DESC:TECHNICAL FIELD OF THE INVENTION:
The present invention relates to method for improving peptides or proteins production using yeast as the host.

The present invention relates to host genome modification of a yeast for enhanced redox potential (maintenance of NADH/NAD ratio) and increased ATP availability by incorporating the gene corresponding to NADH oxidase and ADP Cyclase respectively for higher expression of homologous or heterologous peptide or protein production.

Further, the present invention also relates to a yeast strain improvement making it a protease deficient host, so that expressed homologous or heterologous proteins do not get degraded by the proteases.

The present invention also relates to insertion of multicopy of the desired gene in Yeast genome.

The present invention also relates to the construct containing the UNA sequences (transcriptional enhancers) to increase protein production.

The present invention also relates to the construct containing the UNB sequences (translational enhancers) to increase protein production.

Further, these translational enhancers increase the chances of enhanced expression of the target heterologous protein. They may give better access to the Kozak Scanning sequence. The Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation.

The present invention also relates to the N-glycosylation sites that may affect adversely in enhancing protein secretion and expression, thus an absence of near-

zero N-glycosylation motifs leads to better post-translational modifications, secretion, and enhanced expression.

The present invention also relates to the insertion of Alpha mating factor (AMF) signal sequence for increased soluble expression of the target protein.

The present invention also relates to media, feed, growth conditions and high cell cell density fermentation process for production of peptides or proteins

BACKGROUND AND PRIOR ART OF THE INVENTION:
Different expression systems are used for the production of recombinant proteins such as bacteria, yeasts, mammals, plants, and insects. Yeast expression systems Saccharomyces cerevisiae, Kluyveromyces and Pichia are more popular. The Pichia expression system is one of the most popular and standard tools for the production of recombinant proteins.

Pichia is a species of methylotrophic yeast. It was found in the 1960s, with its feature of using methanol as a source of carbon and energy. After years of study, Yeast was widely used in biochemical research and biotech industries. Pichia is a cost-effective eukaryotic protein expression system for both secretion and intracellular expression. It is ideally suitable for the large-scale production of recombinant eukaryotic proteins.

Pichia can able to grow on a simple, inexpensive medium, with a high growth rate. It can grow either in shake flasks or a fermenter, which makes it suitable for both small- and large-scale production.

Pichia has two alcohol oxidase genes, Aox1 and Aox2, which include strongly inducible promoters. These two genes allow Pichia to use methanol as a carbon and energy source. The AOX promoters are induced by methanol and repressed by glucose. Usually, the gene for the desired protein is introduced under the control of the Aox1 promoter, which means that protein production can be induced by the addition of methanol to a medium. After several types of researches, scientists found that the promotor derived from the AOX1 gene in P. pastoris is extremely suitable to control the expression of foreign genes, which had been transformed into the P. pastoris genome, producing heterologous proteins.
With a key trait, P. pastoris can grow with extremely high cell density on the culture. This feature is compatible with heterologous protein expression, giving higher yields of production.

The technology required for genetic manipulation of P. pastoris is similar to that of Saccharomyces cerevisiae, which is one of the most well-studied yeast model organisms. As a result, the experiment protocol and materials are easy to build for P. pastoris.

The presence of multi-copy of the heterologous gene increase the chances of production of multiple copies of mRNAs which may produce much enhanced heterologous protein production.

However, if the multiple copies of mRNA that’s been produced by multiple gene copies are not being able to get translated due to limitations of tRNAs, amino acids (building blocks of proteins), folding machinery (chaperons) then the potential chances of all the copies of multiple mRNAs being converted to proteins are not possible.

The presence of multi copies of the gene to be expressed generally tends to increase the expression level of the target protein. Technically more copy number is possible as the paper reported up to 12 copies. The selection marker used is Zeocin from 0.1 to 2 mg/ml (single copy to multiple copies). Markers like hygromycin and His-/+ supplementations may also be used for checking the insertion of copies.

Researchers have used panARS-(A 452-nt fragment, corresponding to the panARS sequence) based episomal vector pSEC-SUMO (Lifesensors, USA) was used as a

backbone vector to generate pARS vector for P. pastoris for transformation in GS115 host. Up to 18 copies of genes have been reported in the article.

The strategy of enhanced protein production in Pichia should be cost-effective and growth medium and the expression vector should be chosen such that the use of Histidine (expensive amino acid) as a selection marker will not be used for ascertaining insertion of target gene in the host Pichia.

Patent No WO2004024862A3 covers novel prepro-insulin polypeptides. The polypeptides consist of an N-terminal region, derived from N-terminal regions of secretory proteins, and a downstream insulin polypeptide region. The N-terminal region directs the polypeptides efficiently into the secretory pathway of yeasts. Modifications at the N-terminal region, just adjacent to the insulin polypeptide region, further increase the efficiency of secretion and improve the final yield of secreted insulin. The patent also discloses expression systems for the expression of said polypeptides under the regulation of yeast derived alcohol inducible promoters. Thus a combination of such promoters and precursors with the said N-terminal regions appear to function as very high-yielding expression systems in yeasts.

US20150361438A1 describes the method and system for expression systems, based on ade1 and ade2 auxotrophic strains of yeast and fungi, including P. pastoris are disclosed. The expression systems are useful for increased cellular productivity of transformed cell lines and for the production of recombinant glycoproteins at an industrial scale. The method is based on constructing slower-growing ade2 auxotrophic strains of the lower eukaryote cells and using integration vectors that are capable of integrating into the genome of the ade2 auxotrophic strain and which comprises nucleic acids encoding an ADE2 marker gene or open reading frame (ORF) operably linked to a promoter and a recombinant protein, wherein the integration vector integrates into the genome of the ade2 auxotrophic strain, the ADE2 renders the auxotrophic strain prototrophic for adenine, and the recombinant protein is expressed.

Gidijala et al. (Microb Cell Fact (2018) 17:112) describes the impact and necessity of incorporating translation enhancers like UNA1, UNA2, UNB, collectively termed as UNall. Transcription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements.

In addition to transcription and translation enhancers such as UNall, there are short nucleotide sequences whose presence also enhanced multifold expression of the protein, where in the short stretch nucleotide acts in gene level and not in protein level.

OBJECT OF THE INVENTION:

It is an object of the present invention to provide a method for improving peptides or proteins production using yeast as the host system.

It is a further object of the present invention to provide a yeast strain improvement making it protease deficient, co-expression of NADH oxidase and ADP cyclase, in order to maintain the NADH/NAD ratio and ATP generation in the host cells.

It is another object of the present invention to provide an enhanced transcription and translation in a yeast expression system by inserting relevant linkers to its genome.

SUMMARY OF THE INVENTION:
In an aspect, the present invention relates to a method for improving peptides or proteins production using yeast as the host

In another aspect, the present invention relates to a yeast strain improvement making it protease deficient, co-expression of NADH oxidase and ADP cyclase, in order to maintain the NADH/NAD ratio and ATP generation in the host cells.

Accordingly, the present invention also relates to a yeast strain improvement making it a protease deficient host, so that expressed homologous or heterologous proteins do not get degraded by the proteases.

In another aspect, the invention provides host genome modification of yeast for enhanced redox potential (maintenance of NADH/NAD ratio) and increased ATP availability by incorporating the gene corresponding to NADH oxidase and ADP Cyclase respectively for higher expression of homologous or heterologous peptide or protein production.

In one aspect, the present invention relates to the insertion of multicopy of the desired gene into a yeast genome.

In yet another aspect, the present invention relates to enhanced transcription and translation by inserting the relevant linkers to the genome.

In another aspect, the present invention relates to the construct containing the UNA sequences (transcriptional enhancers) to increase protein production.

In yet another aspect, the present invention also relates to the construct containing the UNB sequences (translational enhancers) to increase protein production.

In one aspect of the present invention, these transcriptional enhancers increase the chances of enhanced transcription leading to more numbers of mRNA of the target or homologous or heterologous gene expression.

In another aspect of the present invention, these translational enhancers increase the chances of enhanced expression of the target heterologous protein. They may give better access to the Kozak Scanning sequence. The Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation.

Enhanced secretion and high cell density fermentation conditions also covered in the Invention.

In another embodiment, the present invention also discloses a method of genetic modification in heterologous protein expression host organism and manipulations in gene expression constructs by gene overexpression and/ or muticopy expression and/ or gene deletions, to enhance heterologous protein expression level to multifolds in yeast.

In yet another embodiment, the Yeast for protein expression are selected from the group comprising methylotrophic yeast Pichia pastoris (Khomagotella phaffi), Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Schizosaccharomyces pombe, Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Candida albicans, Candida spp ,Yarrowia spp,and Rhodutorula spp, preferably Pichia pastoris.

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

In order to achieve the objective, the present invention discloses a method for improving peptides or proteins production using yeast as the host.

In one embodiment, the present invention provides a yeast strain improvement making it protease deficient, co-expression of NADH oxidase and ADP cyclase, in order to maintain the NADH/NAD ratio and ATP generation in the host cells.

In another embodiment, the present invention relates to a yeast strain improvement making it a protease deficient host, so that expressed homologous or heterologous proteins do not get degraded by the proteases.

In a preferred embodiment, the present invention provides host genome modification of yeast for enhanced redox potential (maintenance of NADH/NAD ratio) and increased ATP availability by incorporating the gene corresponding to NADH oxidase and ADP Cyclase respectively for higher expression of homologous or heterologous peptide or protein production.

In one embodiment of the present invention, the yeast strain improvement is achieved by inserting multicopy of the desired gene into a yeast genome.

In another embodiment of the present invention, the enhanced transcription and translation is achieved by inserting relevant linkers to the yeast genome.

In some embodiments, the present invention deals with a construct containing the UNA sequences (transcriptional enhancers) to increase protein production.

In yet another embodiment, the present invention deals with a construct containing the UNB sequences (translational enhancers) which increases the protein production.

In another embodiment of the present invention, the insertion of Alpha mating factor (AMF) signal sequences helps to increase the soluble expression of the target proteins.

In yet another embodiment of the present invention, these transcriptional enhancers chances the transcription leading to more numbers of mRNA of the target or homologous or heterologous gene expression.

In another embodiment of the present invention, these translational enhancers chances the expression of the target heterologous protein. They may give better access to the Kozak Scanning sequence. The Kozak consensus sequence is a nucleic acid motif that functions as the protein translation initiation.

In another embodiment of the present invention, the N-glycosylation sites may affect adversely in enhancing protein secretion and expression, thus an absence of near-zero N-glycosylation motifs leads to better post-translational modifications, secretion, and enhanced expression.

Accordingly to the present invention, the Yeast for protein expression are selected from the group comprising but not limited to methylotrophic yeast Pichia pastoris (Khomagotella phaffi), Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Schizosaccharomyces pombe, Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Candida albicans,Candida spp, Yarrowia spp,and Rhodutorula spp.

In a preferred embodiment of the present invention, the enhanced secretion and high proteins are achieved using cell density fermentation conditions.

In another embodiment, the present invention also discloses media, feed, growth conditions, and a high cell-cell density fermentation process for the production of peptides or proteins.

In another embodiment, the present invention also discloses a method of genetic modification in heterologous protein expression host organism and manipulations in gene expression constructs by gene overexpression and/ or muticopy expression and/ or gene deletions, to enhance heterologous protein expression level to multifolds in yeast.

In another embodiment, the expression host modifications for supporting enhanced protein expression includes but not limited to the deletion host secretory Protease and/ or increasing the metabolic rate/ regeneration of energy carriers.

In another embodiment, the said secretory proteases include the proteases which are secreted extracellular and act on heterologous protein and degrade such as and not limited to the proteases coded by pep, prb, yps, etc., meant for degradation of secretory protein.

In yet another embodiment, the method comprises deleting the genes coding for external proteases selected from pep, prb, yps, to inhibit the degradation of secretory protein, thereby increasing protein yield.

In another embodiment, the said increment in metabolic rate and/ or regeneration of energy carriers include enhanced expression and increased regeneration of metabolites such as and not limited to ATP, NADH, Pyruvate and Acetyl CoA.

In another embodiment, the host with modification in increased metabolism is selected from high expression host for any type of heterologous and/ or homologous protein production for in-vitro and in-vivo applications.

In another embodiment, the ATP expression is enhanced by integration of ATP generation enzyme such as and not limited to ADP kinase (Adk), ATP synthase, etc.

In another embodiment, the NADH expression and regeneration is enhanced by integration of enzymes such as and not limited to NADH oxidase (NoxE), Dehydrogenases like ADH linked with NADH generation dehydrogenase such as GAPDH, etc.

In another embodiment, the enhanced regeneration of pyruvate and Acetyl-CoA are achieved through expression of enzymes such as and not limited to mitochondrial pyruvate carrier (MPC), Malic acid enzyme (MalE), pyruvate kinase, and the like.

In another embodiment, the said modifications in the expression plasmid include target gene expression under strong promoter AOX1 as single gene and/ or multicopy gene.

In another embodiment, the said modification in the expression plasmid also includes the addition of translational and transcriptional enhancer sequences in upstream region of gene, such as and not limited to UNA1, UNA2, UNB, Exin 21mer, etc.

In another embodiment, the combined effect of multi gene copy expression, under strong promoter equipped with transcriptional and translational enhancers will yield multifold expression of heterologous protein.

In another embodiment, the heterologous protein expressed from the modified yeast host include but not limited to milk protein such as casein, lactalbumin, lactoferrin; therapeutic proteins such as angiotensin, Hematopoietin, etc; sweet protein and peptides such as Brazzein, curculin, mabinlin, miraculin, monellin, pentadin, thaumatin, etc.; enzymes such as cellulase, amylase, lipase, pectinase, xylanase, etc.

EXAMPLES

Example 1: Enhanced level of Milk protein Casein expression from Modified Pichia expression strain:

Heterologous expression of protein is performed using Pichia or kluveromyces, by expression under the control high active promoter of Alcohol oxidase (AOX) gene. For increased level of protein expression, the overall metabolism of Pichia should be faster which requires lot of metabolic energy in terms of NADH and ATP. Inorder to increase the level of expression in Pichia, additional gene modification were performed in the host organism to increase regeneration of ATP, NADH and pyruvate, to increase the protein turnover.

Gene construct for Expression of Alpha S1-casein was integrated into a yeast genome. The gene corresponding to NADH oxidase and ADP Cyclase were co-expressed along with Alpha S1-casein n the same expression cassette under a strong promoter so that the expressed protein can maintain the NADH/NAD ratio and enhanced ATP production.

Finally, the combination effect of NADH, ATP, Pyruvate regeneration and transcriptional and translational enhancers yielded 8 fold increase in expression, resulting in 80 g/L Casein yield (Table 1).

Table 1
Expression Host Casein yield (g/L) Yield increment fold
Wild type 10 1
Modified host 80 8

Example 2: Enhanced level sweet peptide Brazzein expression from Modified Pichia expression strain:

Gene construct for Expression of sweet peptide, Brazzein was integrated into a yeast genome. The gene corresponding to NADH oxidase and ADP Cyclase were co-expressed along with Brazzein in the same expression cassette under a strong promoter so that the expressed protein can maintain the NADH/NAD ratio and enhanced ATP production.

Finally, the combination effect of NADH, ATP, Pyruvate regeneration and transcriptional and translational enhancers yielded 12 fold increase in expression, resulting in 1.2 g/L Brazzein yield (Table 2).

Table 2
Expression Host Brazzein yield (g/L) Yield increment fold
Wild type 0.1 1
Modified host 1.2 12
,CLAIMS:1. A method of genetic modification in heterologous protein expression host organism and manipulations in gene expression constructs by gene overexpression and/ or muticopy expression and/ or gene deletions, to enhance heterologous protein expression level to multifolds in yeast.
2. The method as claimed in Claim 1, wherein the said Yeast for protein expression are selected from the group comprising methylotrophic yeast Pichia pastoris (Khomagotella phaffi), Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Schizosaccharomyces pombe, Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Candida albicans, Candida spp ,Yarrowia spp,and Rhodutorula spp, and in particular Pichia pastoris.
3. The method as claimed in Claim 1, wherein the expression host modifications for supporting enhanced protein expression includes but not limited to the deletion host secretory Protease and/ or increasing the metabolic rate/ regeneration of energy carriers.
4. The method as claimed in Claim 3, wherein the said secretory proteases include the proteases which are secreted extracellular and act on heterologous protein and degrade such as and not limited to the proteases coded by pep, prb, yps, etc., meant for degradation of secretory protein.
5. The method as claimed in Claim 4, wherein the method comprises deleting the genes coding for externalproteases selected from pep, prb, yps, to inhibit the degradation of secretory protein, thereby increasing protein yield
6. The method as claimed in Claim 3, wherein the said increment in metabolic rate and/ or regeneration of energy carriers include enhanced expression and increased regeneration of metabolites such as and not limited to ATP, NADH, Pyruvate and Acetyl CoA.
7. The method as claimed in Claim 1 and 3, wherein said host with modification in increased metabolism is selected from high expression host for any type of heterologous and/ or homologous protein production for in-vitro and in-vivo applications.
8. The method as claimed in Claim 6, wherein the ATP expression is enhanced by integration of ATP generation enzyme such as and not limited to ADP kinase (Adk), ATP synthase, etc.
9. The method as claimed in Claim 6, wherein NADH expression and regeneration is enhanced by integration of enzymes such as and not limited to NADH oxidase (NoxE), Dehydrogenases like ADH linked with NADH generation dehydrogenase such as GAPDH, etc.
10. The method as claimed in Claim 6, wherein enhanced regeneration of pyruvate and Acetyl-CoA are achieved through expression of enzymes such as and not limited to mitochondrial pyruvate carrier (MPC), Malic acid enzyme (MalE), pyruvate kinase, and the like.
11. The method as claimed in Claim 1, wherein the said modifications in the expression plasmid include target gene expression under strong promoter AOX1 as single gene and/ or multicopy gene.
12. The method as claimed in Claim 1, wherein the said modification in the expression plasmid also includes the addition of translational and transcriptional enhancer sequences in upstream region of gene, such as and not limited to UNA1, UNA2, UNB, Exin 21mer, etc.
13. The method as claimed in Claim 11 and 12, wherein the combined effect of multi gene copy expression, under strong promoter equipped with transcriptional and translational enhancers will yield multifold expression of heterologous protein.
14. The method as claimed in Claim 1, wherein the heterologous protein expressed from the modified yeast host include but not limited to milk protein such as casein, lactalbumin, lactoferrin; therapeutic proteins such as angiotensin, Hematopoietin, etc; sweet protein and peptides such as Brazzein, curculin, mabinlin, miraculin, monellin, pentadin, thaumatin, etc.; enzymes such as cellulase, amylase, lipase, pectinase, xylanase, etc.