDS-PAGE.
FIGURE 5 depicts the impact of time on large scale production of wtBRZ protein by fermentation of wtBRZ/K. phaffi integrant.
FIGURE 6 depicts the SDS-PAGE analysis of wtBRZ protein.
FIGURE 7 depicts results of HPLC analysis of wtBRZ protein.
FIGURE 8 depicts results of screening of recombinant vectors carrying wtBRZ gene cloned in frame with different signal peptides for expression under GAP promoter in Pichia pastoris.
FIGURE 9 depicts results of screening of recombinant vectors carrying wt BRZ gene cloned in frame with different signal peptide for expression under AOX1 promoter in Pichia pastoris.
Figure 10 depicts results of screening of pREV11-wtBRZ/K. phaffii integrants for the expression of wtBRZ protein.
Figure 11 depicts large scale production of wt Brazzein protein by fermentation of pREV11-wtBRZ/K. phaffi integrant.
Figure 12 depicts results of HPLC analysis of wtBRZ protein expressed under AOX1 promoter.
Figure 13 depicts results of screening and confirmation of recombinant plasmid carrying gene encoding for wild type Brazzein protein under the control of GCw14 promoter, mutants and variants thereof. Figure 14 depicts results of screening pREV29-wtBRZ/K. phaffii integrants for the expression of wtBRZ protein.
Figure 15 depicts results of screening and confirmation of recombinant plasmid carrying gene encoding for mutant Brazzein protein under AOX1 promoter.
Figure 16 depicts results of screening pREV11-SEQ ID No. 2/K. phaffii integrants for the expression of mutated Brazzein protein.
Figure 17 depicts results of large scale production of mutated Brazzein protein by fermentation of pREV11-SEQ2/K. phaffi integrant.
Figure 18 depicts results of HPLC analysis of mutant Brazzein protein SEQ ID No. 2 expressed under AOX1 promoter.
Figure 19 depicts results of screening and confirmation of recombinant plasmid carrying gene encoding for mutant Brazzein protein under GAP promoter.
Figure 20 depicts maps of the different expression vectors created and referred to in the working examples.

DETAILED DESCRIPTION OF THE INVENTION
Addressing the aforesaid need in the art pertaining to the large-scale production of Brazzein, the present disclosure provides an expression cassette and expression system for the production of Brazzein, method(s) for Brazzein production employing the same.

Definitions
Brazzein is a protein isolated from the fruit of the African plant, Pentadiplandra brazzeana Bailon, the known sequence of which consists of 54 aa and having data bank access number P56552 (UniProtKB: locus DEF_PENBA). In the context of the present disclosure, the Brazzein protein and nucleotide sequence encoding Brazzein are synthetically prepared in the laboratory. The term “Brazzein” as used in the present disclosure envisages wild type Brazzein protein or any variant or functional fragment thereof. In a non-limiting embodiment, ‘Brazzein’ in the context of the present disclosure, may include any sequence represented by SEQ ID No. 1 or sequences bearing at least about 70% identity thereto.
The term “protein” or “Brazzein” in the context of the present disclosure, refers to a polymer of amino acids, or amino acid analogues that forms the sweet protein Brazzein, regardless of its size or function. The term includes polyaminoacid Brazzein products such as but not limited to naturally occurring proteins, homologs, orthologs, paralogs, functional fragments and other equivalents, variants, and analogues of Brazzein.
The term “functional fragment” of a polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein and has substantially the same functional features as the full-length polypeptide or protein.
The term “variant polypeptide” or obvious variants of the said term such as “mutants” refer to an amino acid sequence that is different from the reference polypeptide by one or more amino acids, e.g., by one or more amino acid substitutions, deletions, and/or additions. The variant or mutant is preferably functional variant or mutant that retains some or all of the functional features of the reference polypeptide.
“Nucleotide sequence” or “gene sequence” is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to refer to a DNA sequence that encodes for a specific amino acid sequence. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Thus, the said term in the context of “Brazzein” envisages any nucleotide sequence that encodes Brazzein including wild type, mutated and codon optimized sequences encoding the said protein.
The term “growing”, “growth” or its obvious variants in the context of the growing recombinant cells for the expression of Brazzein as described in the present disclosure includes providing an appropriate medium, resources and conditions that would allow cells to multiply and divide, to form a cell culture. The choice of medium, resources and conditions may be optimized depending on factors such as but not limited to scale, production facility and availability of resources.
The term “yeast expression system” as used in the present refers to eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. As is well known, recombinant yeast expression systems may be suitably engineered to enable production of proteins of interest at a desired scale.
The term “E. coli expression system” refers to a prokaryotic expression system that leverages Escherichia coli (E. coli), a bacterium commonly used as a host organism for the production of proteins of interest at a desired scale.
The term “isolated” or obvious variants thereof when used in the context of an isolated nucleic acid or an isolated polypeptide refers to a nucleic acid or polypeptide that is separated from its native environment by virtue of human intervention.
The term “promoter” refers to a DNA sequence that controls expression of a nucleic acid encoding a particular protein. The coding sequence is usually located 3' to the promoter.
The term “terminator” encompasses a DNA sequence at the end of a transcriptional unit which signals 3’ processing and polyadenylation of a primary transcript and termination of transcription.
The term “transform” or its obvious variants refer to the transfer of a polynucleotide of interest into a target cell for expression by that cell. The transferred polynucleotide can be incorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal DNA.
The terms “plasmid”, “vector” and “cassette” generally refer to an extra chromosomal element generally carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double- stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction to enable expression of a protein of interest.
The term “selectable marker” includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid construct of the disclosure. These marker genes enable identification of a successful transfer of the nucleic acid molecules into the host cell.
The term “expression” or “gene expression” refers to transcription of a gene and subsequent translation of the RNA leading to synthesis of the encoded protein/enzyme, i.e., protein/enzyme expression.
Reference to “% identity” encompasses the extent to which two aligned DNA or protein sequences are invariant throughout a window of alignment. It is indicative of the number of identical components that are shared by sequences of the two aligned segments.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
As used herein, the terms “include” (any form of “include”, such as “include”), “have” (and “have”), “comprise” etc. any form of “having”, “including” (and any form of “including” such as “including”), “containing”, “comprising” or “comprises” are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps
It is to be noted that the term “a” or “an” entity refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Disclosure
Expression cassette
The present disclosure provides an expression cassette comprising a nucleotide encoding Brazzein or its variants under the control of a promoter, a terminator and optionally, a signal sequence.
In some embodiments, the nucleotide sequence encodes Brazzein having sequence represented by SEQ ID No. 1 or at least about 70% identity thereto.
In some embodiments, SEQ ID No. 1 has the following sequence:
DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY
Non-limiting examples of sequences bearing at least about 70% identity to Brazzein include:
SEQ ID No. 2
DKCCKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICCYCEY
SEQ ID No. 3
DKCRKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICEYCEY
SEQ ID No. 4
DKCEKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICRYCEY
SEQ ID No. 5
DKCKKCYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCCCDYCEY
In some embodiments, the nucleotide sequence encoding Brazzein bears about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% identity to the nucleotide sequence coding for SEQ ID No. 1.
In some embodiments, the nucleotide sequence encoding Brazzein may be optimized for expression in yeast or E.coli. Accordingly, in some embodiments, the nucleotide sequence encoding Brazzein or mutants thereof as contained in the expression cassette of the present disclosure is codon optimized for expression in yeast. In a non-limiting embodiments, the nucleotide sequence(s) encoding Brazzein or mutants thereof i.e. the nucleotide sequences encoding SEQ ID Nos. 1-5, are codon optimized to optimize parameters that are critical to the efficiency of gene expression in yeast, said parameters being selected from but not limited to codon usage bias, codon adaptation index, GC content, repeat sequences (direct repeat, reverse repeat, and dyad repeat), cryptic splice sites, anti-viral motifs, premature poly-A sites and any combinations thereof.
In another non-limiting embodiment, the codon optimized nucleotide sequence of the present disclosure encoding Brazzein or mutants thereof has increased sequence stability. Without intending to be limited by theory, once obtained, the codon optimised nucleotide sequence may be cloned between suitable restriction sites in the multiple cloning site (MCS) of multiple vector backbones for expression in hosts such as but not limited to K. phaffii, K. lactis, S. cerevisea and E.coli.
Accordingly, in some embodiments, the expression cassette comprises the nucleotide sequence as described above under the control of a suitable promoter and a terminator.
In some embodiments, the promoter is a constitutive or inducible promoter.
In some embodiments, when the nucleotide sequence encodes SEQ ID No. 1, it may be expressed under the control of a constitutive promoter. Accordingly, in some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, and a terminator.
In some embodiments, non-limiting examples of the constitutive promoter include but are not limited to GAP promoter, TEF1 promoter, GCW14 promoter and mutant variants thereof.
In some embodiments, the promoter is a constitutive promoter selected from a group comprising GAP promoter represented by SEQ ID No. 17, GCw14 promoter represented by SEQ ID No. 18, mutated GCw14 promoter (mGCw14) represented by SEQ ID No. 19, GAP::mGCw14 hybrid promoter represented by SEQ ID No. 20, TEF1 promoter represented by SEQ ID No. 21.
In an exemplary embodiment, the constitutive promoter is a GAP promoter represented by SEQ ID No. 17.
Accordingly, in some embodiments, the expression cassette comprises a nucleotide sequence encoding a sequence bearing at least about 70% identity to SEQ ID No. 1 under the control of a GAP promoter, and a terminator.
In some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, and a terminator.
In some embodiments, when the nucleotide sequence encodes any of SEQ ID Nos. 2-5, it may be expressed under the control of a constitutive promoter or an inducible promoter. Accordingly, in some embodiments, the expression cassette comprises a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of a constitutive promoter or an inducible promoter, and a terminator.
Non-limiting examples of employable inducible promoters include FLD1, LRA3 and AOX1. When the said promoters are employed in the construct, the protein expression is triggered in presence of a suitable inducer. In a non-limiting embodiment, non-limiting examples of inducers for the said promoters include methylamine, rhamnose and methanol, respectively.
In an exemplary embodiment, the promoter is an inducible promoter selected from a group comprising AOX1 promoter represented by SEQ ID No. 22 and ADH3 promoter represented by SEQ ID No. 23.
Thus, in some embodiments, provided herein is an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a constitutive promoter or an inducible promoter, and a terminator; wherein the constitutive promoter is selected from a group comprising GAP, TEF1, and GCW14; and wherein the inducible promoter is selected from a group comprising FLD1, LRA3, DAS, ADH3 and AOX1.
In some embodiments, the terminator is selected from a group comprising alcohol oxidase 1 (Pp_AOX1t), Pichia pastoris GAPDH terminator (Pp_GAPDHt), Pp_GAP1t and Sc_RPL3t.
In some embodiments, the terminator is selected from a group comprising Pp_AOX1t represented by SEQ ID No. 24 and Pp_GAPDHt represented by SEQ ID No. 25.
In an exemplary embodiment, the terminator is an AOX1 terminator.
Accordingly, in some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, and an AOX1 terminator.
In some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, and an AOX1 terminator.
In some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, and an AOX1 terminator.
In some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, and an AOX1 terminator.
In some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID Nos. 2-5 under the control of an inducible or constitutive promoter, and an AOX1 terminator.
In some embodiments, the expression cassette further encodes a secretion signal sequence.
In some embodiments, the expression cassette further encodes a secretion signal sequence selected from a group comprising FAK - Alpha-factor represented by SEQ ID No. 6, AT – Alpha-factor_T represented by SEQ ID No. 7, AA – Alpha-amylase represented by SEQ ID No. 8, GA – Glucoamylase represented by SEQ ID No. 9, IN – Inulinase represented by SEQ ID No. 10, IV – Invertase represented by SEQ ID No. 11, KP – Killer protein represented by SEQ ID No. 12, LZ – Lysozyme represented by SEQ ID No. 13, SA – Serum albumin represented by SEQ ID No. 14, EG- Major exo-1,3-beta-glucanase represented by SEQ ID No. 15 and FAKS-Alpha-factor full represented by SEQ ID No. 16. Reference to a secretion signal sequence contained in an expression cassette in subsequent embodiments embodies reference to a nucleotide sequence encoding the said signal sequence, as would be understood by a person skilled in the art.
In some embodiments, the signal peptide sequences comprise a stretch of four amino acids (LEKR) for the efficient Kex2 processing of pre-protein. In some embodiments, the said stretch of amino acids is added in the laboratory ahead of or at the time of preparing the expression cassette or, in the alternative, the said stretch of four amino acids is present in the signal peptides sourced commercially. Thus, in some embodiments, the cloning strategy also includes incorporating a stretch of four amino acids (LEKR) at the junction of signal peptide and the codon optimised nucleic acids sequence encoding for the Brazzein protein. In some embodiments, the said stretch of amino acids (LEKR) enables efficient Kex2 processing of the precursor peptide for Brazzein or mutants thereof.
In an exemplary embodiment, the secretion signal sequence is an alpha secretion signal.
Accordingly, in some embodiments, the expression cassette comprises a nucleotide encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and a secretion signal sequence.
In some embodiments, the expression cassette comprises a nucleotide encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and a secretion signal sequence.
In some embodiments, the expression cassette comprises a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible promoter or a constitutive promoter, a terminator and a secretion signal sequence.
In some embodiments, the expression cassette comprises a nucleotide encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and a secretion signal sequence.
In some embodiments, the expression cassette comprises a nucleotide encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and an alpha secretion signal sequence.
In some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator and an alpha signal sequence.
In some embodiments, the expression cassette comprises a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible promoter or a constitutive promoter, a terminator and a secretion signal sequence selected from a group comprising FAK - Alpha-factor, AT – Alpha-factor_T, AA – Alpha-amylase, GA – Glucoamylase, IN – Inulinase, IV – Invertase, KP – Killer protein, LZ – Lysozyme and SA – Serum albumin.
In some embodiments, the expression cassette comprises a nucleotide encoding SEQ ID Nos. 2-5 under the control of an inducible promoter or a constitutive promoter, a terminator and an alpha secretion signal sequence.
In some embodiments, the expression cassette comprises a nucleotide sequence encoding SEQ ID Nos. 2-5 under the control of an inducible or constitutive promoter, an AOX1 terminator and an alpha secretion signal sequence.

Vector
The present disclosure further provides an expression vector for the production of Brazzein protein comprising the expression cassette as described above.
In some embodiments, the vector backbone may be any vector backbone known in the art that is capable of carrying the combination of promoter(s), termination signal(s)/terminator(s) and signal sequence(s) as discussed above in the foregoing embodiments.
Accordingly, provided herein is a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and optionally, a secretion signal sequence.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, a terminator and optionally, a secretion signal sequence.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and a secretion signal sequence.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and an alpha secretion signal sequence.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator and an alpha signal sequence.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible promoter or a constitutive promoter, a terminator and a secretion signal sequence selected from a group comprising FAK - Alpha-factor, AT – Alpha-factor_T, AA – Alpha-amylase, GA – Glucoamylase, IN – Inulinase, IV – Invertase, KP – Killer protein, LZ – Lysozyme and SA – Serum albumin.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide encoding SEQ ID Nos. 2-5 under the control of an inducible promoter or a constitutive promoter, a terminator and an alpha secretion signal sequence.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding SEQ ID Nos. 2-5 under the control of an inducible or constitutive promoter, an AOX1 terminator and an alpha secretion signal sequence.
In some embodiments, the vector may further carry a selectable marker e.g., antibiotic resistance.
In a non-limiting embodiment, examples of such selectable markers include Zeocin. In some embodiments, Zeocin may be employed as selectable marker for all vector backbones mentioned herein except pREV54, wherein the pREV54 vector backbone may contain Ampicillin as selectable marker.
Accordingly, provided herein is a vector comprising the expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator and optionally, an alpha signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector comprises an expression cassette comprising a nucleotide sequence encoding SEQ ID Nos. 2-5 under the control of an inducible or constitutive promoter, an AOX1 terminator and an alpha secretion signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector may comprise one or more copies of the expression cassette as described above.
In an exemplary embodiment, the vector comprises a single copy of the expression cassette. Without wishing to be limited by theory, one of the key advantages of the present disclosure lies in the fact that despite employing only a single copy of the expression cassette, the present disclosure is able to provide a relatively high yield of the protein of interest i.e. Brazzein.
Accordingly, in some embodiments, the vector comprises a single copy of an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector comprises a single copy of an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator, and optionally, an alpha signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector comprises a single copy of an expression cassette comprising a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, an AOX1 terminator, and optionally, an alpha signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the vector backbone is selected from a group comprising pREV11, pREV21, pREV45, pREV46, pREV47, pREV48, pREV49, pREV44, pREV33, pREV34, pREV36, pREV38, pREV54, pREV29, pREV42 and pREV23.
In a non-limiting exemplary embodiment, vector backbone is pREV11 or pREV21
In another embodiment, vector backbone is pREV21.
Accordingly, in some embodiments, envisaged herein is a pREV11 or pREV21 vector comprising a single copy of an expression cassette that in turn comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the pREV11 or pREV21 vector comprises a single copy of an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a AOX1 and GAP promoter respectively, an AOX1 terminator, and optionally, an alpha signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the pREV11 or pREV21 vector comprises a single copy of an expression cassette comprising a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, an AOX1 terminator, and optionally, an alpha signal sequence, wherein the vector may further comprise a selectable marker.
In a non-limiting embodiment, conventional methods of molecular biology may be used to create the Brazzein cassette and expression vector as described above. Such methods are described, for example, in Molecular cloning: a laboratory manual, 3rd ed., Sambrook, Joseph; Russell, David W., New York: Cold Spring Harbor Laboratory, 2001; Molecular Cloning: A laboratory manual, 2d ed., Sambrook, J., Fritsch, E. F., and Maniatis, T. New York: Cold Spring Harbor Laboratory Press, 1989; Cold Spring Harbor Protocols, cshprotocols.cshlp.org; Current Protocols in Molecular Biology, currentprotocols.com.

Recombinant protein expression system
The present disclosure further provides a recombinant protein expression system comprising the expression cassette as described above to enable production of Brazzein. Accordingly, provided herein are recombinant protein expression system(s) comprising the expression cassette as described above to enable production of Brazzein.
In some embodiments, the recombinant protein expression system includes but is not limited to yeast and bacterial expression systems.
In some embodiments, the yeast expression system is selected from a group comprising K. phaffii, K. lactis and S. cerevisea. In some embodiments, the bacterial expression system is E.coli.
In some embodiments, yeast expression system is K. phaffii.
While the subsequent embodiments elaborate on a K. phaffii based expression system, said embodiments are equally applicable to alternative equivalents of the said cell employable in place of K. phaffii, such as but not limited to K. lactis and S. cerevisea. The said embodiments, further, are also extrapolatable to an E.coli based expression system as defined above. Such embodiments of the present disclosure wherein K. phaffii can be alternatively replaced by expression systems such as but not limited to K. lactis, S. cerevisea and E.coli, thus can be associated with an equal expectation of success and are not repeated herein for the sake of brevity.
Accordingly, in some embodiments, the present disclosure provides a recombinant K. phaffii cell comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator, and optionally, a secretion signal sequence.
In some embodiments, the present disclosure provides a recombinant K. phaffii cell comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and optionally, a secretion signal sequence.
In some embodiments, the present disclosure provides a recombinant K. phaffii cell comprising an expression cassette that comprises a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, a terminator and optionally, a secretion signal sequence.
In some embodiments, the present disclosure provides a recombinant K. phaffii cell comprising an expression cassette that comprises a nucleotide encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator, and optionally, an alpha secretion signal sequence.
In some embodiments, the present disclosure provides a recombinant K. phaffii cell comprising an expression cassette that comprises a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible or constitutive promoter, an AOX1 terminator, and optionally, an alpha secretion signal sequence
In some embodiments, the recombinant K. phaffii cell comprises a single copy of an expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator, and optionally, an alpha signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the recombinant K. phaffii cell comprises a single copy of an expression cassette comprising a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, an AOX1 terminator, and optionally, an alpha signal sequence, wherein the vector may further comprise a selectable marker.
As mentioned above, it is one of the key advantages of the present disclosure that despite employing only a single copy of the gene of encoding the protein of interest i.e. Brazzein, the present disclosure is able to provide a relatively high yield of the protein of interest i.e. Brazzein.
In some embodiments, provided herein is a recombinant K. phaffii cell comprising the expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally a secretion signal sequence, wherein the expression cassette is contained in a vector.
In some embodiments, the recombinant K. phaffii cell comprises the expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally a secretion signal sequence, wherein the expression cassette is contained in a vector and wherein optionally, the vector comprises a selectable marker.
In some embodiments, the recombinant K. phaffii cell comprises the expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally a secretion signal sequence, wherein the expression cassette is contained in a vector as a single copy.
In some embodiments, the recombinant K. phaffii cell comprises the expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the expression cassette is contained in a vector as a single copy.
In some embodiments, the recombinant K. phaffii cell comprises the expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator and optionally, a secretion signal sequence, wherein the expression cassette is contained in a vector as a single copy.
In some embodiments, the recombinant K. phaffii cell comprises the expression cassette comprising a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or constitutive promoter, an AOX1 terminator and optionally, a secretion signal sequence, wherein the expression cassette is contained in a vector as a single copy.
In some embodiment, the vector as described under the previous header is used to transform suitable host cells such as but not limited to yeast or bacterial cells. Thus, in some embodiments, the present disclosure provides a recombinant K. phaffii, K. lactis, S. cerevisea or E.coli cell comprising the vector as described above. As mentioned above, while the subsequent embodiments elaborate on a K. phaffii based expression system comprising the vector of the present disclosure, said embodiments are equally applicable to alternative equivalents of the said cell employable in place of K. phaffii, such as but not limited to K. lactis and S. cerevisea. The said embodiments, further, are also extrapolatable to an E.coli based expression system as defined above. Such embodiments of the present disclosure wherein K. phaffii can be alternatively replaced by expression systems such as but not limited to K. lactis, S. cerevisea and E.coli, thus can be associated with an equal expectation of success and are not repeated herein for the sake of brevity.
In some embodiments, the recombinant K. phaffii cell is transformed with a vector comprising the expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the vector may further comprise a selectable marker.
In some embodiments, the recombinant K. phaffii cell is transformed with a vector comprising the expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator, and optionally a secretion signal sequence, wherein the vector may further comprise a selectable marker and wherein the expression cassette is contained in the vector as a single copy.
In some embodiments, the recombinant K. phaffii cell is transformed with a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a GAP promoter, an AOX1 terminator, and optionally, an alpha secretion signal sequence; wherein the expression cassette is contained in the vector as a single copy.
In some embodiments, the recombinant K. phaffii cell is transformed with a vector comprising an expression cassette that comprises a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or constitutive promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence; wherein the expression cassette is contained in the vector as a single copy.
In some embodiments, the K. phaffii cell is of a strain selected from a group comprising strains KM71, SMD1168, GS115, SMD1168H and KM71H.
In some embodiments, the expression cassette or the vector are integrated into the K. phaffii cell at a suitable locus depending on the choice of promoter. Some non-limiting examples for the integration locus for constructs comprising promoter(s) such as AOX1, FLD1,LRA3, DAS and ADH3 include Alcohol oxidase 1, Formaldehyde dehydrogenase L-Rhamnonate dehydratase, Dihydroxyacetone phosphate and Alcohol dehydrogenase, respectively. Some non-limiting examples for the integration locus for constructs comprising promoter(s) such as GAP, GCW14 and TEF1 include Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Glycosylphosphatidylinositol and Translation elongation factor-1 alpha, respectively. In some embodiments, the expression cassette or the vector, irrespective of the choice of promoter, are integrated into the K. phaffii cell at the GAPDH locus.
In some embodiments, the expression cassette or the vector comprising the GAP promoter are integrated into the K. phaffii cell at the GAPDH locus.
Thus, in some embodiments, the recombinant K. phaffii cell comprises an expression cassette or vector comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus.
In some embodiments, the present disclosure provides a recombinant K. phaffii cell comprising an expression cassette or vector comprising a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus.
In some embodiments, the present disclosure provides a recombinant K. phaffii cell comprising an expression cassette or vector comprising a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, a terminator and optionally, a secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus.
In some embodiments, the recombinant K. phaffii cell comprises an expression cassette or vector comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus.
In some embodiments, the recombinant K. phaffii cell comprises an expression cassette or vector comprising a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus.
In some embodiments, the recombinant K. phaffii cell comprises an expression cassette or vector comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, a terminator and optionally, an alpha secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus as a single copy.
In some embodiments, the recombinant K. phaffii cell comprises an expression cassette or vector comprising a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, a terminator and optionally, an alpha secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus as a single copy.
In some embodiments, the recombinant K. phaffii cell comprises an expression cassette or vector comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus as a single copy.
In some embodiments, the recombinant K. phaffii cell comprises an expression cassette or vector comprising a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus as a single copy.
In a non-limiting embodiment, any appropriate method of transformation may be used including electroporation and chemical-mediated transformation as described, for example, in Cregg, Methods in Molecular Biology 389: 27-42, 2007 and in Becker and Guarenta, Methods in Enzymology 194: 182-187, 1991. Transformed yeast cells may be stored at -80° C. in a solution containing 10% glycerol.
Brazzein production
The present disclosure further provides a method for producing Brazzein or mutants thereof, said method comprising growing the recombinant expression system(s) as described above in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein or mutants thereof comprises growing the recombinant expression system(s) as described above in a fermentation medium under suitable conditions.
While subsequent embodiments of the present disclosure refer to the production of Brazzein, said embodiments are equally applicable to the production of mutants of Brazzein such as those described above, and the said embodiments are not repeated herein for the sake of brevity.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with the vector as described above; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally a secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprising steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and optionally a secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprising steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, a terminator and optionally a secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii with a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising a single copy of an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii with a vector comprising a single copy of an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, a terminator and optionally, an alpha secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii with a vector comprising a single copy of an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii with a vector comprising a single copy of an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, a terminator and optionally, an alpha secretion signal sequence , wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising a single copy of an expression cassette that comprises a nucleotide sequence encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, an AOX1 terminator and optionally, an alpha secretion signal sequence; and
b) growing the transformed cell(s) in a fermentation medium under suitable conditions.
In some embodiments, the fermentation medium is selected from a group comprising basal salt medium.
In an exemplary embodiment, the fermentation medium is basal salt medium.
In some embodiments, the growth or fermentation is performed in fed-batch mode.
In some embodiments, the growth or fermentation is performed under aerobic conditions.
In some embodiments, the growth or fermentation is performed while maintaining dissolved oxygen (DO) levels of at least about 40%.
In some embodiments, the DO level during the growth or fermentation is maintained in the range of about 40% to about 80%.
In a non-limiting embodiment, the DO level is maintained at about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or about 80%.
In some embodiments, the growth or fermentation is performed while maintaining aeration of about 0.2vvm to about 2.2vvm.
In a non-limiting embodiment, the growth or fermentation is performed while maintaining aeration of about 0.2vvm, about 0.4vvm, about 0.6vvm, about 0.8vvm, about 1vvm, about 1.2vvm, about 1.4vvm, about 1.6vvm, about 1.8vvm, about 2vvm or about 2.2vvm.
In some embodiments, the growth or fermentation is performed while maintaining agitation in the range of about 70 rpm to about 500 rpm.
In a non-limiting embodiment, the growth or fermentation is performed while maintaining agitation of about 70 rpm, about 120 rpm, about 170 rpm, about 220 rpm, about 270 rpm, about 320 rpm, about 370 rpm, about 420 rpm or about 500 rpm.
In some embodiments, the growth or fermentation is performed at a temperature of about 25°C to about 35°C.
In some embodiments, the growth or fermentation is performed at a temperature of about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C or about 35°C.
In some embodiments, the growth or fermentation is performed while maintaining pH of about 2.5 to about 7.
In a non-limiting embodiment, the growth or fermentation is performed while maintaining pH of about 2.5, about 3, about 4, about 5, about 6 or about 7.
In a non-limiting embodiment, the growth or fermentation comprises a growth phase and feeding phase.
In some embodiments, the growth phase of the fermentation is performed while maintaining DO of at least about 40%, temperature of about 28°C to about 32°C, pH of about 3 to about 7, aeration of about 0.2vvm to about 2.2vvm and/or agitation of about 70rpm to about 150rpm. In a non-limiting embodiment, the growth phase is accompanied by polyol feeding to compensate for depletion of carbon source. In some embodiments, the polyol is glycerol.
In some embodiments, the feeding phase of fermentation is performed while maintaining DO of at least about 40%, temperature of about 23°C to about 27°C, pH of about 2.5 to about 6.5, aeration of about 0.2vvm to about 2.2vvm and/or agitation of about 70rpm to about 150rpm. In a non-limiting embodiment, the feeding phase comprises replacement of polyol feeding with dextrose feeding.
Without intending to be limited by theory, in some embodiments, during fermentation, the yeast culture may be maintained and fed until a cell density corresponding to a wet (packed) cell volume that is about 35% to about 45%, preferably about 40% of the total fermentation volume is achieved.
In some embodiments, the isolation is performed by centrifugation of the fermentation medium followed by one or more of microfiltration, ultrafiltration and diafiltration.
In some embodiments, the method further comprises isolation of Brazzein from the fermentation medium and optionally, spray drying or freeze drying the isolated Brazzein.
Accordingly, in some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, an alpha secretion signal sequence;
b) growing the transformed cell(s) in a fermentation medium under suitable conditions;
c) isolation of Brazzein from the fermentation medium; and
d) optionally, spray drying or freeze drying the isolated Brazzein.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising a single copy of an expression cassette that comprises a nucleotide encoding SEQ ID No. 1 under the control of a constitutive promoter, a terminator and optionally, a secretion signal sequence;
b) growing the transformed cell(s) in a fermentation medium under suitable conditions;
c) isolation of Brazzein from the fermentation medium; and
d) optionally, spray drying or freeze drying the isolated Brazzein.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising a single copy of an expression cassette that comprises a nucleotide encoding any of SEQ ID Nos. 2-5 under the control of an inducible or a constitutive promoter, a terminator and optionally, a secretion signal sequence;
b) growing the transformed cell(s) in a fermentation medium under suitable conditions;
c) isolation of Brazzein from the fermentation medium; and
d) optionally, spray drying or freeze drying the isolated Brazzein.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising a single copy of an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence;
b) growing the transformed cell(s) in a fermentation medium under suitable conditions; and
c) isolation of Brazzein from the fermentation medium, wherein the isolation is performed by centrifugation of the fermentation medium followed by one or more of microfiltration, ultrafiltration and diafiltration.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising a single copy of an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence;
b) growing the transformed cell(s) in a fermentation medium under suitable conditions;
c) isolation of Brazzein from the fermentation medium by centrifugation of the fermentation medium followed by one or more of microfiltration, ultrafiltration and diafiltration; and
d) spray drying or freeze drying the isolated Brazzein.
In some embodiments, the microfiltration is performed through spiral wound TFF membrane.
In some embodiments, the ultrafiltration and diafiltration are performed using spiral wound TFF membrane.
In some embodiments, the isolated protein, post microfiltration, ultrafiltration and/or diafiltration, may be further subjected to dialysis to remove any remaining impurities. In a non-limiting embodiment, the dialysis is performed against water.
In some embodiments, the isolated Brazzein after isolation and optionally, dialysis, may be obtained in the form of a solution. In some embodiments, the said Brazzein solution is subsequently subjected to spray drying or freeze drying to obtain the Brazzein in the form of a fine powder.
Thus, in some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence;
b) growing the transformed cell(s) in a fermentation medium under suitable conditions;
c) isolation of Brazzein from the fermentation medium by centrifugation of the fermentation medium followed by one or more of microfiltration, ultrafiltration and diafiltration to obtain a Brazzein solution;
d) dialysis of the Brazzein solution; and
e) spray drying or freeze drying the Brazzein solution.
In some embodiments, the spray drying is performed at an inlet temperature of about 100°C to about 140°C and outlet temperature of about 70°C to about 82°C.
In an exemplary embodiment, the spray drying is performed at an inlet temperature of about 115°C to about 125°C and outlet temperature of about 76°C to about 78°C.
In some embodiments, the method for producing Brazzein comprises steps of:
a) transforming one or more cells of K. phaffii, K. lactis, S. cerevisea or E.coli with a vector comprising an expression cassette that comprises a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of an inducible or constitutive promoter, a terminator and optionally, a secretion signal sequence;
b) growing the transformed cell(s) in a fermentation medium under suitable conditions;
c) isolation of Brazzein from the fermentation medium by centrifugation of the fermentation medium followed by one or more of microfiltration, ultrafiltration and diafiltration; and
d) spray drying or freeze drying the isolated Brazzein, wherein the spray drying may be performed at an inlet temperature of about 100°C to about 140°C and outlet temperature of about 70°C to about 82°C.
In some embodiments, the obtained Brazzein or mutant(s) thereof has a purity of at least about 80%.
In some embodiments, the obtained Brazzein or mutant(s) thereof has a purity of at least about 85%.
In some embodiments, the obtained Brazzein or mutant(s) thereof has a purity of at least about 90%.
In some embodiments, the obtained Brazzein or mutant(s) thereof has a purity of at least about 95%.
In some embodiments, the obtained Brazzein or mutant(s) thereof has a purity of about 97% to about 99%.
In some embodiments, the obtained Brazzein or mutant(s) thereof has a purity of about 95%, about 96%, about 97%, about 98% or about 99%.
As can be observed from the above, the method of the present disclosure does not employ any harmful chemicals. Accordingly, it is one of the advantages of the method of the present disclosure that it is fit for consumption and can be incorporated in food products due to lack of reliance on harsh chemicals for its production.
Brazzein and its mutants
Further envisaged in the present disclosure are Brazzein and its mutants as obtained by the above-described method(s) and construct(s).
Accordingly, in some embodiments, envisaged herein is Brazzein having sequence represented by SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto produced by the aforementioned method(s) of the present disclosure.
SEQ ID No. 1 (wild type Brazzein)
DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY
Non-limiting examples of Brazzein mutants envisaged in the present disclosure bearing at least about 70% identity to SEQ ID No. 1 include:
SEQ ID No. 2
DKCCKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICCYCEY
SEQ ID No. 3
DKCRKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICEYCEY
SEQ ID No. 4
DKCEKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICRYCEY
SEQ ID No. 5
DKCKKCYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCCCDYCEY
Composition comprising Brazzein and its mutants
The present disclosure further provides compositions comprising Brazzein and/or its mutants as described above, in combination with one or more sweetener(s) and/or bulking agent(s).
Thus, in some embodiments, provided herein is a composition comprising Brazzein and/or its mutants having sequence represented by SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto, in combination with one or more sweetener(s) and/or bulking agent(s).
In some embodiments, the one or more sweeteners are selected from a group comprising low calorie sweeteners and non-calorific sweeteners. Non-limiting examples of such low-calorie sweeteners and non-calorific sweeteners, and/or bulking agents include but are not limited to stevia, monk fruit, _Allulose, FOS, Isomaltulose, Maltodextrin and Dextrin or any combination thereof.
In some embodiments, the one or more bulking agent(s) are selected from a group comprising FOS, Maltodextrin and Dextrin or any combination thereof.
Accordingly, in some embodiments, provided herein is a composition comprising Brazzein and/or its mutants having sequence represented by SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto, in combination with one or more sweeteners selected from a group comprising low calorie sweeteners and non-calorific sweeteners.
In some embodiments, the composition comprises Brazzein and/or its mutants having sequence represented by SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto, in combination with one or more bulking agent(s) selected from a group comprising FOS, Maltodextrin and Dextrin or any combination thereof.
In some embodiments, the composition comprises Brazzein and/or its mutants having sequence represented by SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto, in combination with one or more sweetener(s) and/or bulking agent(s) selected from a group comprising stevia, monk fruit, Allulose, Fos, Isomaltulose, Maltodextrin and Dextrin or any combination thereof.
The subject matter of embodiments of the present invention is described here with specificity to meet statutory requirements, but this description is not necessarily intended to limit the scope of the disclosure. The disclosed subject matter may be embodied in other ways, may include different elements or steps, and may be used in conjunction with other existing or future technologies. This description should not be interpreted as implying any particular order or arrangement among or between various steps or elements except when the order of individual steps or arrangement of elements is explicitly described. The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.
EXAMPLES:
Example 1: Construction of an expression vector for the constitutive expression of Brazzein
The nucleotide sequence encoding wild type Brazzein (wtBRZ - SEQ ID No. 1) was prepared synthetically. The gene encoding wild type Brazzein protein (wtBRZ) was assembled through primer walking using four primers. The gene wtBRZ was finally obtained as ~185 bp fragment through PCR amplification (Figure 1).
The obtained nucleic acid was codon optimised according to the expression hosts to be employed i.e. K. phaffii or E. coli. The codon optimisation was done using publicly available software. To keep the cloning strategy compatible, sites for multiple restriction enzymes were filtered during codon optimisation.
The said sequence was cloned into the pREV21 plasmid vector flanked by the GAP promoter (SEQ ID No. 17) and the AOX1 terminator (SEQ ID No. 24). The vector map is depicted in Figure 20 (a). The vector was then transformed into E. coli cells. Transformed clones were selected, the plasmid was isolated from the transformed E. coli clones and confirmed by PCR, restriction analysis and sequencing. Upon restriction with a pair of enzymes, the recombinant plasmid pREV21-wtBRZ resulted in desired size DNA fragments (Figure 2).
Example 2: Transformation of the cells of K. phaffii with the vectors for the expression of Brazzein by electroporation
The plasmid pREV21-wtBRZ was linearised with restriction enzyme AvrII and K. phaffii was transformed with the said plasmid by electroporation. The pREV21-wtBRZ/K. phaffii integrants were selected and screened for the presence of integrated DNA. For screening of the pREV21-wtBRZ/K. phaffii integrants, genomic DNA (gDNA) was isolated and used as template in PCR with a primer pair. The forward and reverse primers annealed to the vector backbone at upstream and downstream positions with respect to wtBRZ gene, respectively. True integrants resulted in amplification of ~500 bp size DNA in PCR encompassing the wtBRZ gene (Figure 3).
The pREV21-wtBRZ/K. phaffii integrants were further subjected to growth for expression of wtBRZ protein. The cell free culture broth was analyzed for the presence of wtBRZ protein by SDS-PAGE analysis. Almost all the K. phaffi integrants tested resulted in expression of wtBRZ protein (Figure 4). One such K. phaffi cell line was further taken up for the large-scale production of wt Brazzein protein by fermentation.
Example 3: Fermentation Process
Preparation of pre-seed and seed inoculum -
The pre-seed was generated by inoculating about 1.5 ml of the glycerol stock in about 25 mL of sterile BMGY medium and growing for about 24 h-36 h at about 28±2°C with agitation of about 150-200 rpm. Further, the seed flask was inoculated with the inoculum grown in pre-seed flask and the seed culture was grown for about 24-30 hrs at about 28±2°C with agitation of about 150-200 rpm.
The final seed was generated through fermentation of about 30L broth. After inoculation, the seed fermenter was run for about 16-20 hrs at about 28±2°C temperature. The agitation and aeration were controlled based on the final dissolved oxygen (DO) concentration where any drop below 50 in DO was compensated with an increase in agitation and aeration maintained at about 150 rpm-400 rpm and about 0.2-2vvm, respectively. The pH was maintained at about 5±2.

Growth phase -
Basal salt medium was prepared and sterilized in-situ in the fermenter. The growth phase was started by inoculating basal salt medium in a 2.5kL fermenter with 5% seed culture volume and allowing fermentation to proceed for about 24 hours. The dissolved oxygen (DO) levels were continuously monitored and not allowed to drop below about 40%. The aeration and agitation were adjusted based on the DO levels where once the DO dropped below 50 the aeration and agitation were maintained at about 0.2vvm-2.2vvm and about 70-150 rpm, respectively. The overall pH and temperature in the fermenter were maintained at about 5±2 and about 30±2°C, respectively. The expected optical density OD600 at this phase of growth was about 70 to 100.
Once a DO spike was observed indicating the depletion of carbon source, glycerol feeding was initiated and continued till the OD600 reached about 180-220. The glycerol addition phase was then slowly and gradually terminated while maintaining a temperature of about 25±2°C.
Feeding Phase -
Termination of glycerol feeding was accompanied with simultaneous initiation of dextrose feeding. The dextrose feeding was done based on the DO levels, wherein at the start of dextrose feeding, OD was about 180-220. Overall, the DO was maintained at about 40% and dextrose feed was adjusted, accordingly. The temperature and pH were maintained at about 25±2°C and about 4.5±2, respectively.
The expression of Brazzein was monitored periodically by analysing culture supernatant on an SDS-PAGE. The overall fermentation was continued for about 80-130 hours. The fermentation broth sampled at different time intervals during fermentation were analyzed by SDS-PAGE. The amount of expressed wtBRZ protein was found to be increasing with time (Figure 5).
The total amount of recombinant Brazzein protein in the culture broth was estimated by standard protein estimation methods. Multiple fermentation batches were processed for expressing Brazzein. On an average, across different batches, the concentration of expressed Brazzein protein was found to be about 5-15 g/L.

Example 4: Harvesting and spray or freeze drying
Harvesting of the Brazzein protein was done by continuous centrifugation of the culture broth at about 8000 rpm. Clear supernatant obtained after the centrifugation was subjected to microfiltration using a spiral wound TFF membrane. The filtrate was further subjected to ultrafiltration and diafiltration and sufficiently concentrated to reach the desired amounts. The protein thus obtained was collected and dialyzed against water for removing other media components.
The obtained solution of Brazzein protein in water was finally spray dried to a fine powder with the set conditions of inlet temperature & outlet temperature and air pressure. Alternatively, the protein was freeze dried using a lyophilizer. The final product was collected in the powder form.
The final powder was stored at room temperature. The powder was analysed for overall purity and net protein content by HPLC analysis. The wtBRZ powder was dissolved in water and subjected to SDS-PAGE (Figure 6) and HPLC analysis. A single peak corresponding to wtBRZ and not any other protein was obtained in HPLC analysis (Figure 7). In the final powder, the purity of the recombinant Brazzein protein on dry weight basis was observed to be greater than 97%.
Example 5: Use of different signal peptides for expression of wild type Brazzein gene (wtBRZ) under control of GAP promoter
The wild type Brazzein gene (wtBRZ) was cloned in-frame with different signal peptides in respective vector backbones for expression under GAP promoter (SEQ ID No. 17). Different signal peptides carried by the recombinant plasmids were (a): AA (SEQ ID No. 8) in a pREV44 vector backbone; (b): GA (SEQ ID No. 9) in a pREV45 vector backbone; (c): IN (SEQ ID No. 10) in a pREV46 vector backbone; (d): IV (SEQ ID No. 11) in a pREV47 vector backbone; (e): KP (SEQ ID No. 12) in a pREV48 vector backbone; (f): LZ (SEQ ID No. 13) in a pREV49 vector backbone. The vector maps are depicted in Figure 20 (b-g). The recombinant plasmid for each of these expression vectors was isolated from corresponding transformed E. coli clones (prepared as per the protocol in Example 2) and confirmed by PCR, restriction analysis and sequencing. Upon restriction with suitable enzymes (BglII/XhoI, BspHI/SacII, BglII/BamHI BspHI/BamHI, BglII/XhoI and BspHI/SacII for plasmids (a) to (f), respectively as mentioned above) the recombinant plasmids resulted in desired size DNA fragments (boxed in the different panels of Figure 8).
Example 6: Use of different signal peptides for expression of wild type Brazzein gene (wtBRZ) under control of AOX1 promoter
The wild type Brazzein gene was cloned in-frame with different signal peptides for expression under AOX1 promoter. Different signal peptides carried by the recombinant plasmids were (a): FAK in pREV11 vector backbone; (b): AA (SEQ ID No. 8) in pREV33 vector backbone; (c): GA (SEQ ID No. 9) in pREV34 vector backbone; (d): IV (SEQ ID No. 11) in pREV36 vector backbone; (e): LZ (SEQ ID No. 13) in pREV38 vector backbone; (f): KP (SEQ ID No. 12) in pREV54 vector backbone. The vector maps are depicted in Figure 20 (h-m). Recombinant plasmid for each of these expression vectors was isolated from corresponding transformed E. coli clones and confirmed by PCR, restriction analysis and sequencing. Upon restriction with mentioned enzymes (XhoI/SacII, BstBI/SacII, BstBI/SacII, BglII/XhoI, BstBI/SacII and XhoI/SacII for plasmids (a) to (f), respectively as mentioned above) the recombinant plasmids resulted in desired size DNA fragments (boxed in the different panels of Figure 9), thus confirming the cloning of gene encoding for the protein of interest i.e. wild type Brazzein as facilitated by the different recombinant plasmids.
Example 7: Preparation of pREV11-wtBRZ/K. phaffi integrant cell line
Plasmid pREV11-wtBRZ was prepared and linearised with restriction enzyme SacI and K. phaffii was transformed with the said plasmid by electroporation. The pREV11-wtBRZ/K. phaffii integrants were subjected to growth for expression of wt Brazzein protein. The cell free culture broth was analyzed for the presence of wt Brazzein protein by SDS-PAGE analysis. All most all the K. phaffi integrants tested resulted in expression of wt Brazzein protein (Figure 10). One such K. phaffi cell line was further taken up for the large-scale production of wt Brazzein protein by fermentation.
The selected pREV11-wtBRZ/K. phaffi integrant cell line was subjected to large scale fermentation. The fermentation broth sampled at different time intervals during fermentation were analyzed by SDS-PAGE. The amount of expressed wt Brazzein protein was found to be increasing with time (Figure 11).
The expressed wt Brazzein protein was spray dried to a fine powder. The wt Brazzein powder was dissolved in water and analyzed through HPLC. A single peak corresponding to wt Brazzein and not any other protein was obtained in HPLC analysis. This confirmed the purity of the spray dried wt Brazzein powder (Figure 12).
Example 8: Use of FAK signal peptide for expression of wild type Brazzein gene (wtBRZ) under control of GCw14 promoter, mutants and variants thereof
The wild type Brazzein gene (encoding SEQ ID No. 1) was cloned in-frame with FAK signal peptide for expression under promoters: (a) wild type GCw14 (SEQ ID No. 18); (b) mutated GCw14 (SEQ ID No. 18). The vector maps are depicted in Figure 20 (n, o). Recombinant plasmid for each of these expression vectors was isolated from corresponding transformed E. coli clones and confirmed by PCR, restriction analysis and sequencing. Upon restriction with mentioned enzymes the recombinant plasmids resulted in desired size DNA fragments (boxed in the left and right panels of Figure 13(a), 13(b)), thus confirming the cloning of gene encoding for the protein of interest i.e. wild type Brazzein under the control of wild type GCw14 and mutated GCw14 promoters.
In a subsequent experiment, the wild type Brazzein gene (encoding SEQ ID No. 1) was cloned in-frame with FAK signal peptide for expression under hybrid GAP::mGCw14 promoter (SEQ ID No. 20). The recombinant expression vector was isolated from transformed E. coli clones and confirmed by PCR, restriction analysis and sequencing. Upon restriction with BglII/XhoI the recombinant plasmid resulted in desired size DNA fragments (boxed in the Figure 13(c)), thus confirming the production of the protein of interest i.e. wild type Brazzein under the control of different promoters.
Example 9: Use of pREV29-wtBRZ/K. phaffii integrants for expression of wild type Brazzein gene (wtBRZ)
pREV29-wtBRZ/K. phaffii integrants, wherein the plasmid contained wild type Brazzein gene under the control of GCw14 promoter (SEQ ID No. 18), were prepared and subjected to growth for expression of wt Brazzein protein. The cell free culture broth was analyzed for the presence of wt Brazzein protein by SDS-PAGE analysis. Most of the K. phaffi integrants tested resulted in expression of wt Brazzein protein (Figure 14).
Example 10: Expression of mutated Brazzein protein under control of AOX1 promoter
The gene for mutated Brazzein proteins were cloned in-frame with FAK signal peptide for expression under AOX1 promoter (SEQ ID No. 22). Different mutated Brazzein protein carried by the recombinant plasmids (pREV11) were those represented by (a): SEQ ID No. 2; (b): SEQ ID No. 3; (c): SEQ ID No. 4; and (d): SEQ ID No. 5. The vector maps are depicted in Figure 20 (p-s). Recombinant plasmid for each of these expression vectors was isolated from corresponding transformed E. coli clones and confirmed by PCR, restriction analysis and sequencing. Upon restriction with XhoI/SacII the recombinant plasmids resulted in desired size DNA fragments (boxed in yellow in Figure 15), thus confirming the cloning of gene encoding mutated Brazzein proteins under the control of AOX1 promoter.
For the purposes of further experimentation, pREV11-SEQ ID No. 2/K. phaffii integrants were prepared and subjected to growth and induced for expression of the mutated Brazzein protein (represented by SEQ ID No. 2). The cell free culture broth was analysed for the presence of mutated Brazzein protein by SDS-PAGE analysis. All most all the K. phaffi integrants tested resulted in expression of mutated Brazzein protein (Figure 16). One such K. phaffi cell line was further taken up for the large-scale production of mutated Brazzein protein by fermentation.
The selected pREV11- SEQ ID No. 2/K. phaffi integrant cell line was subjected to large scale fermentation. The fermentation broth sampled at different time intervals during fermentation were analyzed by SDS-PAGE. The amount of expressed mutated Brazzein protein was found to be increasing with time (Figure 17).
The expressed SEQ ID No. 2 mutated Brazzein protein was spray dried to a fine powder. The mutated Brazzein powder (SEQ ID No. 2) was dissolved in water and analysed through HPLC. A single peak corresponding to the mutated Brazzein and not any other protein was obtained in HPLC analysis, thus confirming the purity of the spray dried mutated Brazzein powder (represented by SEQ ID No. 2) (Figure 18).
Example 11: Expression of mutated Brazzein protein under control of GAP promoter
The gene encoding for mutated Brazzein protein (SEQ ID No. 2) was cloned in-frame with FAK signal peptide for expression under GAP promoter (SEQ ID No. 17). The recombinant expression vector (pREV21) was isolated from transformed E. coli clones and confirmed by PCR, restriction analysis and sequencing. Upon restriction with XhoI/SacII the recombinant plasmid resulted in desired size DNA fragments (boxed in the gel depicted in Figure 19), thus confirming the cloning of gene encoding for mutated Brazzien protein under the control of GAP promoter.
The subject matter of embodiments of the present invention is described here with specificity to meet statutory requirements, but this description is not necessarily intended to limit the scope of the disclosure. The disclosed subject matter may be embodied in other ways, may include different elements or steps, and may be used in conjunction with other existing or future technologies. This description should not be interpreted as implying any particular order or arrangement among or between various steps or elements except when the order of individual steps or arrangement of elements is explicitly described. ,CLAIMS:1. An expression cassette comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a constitutive promoter or an inducible promoter, and a terminator; wherein the constitutive promoter is selected from a group comprising GAP, TEF1, and GCW14; and wherein the inducible promoter is selected from a group comprising FLD1, LRA3, AOX1, DAS and ADH3.
2. The expression cassette as claimed in claim 1, wherein the nucleotide sequence is selected from a group comprising nucleotide sequences encoding SEQ ID Nos. 2-5.
3. The expression cassette as claimed in claim 1, wherein when the nucleotide sequence encodes SEQ ID No. 1, it is under the control of a constitutive promoter, and a terminator.
4. The expression cassette as claimed in claim 2, wherein when the nucleotide sequence encodes any of SEQ ID Nos. 2-5, it is under the control of a constitutive promoter or an inducible promoter, and a terminator.
5. The expression cassette as claimed in any of claims 1-4, wherein the terminator is selected from a group comprising alcohol oxidase 1 (Pp_AOX1t), Pp_GAPDHt, Pp_GAP1t and Sc_RPL3t.
6. The expression cassette as claimed in any of claims 1-5, wherein the expression cassette further encodes a secretion signal sequence selected from a group comprising FAK - Alpha-factor, AT – Alpha-factor_T, AA – Alpha-amylase, GA – Glucoamylase, IN – Inulinase, IV – Invertase, KP – Killer protein, LZ – Lysozyme, SA – Serum albumin. EG- Major exo-1,3-beta-glucanase and FAKS-Alpha-factor full.
7. A vector comprising the expression cassette as claimed in any of claims 1-6.
8. The vector as claimed in claim 7, wherein the vector is selected from a group comprising is selected from a group comprising pREV11, pREV21, pREV45, pREV46, pREV47, pREV48, pREV49, pREV44, pREV33, pREV34, pREV36, pREV38, pREV54, pREV29, pREV42 and pREV23.
9. The vector as claimed in any of claims 7 or 8, wherein the vector comprises a single copy of the expression cassette.
10. The vector as claimed in any of claims 7-9, wherein the vector further comprises a selectable marker.
11. A recombinant yeast expression system comprising the expression cassette as claimed in any of claims 1-7 or the vector as claimed in any of claims 7-10.
12. The recombinant protein expression system as claimed in claim 11, wherein the recombinant protein expression system comprises a single copy of the expression cassette.
13. The recombinant protein expression system as claimed in claim 11, wherein the recombinant protein expression system is a yeast or bacterial expression system.
14. The recombinant protein expression system as claimed in any of claims 11-13, wherein the yeast expression system is selected from a group comprising K. phaffii, K. lactis and S. cerevisea; and wherein the bacterial expression system is E.coli.
15. The recombinant protein expression system as claimed in any of claims 11-14, wherein the expression cassette or the vector are integrated at an integration locus selected from a group comprising Alcohol oxidase 1, Formaldehyde dehydrogenase L-Rhamnonate dehydratase, Dihydroxyacetone phosphate, Alcohol dehydrogenase, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Glycosylphosphatidylinositol and Translation elongation factor-1 alpha.
16. The recombinant protein expression system as claimed in any of claims 11-13, wherein the recombinant protein expression system is a K. phaffii cell comprising the expression cassette or the vector comprising a nucleotide sequence encoding SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto under the control of a GAP promoter, a terminator and optionally, an alpha secretion signal sequence, wherein the expression cassette is integrated into the K. phaffii cell at the GAPDH locus as a single copy.
17. A method for producing Brazzein or mutants thereof, said method comprising growing the recombinant protein expression system as claimed in any of claims 11-16 in a fermentation medium under suitable conditions.
18. The method as claimed in claim 17, wherein the growing of the recombinant protein expression system comprises a growth phase and a feeding phase; wherein the growth phase comprises maintaining one or more of DO of at least about 40%, temperature of about 28°C to about 32°C, pH of about 3 to about 7, aeration of about 0.2vvm to about 2.2vvm and/or agitation of about 70rpm to about 150rpm; and wherein the feeding phase comprises maintaining one or more of DO of at least about 40%, temperature of about 23°C to about 27°C, pH of about 2.5 to about 6.5, aeration of about 0.2vvm to about 2.2vvm and/or agitation of about 70rpm to about 150rpm.
19. The method as claimed in claim 18, wherein the growth phase comprises polyol feeding to compensate for depletion of carbon source and the feeding phase comprises replacement of polyol feeding with dextrose feeding.
20. The method as claimed in any of claims 17-19, wherein the growth or fermentation is performed in fed-batch mode and/or wherein the growth or fermentation is performed under aerobic conditions.
21. The method as claimed in any of claims 17-20, wherein the fermentation medium is a basal salt medium.
22. The method as claimed in any of claims 20-21, wherein the culture is maintained and fed until a cell density corresponding to a wet (packed) cell volume that is about 35% to about 45% of the total fermentation volume is achieved.
23. The method as claimed in any of claims 20-22, further comprising isolation of the Brazzein by centrifugation of the fermentation medium followed by one or more of microfiltration, ultrafiltration and diafiltration.
24. The method as claimed in claim 23, further comprising spray drying or freeze drying the isolated Brazzein.
25. The method as claimed in claim 24, wherein the spray drying is performed at an inlet temperature of about 115°C to about 125°C, and outlet temperature of about 76°C to about 78°C.
26. The method as claimed in any of claims 17-25, wherein the obtained Brazzein or mutant(s) thereof has a purity of at least about 80%.
27. A Brazzein mutant represented by sequence(s) selected from a group comprising SEQ ID Nos. 2-5.
28. A composition comprising Brazzein and/or its mutants having sequence represented by SEQ ID No. 1 or a sequence bearing at least about 70% identity thereto, in combination with one or more sweeteners and/or bulking agents; wherein the sweeteners are selected from a group comprising low calorie sweeteners and non-calorific sweeteners.