Inducible promoter for high expression of proteins in kluyveromyces marxianus

Abstract

The present invention relates to a new high-expression vector having novel inducible promoter identified and isolated from Kluyveromyces marxianus for improved and regulated gene expression. The identified novel inducible IMTIP1 promoter having >890 fold induction in the presence of xylose as compared to that in dextrose. This high-expression vector could be used for homologous and heterologous genes expression in K. marxianus. The developed vector can be used for economic and efficient inducible expression of proteins for industrial and therapeutic applications.

Description

The present invention relates to a new high-expression vector having novel inducible promoter identified and isolated from Kluyveromyces marxianus for improved and regulated gene expression. This high-expression vector could be used for homologous and heterologous genes expression in K. marxianus. The developed vector can be used for economic and efficient inducible expression of proteins for industrial and therapeutic applications.BACKGROUND OF THE INVENTIONThere is a constant need to develop new host organism to express range of proteins required for various biotechnological or biotherapeutic applications. Kluyveromyces marxianus (K. marxianus), classified as Generally Recognized As Safe (GRAS), is one of the emerging industrially important microorganism for protein production. Some of the unique and also highly desired properties of K. marxianus are its thermo tolerance with ability to grow until 52°C, fast growth rate with doubling time of ~52 min, ability to utilize different carbon sources such as pentose as well as hexose sugar, and high secretory capacity. The combination of these unique properties make K. marxianus one of the best choices for biotechnological applications such as production of enzymes as well as alcohol fermentation (Nonklang et al., 2008), (Fonseca et al., 2008) lactic acid and xylitol (Bae et al., 2017; Kim et al., 2015; Zhang et al., 2015; Zhang et al., 2014). The identification of a suitable strong promoter is critical for the protein production in large amount in an organism. Proteins, that are non-toxic, are generally expressed using a constitutive promoter however those that have significant adverse impact on organism growth are expressed from an inducible promoter. Inducible promoters are even preferred for non-toxic proteins, as a higher cell mass could be achieved by growing cells without expressing the protein of interest until a desired cells density is achieved. For expression into K. marxianus, most of the currently used promoters for heterologous protein expression are those derived from Saccharomyces cerevisiae genome such as promoter of glyceraldehyde-3-dehydrogenase (TDH3) (Nonklang et al., 2009), galactokinase (GAL1) (Almeida et al., 2003), and 3-phosphoglycerate kinase (PGK1) (Ball et al., 1999; Pecota et al., 2007). A previous study compared the protein expression using promoters derived from S. cerevisiae with the homologous native promoter derived from K. marxianus. The study shows that the promoters from S. cerevisiae are relatively weaker than those of the corresponding native promoters of K. marxianus (Yang et al., 2015) paving the need for further exploration of native constitutive and inducible promoters of K. marxianus. Some of the inducible promoters of K. marxianus origin have been attempted for protein expression like Inulinase (INU1) and GAL1 promoter, however these promoter still have the limitation of leaky expression in non-inducible condition (Bergkamp et al., 1993, Gao et al., 2015, Liu et al., 2013, Akada et al., 2014). Additionally, there is a need of more inducible promoters regulated by different inducers to meet the growing need of wide range of proteins with different biochemical and biophysical properties that could be expressed in K. marxianus.In the present invention, we analyzed available large scale transcriptomic data obtained under different growth conditions of K. marxianus (Lertwattanasakul et al., 2015) and identified a strong inducible promoter. The invention reports the identification of novel xylose inducible promoter having highest strength among any other known inducible promoters used in K. marxianus until now. The invention provides method for economic and efficient large scale production of both homologous and heterologous proteins for biotechnological and biotherapeutic purposes.OBJECTIVES OF THE INVENTIONThe primary objective of the invention is to identify a strong novel promoter for i) high level protein expression in K. marxianus, ii) that does not provide constitutive expression, iii) inducible by a low cost inducer and thus economically relevant for industrial applications, and iv) could be used at ambient growth conditions v) could be used at higher temperature of 37°C and 42°C.Another objective of the invention is to develop vectors for high level expression of homologous and heterologous proteins in K. marxianus using growth media with xylose as carbon source. Yet another objective of the invention is to develop inducible high expression promoter with low cost inducible substrate and high gene expression. Yet another objective of the invention is to develop a simpler, economical, and efficient mode of inducible protein expression in K. marxianus.SUMMARY OF THE INVENTIONAccordingly, the present invention relates to identification of inducible promoters for high expression of homologous or heterologous proteins, a large-scale analysis of K. marxianus transcriptomic data was carried out. The available transcriptomic data of K. marxianus, (Lertwattanasakul et al., 2015) grown in the presence of either dextrose or xylose as carbon source in growth media, was rigorously analyzed. The promoter of the genes that transcribed strongly in the presence of xylose and repressed in the presence of dextrose were further sub-cloned into plasmid for use in K. marxianus. The reporter gene encoding eGFP was sub-cloned under the identified promoter. Further S. cerevisiae CYC terminator DNA sequence was subcloned downstream of 3’end of eGFP gene. The plasmid encoding from 5’ to 3’, identified promoter, gene encoding eGFP and the CYC terminator was transformed into K. marxianus. Transformants were grown in liquid synthetic growth media containing dextrose or xylose as carbon source. The cells were lysed, and the lysate was immunoblotted with anti-GFP antibodies to measure the level of expression of eGFP (Figure 3). Further, another plasmid was constructed that encode from 5’ to 3’, identified promoter, gene encoding firefly luciferase, and the CYC terminator, and transformed into K. marxianus. The transformants were grown in liquid growth media containing dextrose or xylose as carbon source, and luciferase expression was examined by monitoring the activity of expressed luciferase enzyme as increase in luminescence (Figure 4). The results from both assays, the expression of eGFP and firefly luciferase activity, showed that the identified promoter is first tightly controlled inducible promoter that induces >800-fold gene expression in the presence of xylose than dextrose as a carbon source in K. marxianus. Further the expression from the identified promoter is >2 fold higher than the previously identified strong GAL1 promoter.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1. Scheme of plasmid construction and restriction sites used for cloning. The restriction sites used for cloning of promoter are SacI and XbaI. The restriction sites for cloning of terminator are XhoI and KpnI. The gene encoding desired protein is cloned using restriction site BamHI and XhoI. Figure 2. New vector (MTCC 25287; pKMDSH5) map encoding newly identified IMTIP1 promoter and CYC terminator. The promoter was subcloned between restriction enzyme sites Sac1 and Xba1. The terminator was subcloned between restriction sites XhoI and Kpn1. The gene encoding enhanced green fluorescent protein (eGFP) was subcloned between restriction sites BamHI and XhoI.Figure 3. The expression level of eGFP under control of GAL1 or IMTIP1 promoter in K. marxianus. The K. marxianus strain harboring plasmid encoding eGFP under the control of indicated promoters was grown in synthetic media containing different sugars as carbon source. For Galactose inducible promoter, either dextrose, raffinose + galactose or galactose were used as carbon source. For our newly identified promoter IMTIP1, either dextrose or xylose was used as carbon source. Overnight grown cells were subcultured in secondary synthetic growth media containing different sugars as the carbon source at 30?C. The cells were grown until O.D.600nm reaches ~ 1. Cells were harvested by centrifugation at 4000g for 10 min. The cells were lysed, and the lysate was probed on immunoblot using anti-GFP antibodies. (cat. No. MA5-15256; Thermo Fisher Scientific)Figure 4. Activity of luciferase expressed from IMTIP1 or GAL1 promoter in K. marxianus. The K. marxianus cells were transformed with plasmid encoding firefly luciferase under control GAL1 or IMTIP1 promoter. The overnight grown cells (at 30?C) in synthetic media containing dextrose (S.D.) were subcultured further into liquid synthetic media with carbon source based upon the type of promoter. For GAL1 promoter, cells were grown into synthetic media containing dextrose, raffinose + galactose or galactose whereas for IMTIP1 promoter, dextrose or xylose was added as carbon source. The cells were grown at 30°C until O.D.600nm reaches around ~ 1. The cells were collected by centrifugation, washed with distilled H2O and resuspended into synthetic media. To monitor luciferase activity, 50 µl of 1mg / ml solution of D-Luciferin (Sigma,USA) was added to 200 µl of 0.3 O.D.600nm cell suspension, and increase in luminescence was monitored in Multimode Plate Readers (TECAN Infinite M200 PRO, Switzerland).DETAILED DESCRIPTION OF THE INVENTIONThe present invention is related to the identification of strong inducible promoter that is induced by an economical and safe small molecule. The identified promoter in the present invention supports tight regulation of protein expression only under inducible molecule xylose, and not in dextrose. Thus, the identified promoter is the first promoter for such tightly controlled expression of protein in K. marxianus. For identification of strong inducible promoter sequences, a large-scale transcriptomic data from K. marxianus cells grown into liquid growth media in the presence of dextrose or xylose was extensively analyzed. The different growth conditions examined are (i) growth using dextrose as carbon source at 30°C under shaking and (ii) growth using xylose as carbon source at 30°C under shaking conditions (Lertwattanasakul et al., 2015). A total of more than 5000 gene expression was analyzed at transcriptomic level. To examine promoter strength, the eGFP was used as a reporter gene and expressed under the control of the identified inducible promoter. The eGFP abundance was examined by monitoring fluorescence as well as its expression level by immunoblotting with anti-GFP antibody. It was found that the results provided us with promoter IMTIP1, 480 bp upstream of Gene ID: 34714698 having strongest strength and inducible property as compared to the other known native inducible GAL1 promoter of K. marxianus (Figure 3)To further validate and quantitate gene expression from identified inducible promoter we use another reporter gene encoding firefly luciferase. The cellular abundance of firefly luciferase expressed under the control of IMTIP1 or GAL1 promoters was compared by measuring increase in luminescence as reporter of luciferase activity (Figure 4). More specifically, the present invention relates to a strong inducible promoter consisting of polynucleotides:The IMTIP1 promoter represented by SEQ ID NO: 1 or having an identity at least about 70% or more to SEQ ID NO: 1 and having a promoter activity in K. marxianus.A DNA sequence hybridizing with a polynucleotide complementary to the polynucleotide represented by SEQ ID NO: 1 and having a promoter activity in K. marxianus.A polynucleotide having a deletion, substitution or addition of at least one or more nucleotides in the polynucleotide represented by SEQ ID NO: 1 and having a promoter activity in K. marxianus.A hybrid vector in present invention having one or multiple DNA inserts or DNA sequence encoding a desired polypeptide or a gene under control of promoter of invention of SEQ ID 1. The vector of invention encodes features such as suitable restriction sites for cloning DNA, replication in host, a selective genetic marker and yeast replication origin.The plasmids of invention possess autonomously replicating segment (ARS) of K. marxianus origin for maintenance in the host cell post transformation. The promoter activity in a polynucleotide sequence is evaluated using a reporter assay measuring steady state level of gene product subcloned between the promoter and terminator. In the reporter assay, a reporter gene encoding a protein product is expressed under the control of the promoter with terminator sequence at its 3’ end. A reporter gene (encoding a reporter protein, for example green fluorescent protein (GFP) (Bierhuizen et al., 1997) or firefly luciferase (Himes and Shannon, 2000) is subcloned in between promoter and terminator in a hybrid plasmid. The plasmid is transformed into a yeast host, and the abundance of the expressed protein is measured using a biochemical assay or on immunoblot with appropriate antibody. In this present invention, we have invented new K. marxianus promoter IMTIP1 (SEQ ID NO: 1) and compared its strength with previously known galactose inducible GAL1 promoter (PGAL1) of K. marxianus. The promoter strength was compared by measuring the steady state level of reported protein eGFP. First, gene encoding eGFP was subcloned upstream of gene encoding S. cerevisiae CYC terminator. Further, the DNA sequence encoding promoter IMTIP1 or GAL1 was subcloned upstream of gene encoding eGFP resulting in the design of expression vector harboring nucleotide cassettes P IMTIP1-eGFP-TCYC or PGAL1-eGFP-TCYC. The designed vectors were transformed in a URA3 auxotrophic strain of K. marxianus. The transformants were selected onto solid SD agar plate without uracil. 4-5 transformants were pooled, and grown in selective synthetic media with glucose as carbon source at 30°C. The overnight grown culture was centrifuged, and cell pellet was washed three times with sterile water. The cells were diluted 100-fold into fresh growth media containing different source of carbon. For cells harboring plasmid encoding GAL1 promoter, dextrose, raffinose + galactose or galactose was used as carbon source. For cells harboring plasmid encoding IMTIP1 promoter, dextrose or xylose was used as carbon source. The culture was grown at 30°C under shaking conditions. Cells were collected at mid-log phase and washed with water. The cells were lysed and equal amount of total proteins in whole cell lysate was used for detection of eGFP expression on an immunoblot using anti- GFP antibody.Accordingly, one of embodiment of the present study is that strong tight control of identified promoter than any of the other previously known inducible K. marxianus promoters (Figure 3) Furthermore, the present invention validates and quantifies the strength of inducible promoter using in vivo luciferase assay. We replaced gene encoding eGFP with that encoding Luciferase (FLuc) from the firefly Photinus pyralis to generate pPKM316-PIMTIP1-FLuc-TCYC or pPKM316-PGAL1-FLuc-TCYC plasmid. The resulting plasmids were individually transformed into K. marxianus. The transformants were grown in liquid growth media. The overnight grown culture was centrifuged, and cell pellet was washed three times with sterile water. The cells were diluted 100-fold into fresh growth media containing varying source of carbon. For cells harboring plasmid encoding GAL1 promoter, dextrose, raffinose + galactose or galactose was used as carbon source. For cells harboring plasmid encoding IMTIP1 promoter, dextrose or xylose was used as carbon source. The culture was grown at 30°C under shaking conditions until mid-log phase. Cells were collected by centrifugation and re-suspended into 1 ml synthetic media. 50 µl of 1mg / ml D-Luciferin was added to 200µl of 0.3 O. D600 cells, and luminescence was measured in Tecan Infinite 200 PRO. Accordingly, the other embodiment of the present study is that IMTIP1 promoter identified in the present invention has higher strength than previous known inducible promoter of K. marxianus. (figure 4) EXAMPLESThe following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.Example 1Construction of K. marxianus strain with uracil auxotrophy The uracil auxotroph in K. marxianus was constructed using uracil as a selectable marker. To construct uracil auxotrophy, a DNA sequence having homology 250 base pair (bp) upstream and 250bp downstream of K. marxianus URA3 gene was designed and synthesized from GenScript USA, Inc. This cassette was transformed in K. marxianus (NBRC 1777) and desired uracil auxotroph’s were selected on the basis of their ability to grow onto solid growth media containing 5-fluoroorotic acid (5-FOA).Example 2Preparation of vector for expression of reporter geneThe integrating shuttle vector pRS306 (ATCC 77141) was used as a backbone for construction of recombinant yeast expression vector. The ARS / CEN sequence of K. marxianus strain NBRC1777 was PCR amplified and inserted into the vector pRS306 by Transfer-PCR method (Erijman et al., 2011). The resultant expression vector is referred to as pPKM316.The eGFP was used as reporter gene. First, the gene encoding eGFP was subcloned into plasmid pKM316 using restriction sites BamHI and XhoI to construct pPKM316-eGFP. The CYC terminator from S. cerevisiae was digested with restriction enzymes XhoI and KpnI, and further cloned into pPKM316-eGFP at the 3’end of gene encoding eGFP to construct pPKM316-eGFP TCYC.The DNA sequence encoding promoters such as IMTIP1 and GAL1 were PCR amplified from genomic DNA of K. marxianus strain NBRC1777. The amplified DNA sequences were digested with restriction enzymes SacI and XbaI. The digested product was ligated into pPKM316-eGFP-TCYC to construct vectors (pKMDSH5) pPKM316-PIMTIP1-eGFP-TCYC or pPKM316-PGAL1-eGFP-TCYC. To further examine the promoter strength, the luciferase gene (FLuc) from firefly Photinus pyralis was subcloned downstream of IMTIP1 or GAL1 promoter by replacing gene encoding eGFP with that of FLuc using restriction sites BamHI and XhoI to obtain pPKM316-PIMTIP1-FLuc-TCYC or pPKM316-PGAL1-FLuc-TCYC respectively.Example 3Promoter strength monitored using expression of eGFP as reporter proteinNBRC1777 cells lacking URA3 were transformed with plasmid (pKMDSH5) pPKM316-PIMTIP1-eGFP-TCYC or pPKM316-PGAL1-eGFP-TCYC. Transformants were grown overnight, and further subcultured into liquid synthetic media lacking uracil with different carbon sources. Cells with plasmid encoding GAL1 promoter were grown in the presence of either glucose, raffinose + galactose or galactose. Similarly, cells harboring plasmid encoding IMTIP1 promoter were grown in liquid synthetic media containing glucose or xylose as carbon source. Cells were grown at 30°C until mid-log phase. Cells were collected and lysed. The cellular lysate was normalized for same amount of total protein and loaded onto 12% SDS–polyacrylamide gel. The immunoblotting was carried out with antibodies against GFP (cat. No. MA5-15256; Thermo Fisher Scientific) as primary antibody, and goat anti-mouse antibody conjugated to horseradish peroxidase was used as secondary antibody (cat. no. 7076; Cell Signaling Technology, Inc.).Example 4Promoter strength evaluation using luciferase assayThe plasmid pPKM316-PIMTIP1-FLuc-TCYC or pPKM316 PGAL1-FLuc-TCYC was transformed into K. marxianus strain NBRC1777 lacking URA3. The 3-4 transformants were pooled and grown overnight into liquid growth media lacking uracil with dextrose as source of carbon. Overnight grown primary culture re-inoculated at 0.05 O.D.600nm into synthetic media with either glucose, raffinose + galactose or galactose as carbon source for GAL1 promoter, and either glucose or xylose for cells expressing FLuc from IMTIP1 promoter. Cells were a grown at 30°C until mid-log phase. Cells were harvested by centrifugation and re-suspended into 1 ml of synthetic media. The luciferase expression was monitored as a function of luciferase activity. For measuring luciferase activity, 200µl of 0.3 OD600 cells were added with 50 µl of 1mg / ml D-Luciferin (Sigma-Aldrich, USA), and the luminescence was measured in Multimode Plate Reader (TECAN Infinite M200 PRO, Switzerland). All reagents were prepared as described by the manufacturer (Sigma-Aldrich, USA).ADVANTAGES OF INVENTION1. The identified novel inducible promoter have higher protein expression in diverse yeast species. 2. The identified novel inducible promoter IMTIP1 and developed expression vector (pKMDSH5) having >2-fold higher expression then previously known inducible GAL1 promoter in K. marxianus. 3. The identified novel inducible IMTIP1 promoter having >890-fold induction in the presence of xylose as compared to that in dextrose, whereas known inducible promoter GAL1 having around 2.2-fold induction in the presence of galactose as compared to that in dextrose4. The identified novel IMTIP1 promoter is a xylose inducible promoter. Since xylose is less expensive than galactose, the overall protein production is cost effective. 5. The identified novel IMTIP1 promoter is valuable for production of toxic proteins.REFERNCES1. Akada, R., Hoshida, H., and Ide, M. (2014). High-expression promoter derived from Kluyveromyces marxianus. U.S. Patent 8,846,343.2. Almeida, C., Queiros, O., Wheals, A., Teixeira, J., Moradas, F. P. (2003) Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase. J MicrobiolMethods 55(2):433–440.3. Bae, J.H., Kim, H.J., Kim, M.J., Sung, B.H., Jeon, J.H., Kim, H.S., Jin, Y.S., Kweon, D.H., Sohn, J.H. (2017) Direct fermentation of Jerusalem artichoke tuber powder for production of L-lactic acid and D-lactic acid by metabolically engineered Kluyveromyces marxianus. J. Biotechnol. 266, 27-33.4. Ball, M.M., Raynal, A., Guerineau, M., Iborra, F. (1999) Construction of efficient centromeric, multicopy and expression vectors for the yeast Kluyveromyces marxianus using homologous elements and the promoter of a purine-cytosine-like permease. J Mol Microbio Biotechnol 1(2):347–353.5. Bergkamp, R.J., Bootsman, T.C., Toschka, H.Y., Mooren, A.T., Kox, L.,Verbakel, J.M., Geerse, R.H., Planta, R.J. (1993) Expression of an a-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus. Appl MicrobiolBiotech 40(2–3):309–317.6. Bierhuizen, M.F., Westerman, Y., Visser, T.P., Wognum, A.W., Wagemaker, G. (1997) Green fluorescent protein variants as markers of retroviral-mediated gene transfer in primary hematopoietic cells and cell lines. Biochem Biophy's ResCommun, 234, 371-375.7. Erijman, A., Dantes, A., Bernheim, R., Shifman, J.M. and Peleg, Y. (2011) Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering. J. Struct. Biol., 175, 171–177.8. Fonseca, G.G., Heinzle, E., Wittmann, C., Gombert, A.K. (2008). The yeast Kluyveromyces marxianus and its biotechnological potential. Appl. Microbiol. Biotechnol. 79: 339–345.9. Gao, J.Q., Yuan, W.J., Kong, L., Xiang, R.J., Zhong, S.J. (2015) Efficient ethanol production from inulin by two-stage aerate strategy. Biomass Bioenerg 80: 10-16.10. Gough, S.B.D., Nigam, P., Marchant, R., McHale, A.P. (1997). Production of ethanol from molasses at 45°C using alginate immobilized Kluyveromyces marxianus IMB3. Bioprocess Biosyst. Eng. 16:389–392.11. Himes, S. R., Shannon, M. F. (2000) Assays for transcriptional activity based on the luciferase reporter gene. Methods Mol Biol, 130, 165-174.12. Kim, J. S., Park, J. B., Jang, S. W., Ha, S. J. (2015). Enhanced xylitol production by mutant Kluyveromyces marxianus 36907-FMEL1 due to improved xylose reductase activity. Applied biochemistry and biotechnology, 176(7), 1975-1984.13. Lee, K.S., Kim, J.S., Heo, P. (2013). Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus. Appl. Microbiol. Biotechnol. 97(5):2029–2041.14. Lertwattanasakul, N., Kosaka, T., Hosoyama, A., Suzuki, Y., Rodrussamee, N., Matsutani, M. (2015) Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses. Biotechnol Biofuels 8:1–14.15. Liu, G.L., Chi, Z., Chi, Z.M. (2013) Molecular characterization and expression of microbial inulinase genes. Crit Rev Microbiol 39(2):152–165.16. Nonklang, S., Abdel-Banat, B.M., Cha-aim, K., Moonjai, N., Hoshida, H., Limtong, S., Yamada, M., Akada, R. (2008) High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042. Appl Environ Microbiol 74: 7514-7521.17. Nonklang, S., Ano, A., Abdel-Banat, B.M.A., Saito, Y., Hoshida, H., Akada, R. (2009) Construction of flocculent Kluyveromyces marxianus strains suitable for high-temperature ethanol fermentation. Biosci Biotech Bioch 73(5):1090–109518. Pecota, D.C., Rajgarhia, V., Da Silva, N.A. (2007) Sequential gene integration for the engineering of Kluyveromyces marxianus. J Biotech 127(3):408–416.19. Walther, T., Hensirisak, P., Agblevor, F.A. (2001). The in?uence of aeration and hemicellulosic sugars on xylitol production by Candida tropicalis. Bioresour. Technol. 76, 213–220.20. Yang, C., Hu, S., Zhu, S., Wang, D., Gao, X., Hong, J. (2015). Characterizing yeast promoters used in Kluyveromyces marxianus. 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Claims

We claim1. An inducible promoter IMTIP1 having Seq Id no. 1 for expression of homologous and heterologous proteins.

2. The promoter as claimed in claim 1 having at least 70% sequence homology to the SEQ ID NO. 1.

3. The promoter as claimed in claim 1 is cloned in an expression vector wherein the said promoter is linked to a DNA encoding reporter gene.

4. The reporter gene as claimed in claim 3 is selected from the group comprising of GFP, Luciferase.

5. The promoter as claimed in claim 1 wherein the inducer is selected from the group comprising of xylose, pentose and hexose sugar.

6. The promoter as claimed in claim 1 wherein the host cell for expression is selected from the group comprising of K. marxianus, S. cerevisiae.AbstractInducible promoter for high expression of proteins in Kluyveromyces marxianusThe present invention relates to a new high-expression vector having novel inducible promoter identified and isolated from Kluyveromyces marxianus for improved and regulated gene expression. The identified novel inducible IMTIP1 promoter having >890 fold induction in the presence of xylose as compared to that in dextrose. This high-expression vector could be used for homologous and heterologous genes expression in K. marxianus. The developed vector can be used for economic and efficient inducible expression of proteins for industrial and therapeutic applications.Claims:We claim