WO2018236294A1

WO2018236294A1 - Gene expression product for producing target proteins or bio-products from heat-tolerant yeast by methanol induction and non-induction including its process of product usage

Info

Publication number: WO2018236294A1
Application number: PCT/TH2018/000030
Authority: WO
Inventors: Sutipa TANAPONGPIPAT; Peerada PROMDONKOY; Niran Roongsawang; Piyanun Harnpicharnchai; Sriwan WONGWISANSRI; Lily Eurwilaichitr
Original assignee: National Science And Technology Development Agency
Priority date: 2017-06-23
Filing date: 2018-06-05
Publication date: 2018-12-27

Abstract

This invention is a gene expression product for producing target proteins or bio- products from heat-tolerant yeast by methanol induction and non-induction including its process of product usage. The product in this invention consists of Ogataea thermomethanolica yeast cells heat-tolerant species and proper plasmid vectors. For plasmid vector using methanol induction, the important part for gene expression is methanol inducible promoter from O. thermomethanolica, which is AOX promoter. While, the important part for gene expression in non-induction plasmid vector is GAP promoter which is a non-inducible promoter isolated from O. thermomethanolica. Both promoters can work under high temperature which is appropriate to apply with protein production in larger scale. Moreover, using O. thermomethanolica heat- tolerant species as host cell yeast can reduce the cost of production because it does not need an extra cooling system. In addition, there are other advantages, for example, the recombinant proteins can be produced extracellularly as the main proteins without or small amounts of host cell proteins. This makes it easier for purification step. In the case of the target protein is an enzyme, gene expression product from this invention also produce high level of enzyme activity.

Description

GENE EXPRESSION PRODUCT FOR PRODUCING TARGET PROTEINS OR BIO- PRODUCTS FROM HEAT-TOLERANT YEAST BY METHANOL INDUCTION AND NON-INDUCTION INCLUDING ITS PROCESS OF PRODUCT USAGE.

Technical Field This invention relates to biotechnology in gene expression product for producing target proteins or bio-products from heat-tolerant yeast by methanol induction and non-induction including its process of product usage.

Background Art

Microbes are important sources of beneficial proteins and bio-products. However, the production of proteins or biological products from each microorganism may be limited because of the different ability to produce products in each microorganism. This is a barrier for the desired amount of production in various industries. Therefore, the target protein and biological material production, as a recombinant product using potential host to express target genes, is important and beneficial in biotechnology. Potential microorganism hosts must have the ability to grow rapidly in readily available and inexpensive culture media. The recombinant products can be produced from both prokaryote and eukaryote with the different advantages or limitations in recombinant protein production. For example, the prokaryotic Escherichia coli E. coli) has many advantages including rapid growth in simple and cheap culture media and also has complete genetic information. This makes it possible to genetically modify strain to produce target recombinant proteins. However, the major limitation of using prokaryotic as a host cell is that there is no post-translational modification process such as glycosylation which is an important process for the protein production from eukaryotic microorganisms. Therefore, the host cell selection from eukaryote is essential for eukaryotic protein production.

Yeast is a eukaryotic microorganism that has been used in both industry and biotechnology for a long time. Yeast can be divided into two groups: conventional yeast and non-conventional yeast. The efficient host cells have been classified in a group of non-conventional yeast. It consists of several yeast strains such as Pichia pastoris (P. pastoris), Pichia methanolica (P. methanolica), Hansenula polymorpha {H. polymorpha), Candida boidinii (C. boidinii), Pichia stipitis (P. stipitis) and Yarrowia lipolytica (Y. lipolytica). P. pastoris, P. methanolica, H. polymorpha and C. boidinii are methylotrophic yeasts which have ability to grow in high cell density and have a regulation system for alcohol oxidase (AOX) production (Hartner and Glieder, 2006). According to the effective methanol oxidization under AOX promoter, this group of yeast has been widely used to produce recombinant proteins under the AOX promoter.

The major component of gene expression for recombinant protein production from yeast host cell is a plasmid vector containing encoding gene and protein production in the host cell. The essential components in plasmid vector consist of: promoter which is located upstream of the gene encoding target protein; and transcriptional termination region, or in short term, terminator, which is located downstream of the target gene for helping the stability of mR A. Moreover, there are other elements such as antibiotic-resistant gene which makes the recombinant yeast resist to the antibiotic and to discriminate between the yeast that harbors the plasmid vector within the cell and the one that does not. The multiple cloning sites (MCSs), which contain the specific position for restriction enzyme to cut and clone genes into the plasmid vector, are also included. In case of secretion of protein outside the cells, another essential part is a gene that encodes the signal peptide or secretion signal, which leads protein to secrete outside the cell. This helps reduce the purification step and makes it more convenient to produce more target proteins. Promoter is an important element that regulates gene expression. It is an important part for the binding of RNA polymerase and transcription factors that can function to either enhance or inhibit the binding of RNA polymerase in the promoter region of each target gene. Promoter can be divided into two types depending on the nature of the expression: constitutive promoter and inducible promoter. The constitutive promoter can express genes at any time without the need for induction and leads to the encoding mRNA. An example of this type of promoter is the GAP (glyceraldehyde 3 -phosphate dehydrogenase) promoter. On the other hand, the inducible promoter is a promoter that needs to be induced under the proper condition. The inducible promoter in yeast is a strong promoter. Example of inducible promoter includes the alcohol oxidase (AOX) promoter that controls alcohol oxidase production as discussed above. AOX promoter is an inducible promoter that will function when methanol is added to culture media containing yeast host cells. The different advantages between both types of promoters are that GAP promoter is a strong promoter and the target proteins can be expressed along with the cell growth making it easy to cultivate yeast cells. On the other hand, when using the AOX promoter, the proteins will be produced after host cells are induced with methanol. The growth rate of yeast cells will decrease while the rate of target protein production will increase. The advantage of using GAP promoter is that the protein can be produced early during culturing period without the need to change the culture media or addition of other reagents to the production process. It is therefore a convenient and easy way to adapt for larger scale production. On the other hand, the advantage of using an inducible promoter is that it can produce proteins which may be toxic to the host cells because the cells slow down or stop growing during the induction phase of the target protein.

To efficiently produce target protein using the plasmid vector, the use of suitable yeast strain with good compatibility with the type of plasmid vector is important. In the past, the development of methylotrophic yeast leads to the development of gene expression products, which are yeast host cells and plasmids for commercial use, to produce target protein. The well-known product is gene expression product in P. pastoris. The methylotrophic yeast can grow rapidly with high cell density. P. pastoris can produce protein using GAP promoter to control target gene expression. Moreover, it can also utilize methanol as a carbon source. This is a reason why P. pastoris has an efficient regulatory system for alcohol oxidase production (AOXl promoter) and get high amount of enzyme. This AOXl promoter has therefore been utilized. The P. pastoris yeast is also widely accepted as a host cell for the recombinant protein production because it secretes recombinant proteins outside the cell using effective alpha-factor secretion signal in the plasmid vector, which is easy and convenient for target protein separation process. In addition, P. pastoris secretes low level of its native protein, thus, the target protein expressed from recombinant plasmid in yeast cell will be produced as major product. However, limitations are different level of each protein production from P. pastoris and not all proteins are not always successfully expressed (Macauley-Patrick et al., 2005). In addition, it still has the limitation in the case of producing protein in large scale of bioreactor. It has often experienced the temperature fluctuations which are result from the accumulated heat generated during the large scale production process. Therefore, it is necessary to use the cooling down system to allow the continued production process. This leads to increase the cost of protein production. Moreover, the quality and quantity of the protein is not consistent. Thus, there are many researches try to develop heat-tolerant yeasts in order to increase the efficiency of target protein production. Based on previous research, there is a screening for yeasts that are methylotrophic. Heat-tolerant yeast, Pichia thermomethanolica (currently grouped as Ogataea thermomethanolica or O. thermomethanolica), especially O. thermomethanolica BCC 16875 or O. thermomethanolica TBRC656 which is the same strain, is a locally identified yeast in Thailand with desired characteristics (Limtong et al. 2005, 2008). This yeast strain has the ability to grow rapidly in standard culture media in the laboratory and grows well at 30 - 40 degrees Celsius. From further study of this yeast strain, it is found that this yeast has ability to produce recombinant protein and has comparable efficiency to transform the plasmid into the cell as that of P. pastoris using integration method into yeast genome. When the plasmid vectors harboring gene encoding phytase and xylanase are integrated into yeast cells (Tanapongpipat et al., 2012; Promdonkoy et al., 2014; Harnpicharnchai et al., 2014), the protein could secrete from yeast cells with high efficiency. Previously, AOX promoter isolated from O. thermomethanolica has been studied. The O. thermomethanolica AOX promoter can function at temperatures up to 45 degrees Celsius. In addition, the induction to produce recombinant protein under the control of AOX promoter has started when yeast cells are cultured with glycerol. This is the growth stage before changing the culture media to methanol which is called de-repression. After yeast cells are cultured in media containing methanol, which is the induction stage, recombinant proteins will be produced at high level. This process is different from the case of the AOX1 promoter from P. pastoris because the AOX1 promoter is only active in the presence of methanol in culture media. Using O. thermomethanolica with its AOX promoter, recombinant proteins can be produced since repression stage which can reduce production time. For a constitutive promoter, GAP promoter, which controls glyceraldehyde 3-phosphate dehydrogenase production and is isolated from O. thermomethanolica, has also been studied. The O. thermomethanolica GAP promoter can function at 30 to 40 degrees Celsius. The types of carbon sources that can be used for cell growth and production of recombinant proteins under GAP promoter operation are such as glucose, glycerol, especially sucrose, which is a source of cheap sugar and good for protein production (Harnpicharnchai et al., 2014; Roongsawang et al., 2016). However, in the research mentioned above, plasmid vector consists of elements that are originated from P. pastoris which is a commercial plasmid. This includes the alpha-factor secretion signal derived from Saccharomyces cerevisiae (S. cerevisiae), the tenninator derived from the P. pastoris AOX gene, the regulatory regions that control drug resistant gene expression are promoters from S. cerevisiae and E. coli. The only exception is the promoter that plays an important part in regulating the expression of the target gene which is isolated from O. thermomethanolica. According to the previous research in Thai petty patent No. 10661 relating to the study of secretion signal sequence in O. thermomethanolica, O. thermomethanolica has a sequence that is similar to alpha-factor secretion signal from S. cerevisiae (Sea). The new alpha-mating factor is called Ota-MF. The Ota-MF has 179 amino acids which is divided into three parts: 18 amino acids for a pre-signal sequence, 20 amino acids of pro-signal sequence and 4 repeats of mature alpha-mating factor (mature a-MF). Each repeat of mature a-MF, of which amino acid sequence is [(R/ G) WGWHGVSRNEAIF], is connected by abridge sequence. Although the Ota is shorter than Sea, the protein secretion out of cells using Ota is more effective than Sea. Nonetheless, this petty patent investigated using only non-inducing promoter (GAP promoter) for target gene expression.

In general, selection of recombinant strains contaiaing plasmid vector in the cell will be based on the difference in growth ability of the recombinant yeast cell on culture media containing antibiotics. Moreover promoter and alpha-factor secretion signal help in the protein secretion out of cells, resistance gene to make recombinant yeast resist the antibiotics, which is used to separate the yeast with and without the plasmid vector in the cell, is also important. According to the previous studies of Thai petty patent no. 11158 and 10661, the development of plasmid vector to apply with O. thermomethanolica uses zeocin resistance gene under promoter from S. cerevisiae. Because promoter, which controls the expression of resistance gene, does not derive from host yeast O. thermomethanolica, the action of the drug resistance gene may not be fully effective.

Therefore, in this invention, alternative plasmids have been developed to make the drug resistance gene work more efficiently. The alternative plasmid vector will use an inducible AOX promoter and non-induction GAP promoter to control the expression of the gene. In addition, TEF1 promoter derived from O. thermomethanolica is used for selection of yeast harboring plasmid vector in the cell. In addition L-ldh promoter is derived from the enzyme L-lactate Dehydrogenase enzyme of Lactobacillus plantarum BCC9546. This promoter that can work both in E. coli and in Bacillus, is used in the plasmid of this invention for plasmid replication in E. coli.

Summary of the invention This invention relates to the development of gene expression product for producing target proteins or bio-products from heat-tolerant yeast by methanol induction and non-induction including its process of product usage. The process of using such products uses O. thermomethanolica heat-tolerant strain as host cell for recombinant protein production. This type of yeast can use the variety of carbon sources for growth such as glucose, glycerol and methanol. It also has the basic properties of host cell to produce proteins. In particular, it can grow at wide temperature ranges, ranging from 25 to 40 degrees Celsius. These properties make this yeast strain suitable for host cells for the production of recombinant protein at the enlarged scale in fermentation tank. In this invention, there are 2 types of plasmid vectors. The first one is a plasmid vector which contains a sequence of promoter that controls the alcohol oxidase production and another one is a plasmid vector which contains a sequence of promoter that controls the glyceraldehyde-3 -phosphate dehydrogenase production. The alternative plasmid vector, containing suitable components for use effectively with O. thermomethanolica heat- tolerant species, is constructed. The product according to the invention comprising: (1) Ogataea thermomethanolica heat-tolerant species as a host cell.

(2) Plasmid vector for target genes expression contains the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) A sequence of promoter is isolated from O. thermomethanolica heat-tolerant species. b) An alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica. c) A sequence of multiple cloning sites (MCSs) is selected from Ecd l, Kpnl, Sad, Nsil, Apal, Sail, Pmel, BamHL, Nhel or combination of them. This site is for cloning of target gene encoding proteins into plasmid vector. d) A sequence of transcriptional terminator of alcohol oxidase gene is isolated from O. thermomethanolica. e) A sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters. The TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546. f) A sequence of transcriptional terminator of glyceraldehyde-3 -phosphate dehydrogenase gene is isolated from O. thermomethanolica. g) A sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli. Brief Description of drawing Fig. 1 shows the construction of the plasmid vectors A) pOtNeo4 and B) pGotNeo4 of this invention, wherein

Ot AOX promoter represents the promoter which controls alcohol oxidase production. This promoter is isolated from O. thermomethanolica heat-resistant species and its length is 801 base pairs. Ot GAP promoter represents a promoter that controls the expression of a gene encoding glyceraldehyde-3 -phosphate dehydrogenase production. This promoter is isolated from O. thermomethanolica.

Ot alpha factor represents the alpha-factor secretion signal, which directly secretes expression of the recombinant protein from O. thermomethanolica. This part is isolated from O. thermomethanolica.

MCS represents multiple cloning site which is the position of restriction enzymes recognition.

Ot AOX TT represents the transcriptional terminator of alcohol oxidase gene which is isolated from O. thermomethanolica.

Neomycin represents the neomycin resistance gene. Ot TEFl promoter represents the promoter which is isolated from O. thermomethanolica with a length of 500 base pairs.

L-ldh promoter represents the promoter which is isolated from L. plantarum BCC9546 with a length of 75 base pairs. Ot GAP TT represents the transcriptional terminator of glycerol-3-phosphate dehydrogenase gene which is isolated from O. thermomethanolica. pUC ori represents pUC origin of replication for plasmid replication in E. coli;

Fig. 2 shows the part of promotor sequence from glyceraldehyde-3 -phosphate dehydrogenase gene which is isolated from O. thermomethanolica;

Fig. 3 shows the part of terminator sequence from alcohol oxidase gene (Ot AOX TT) which is isolated from O. thermomethanolica;

Fig. 4 shows the new promoter sequence for controlling neomycin resistance gene which contains the linked sequence between Ot TEF1 from O. thermomethanolica and L-ldh from L. plantarum BCC9546;

Fig. 5 shows the part of terminator sequence from glyceroldehyde-3 -phosphate dehydrogenase gene (Ot GAP TT) which is isolated from O. thermomethanolica;

Fig. 6 shows the SDS-PAGE expression analysis of A) phytase, B) beta-glucosidase and C) beta- xylosidase which are produced by pOtNeo4 vector in O. thermomethanolica under Ot AOX promoter, wherein

M represents protein marker.

Number represents the individual clone which is selected for protein expression analysis.

(-) represents O. thermomethanolica with plasmid vector pOtNeo4.

(+) represents O. thermomethanolica with recombinant plasmid vector pOtNeo3-phytase; Fig. 7 shows the SDS-PAGE expression analysis of A) phytase, B) beta-glucosidase and C) beta- xylosidase which are produced by pGotNeo4 vector in O. thermomethanolica under Ot GAP promoter, wherein

M represents protein marker.

Number represents the individual clone which is selected for protein expression analysis. (-) represents negative control;

Fig. 8 shows the comparison of expression analysis between enzyme which is produced from new recombinant plasmid A) pOtNeo4 and B) pGotNeo4 in O. thermomethanolica and enzyme which is produced from commercial recombinant plasmid in P. pastoris yeast by SDS-PAGE method, wherein

M represents protein marker.

(-) represents the P. pastoris or O. thermomethanolica yeast species with plasmid vector. Pp represents P. pastoris with plasmid vector containing encoding gene. Ot represents O. thermomethanolica with plasmid vector containing encoding gene. ' Phy represents an example of phytase production.

BGL represents an example of beta-glucosidase production. Xylo represents an example of beta- xylosidase production. Disclosure of Invention

Gene expression product for producing target proteins or bio-products from heat-tolerant yeast comprising:

1) Ogataea thermomethanolica heat-tolerant species as a host cell.

2) Plasmid vectors for target genes expression contain the sequence component for gene expression in the direction from the 5' end to the 3' end as shown in Fig. 1, as follows: a) A sequence of promoter is isolated from O. thermomethanolica heat-tolerant species. For glyceraldehyde-3-phosphate dehydrogenase promoter, the sequence is shown in Fig. 2. b) An alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein isolated from O. thermomethanolica. c) A sequence of multiple cloning sites (MCSs) is selected from EcoKL, Kpnl, Sacl, Nsil, Apal, Soli, Pmel, BamHl, Nhel or combination of them. This site is for cloning of target gene encoding proteins into plasmid vector. d) A sequence of transcriptional terminator of alcohol oxidase gene is isolated from O. thermomethanolica. The sequence is shown in Fig. 3. e) A sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters. The TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546. The sequence is shown in Fig. 4. f) A sequence of transcriptional terminator of glyceraldehyde-3-phosphate dehydrogenase gene is isolated from O. thermomethanolica. The sequence is shown in Fig. 5. g) A sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli.

Wherein a sequence of the promoter, which is isolated from O. thermomethanolica heat-tolerant species, is the promoter that controls the alcohol oxidase production or the glyceraldehyde-3- phosphate dehydrogenase production. In this invention, O. thermomethanolica heat-tolerant species is used as yeast host cell. It grows well in temperature range of 30 to 40 degrees Celsius, and is not resistant to neomycin antibiotic. The selected strain for use in this invention is O. thermomethanolica strain BCC 16875 or O. thermomethanolica strain TBRC656, which is the same strain.

The target gene for applying to gene expression products is isolated from prokaryote or eukaryote. The target gene for applying to gene expression products is gene which encodes potential proteins for using at least in medical, industrial or combination of them. For example, the target gene encodes enzymes from at least one of lignocellulosic enzyme, hemicellulosic enzymes, histidine acid phosphatase or combination of them.

The target gene that encodes enzymes at least one in the group of lignocellulosic enzyme, hemicellulosic enzymes or histidine acid phosphatase is selected from gene that encodes enzymes at least one of beta-glucosidase, beta-cellulase, phytase or combination of them. The next step explains about the detail of the components for each gene expression product, the process for using gene expression production and examples which show the efficiency of product and its process for target protein production as follows:

1. Gene expression product for producing target proteins or bio-products from heat- tolerant yeast by methanol induction according to the invention

The gene expression product by methanol induction comprising:

1) Ogataea thermomethanolica heat-tolerant species as host cell.

2) Plasmid vectors for target genes expression contain the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) A sequence of promoter, which controls the alcohol oxidase production, is isolated from O. thermomethanolica heat-tolerant species. b) An alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica. c) A sequence of multiple cloning sites (MCSs) is selected from EcoRI, Kpnl, Sacl, Nsil, Apal, Sail, Pmel, Bam i, Nhel or combination of them. This site is for cloning of target gene encoding proteins into plasmid vector. d) A sequence of transcription terminator of alcohol oxidase gene is isolated from O. thermomethanolica. e) A sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters. The TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546. f) A sequence of transcriptional terminator of glyceraldehyde-3 -phosphate dehydrogenase gene is isolated from O. thermomethanolica. g) A sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli. The detail of components for gene expression product by methanol induction as follows:

1. Yeast host cell that is a component of gene expression product for producing target proteins or bio-products from heat-tolerant yeast by methanol induction according to the invention.

In this invention, O. thermomethanolica heat-tolerant species is used as yeast host cell. It grows well in temperature range of 30 to 40 degrees Celsius, and is not resistant to neomycin antibiotic. The selected strain for use in this invention is O. thermomethanolica strain BCC 16875 or O. thermomethanolica strain TBRC656, which is the same strain.

2. Plasmid vector that is a component of gene expression products for producing target proteins or bio-products from heat-tolerant yeast by methanol induction according to the invention. Because O. thermomethanolica heat-tolerant species is used as a yeast host cell, and this group of yeasts can utilize methanol as a carbon source. In this invention, the sequence of promoter controlling the alcohol oxidase production, which is isolated from O. thermomethanolica heat- tolerant species, is used as methanol inducible promoter to be a key component for gene expression in plasmid vector in this invention. The detail of the procedure for plasmid vector construction as follows:

2.1. Construction of a plasmid vector for gene expression which consists of Ot AOX promoter from O. thermomethanolica replacing traditional AOX1 promoter from P. pastoris.

The 801 base pairs of Ot AOX promoter is amplified by using initial primers which are PtAOX_NeoF (5'-ATGCGGCCGCGCACAAGCTGGACGA-3 ') and PtAOX_NeoR (5'- CGAGGCCTTTTCGTTTAAAGAATTGTCTTGCTTC-3'). The promoter fragment is cut by restriction enzymes, Notl and Stul, and is then ligated with pPasNeo vector using NotI and Hz^'wdlll. The plasmid vector is named pPasNeo_OtAOX.

2.2 Construction of plasmid vector canying alpha-factor secretion signal which is isolated from O. thermomethanolica. The 114 base pairs of alpha-factor secretion signal from O. thermomethanolica is amplified by using primers which are DAEA-Hindlll-F (5 -

CCAAGCTTACGATGAAATTCAACACTACTCTTC-3' and DAEA-EcoRI-R (5'- ATGAATTCGGCCTCTGCGTCGGCCCCAG-3'). The fragment is cut and ligated with pPasNeo OtAOX vector by using restriction enzymes, HindBI and EcoKi. The plasmid vector is named pPasNeo OTOT.

2.3 Construction of plasmid promoter which regulates neomycin resistant gene. In the original plasmid vector, neomycin resistant gene is controlled by promoters from P. pastoris (TEFl) and from E. coli (GB2), so they may not be efficient when used in the expression system of O. thermomethanolica. In this invention, the initial promoter is replaced with the TEFl promoter which is isolated from O. thermomethanolica for selection in recombinant yeast strains and a new promoter which is isolated from L. plantarum BCC9546 (L-ldh promoter) for selection in E. coli.

Based on chemical synthesis, new promoter is 1,127 base pairs long containing 500 base pairs of TEFl promoter from O. thermomethanolica, 75 base pairs of L-ldh promoter from Lactobacillus plantarum BCC9546, and some part of neomycin resistant gene with cleavage site of Noil and Sphl at the end of 5' and 3', respectively. This new promoter is cut and ligated with pPasNeo_OTOT by using Notl and Sphl to remove the original pieces of TEFl promoter from P. pastoris and GB2 from E. coli. The plasmid vector is named pOt eo2.

2.4. Construction of plasmid vector carrying AOX and GAP transcriptional terminators which are isolated from O. thermomethanolica.

To make the plasmid vector suitable and more effective for use with O. thermomethanolica yeast host cell, the 397 base pairs of terminator, is derived from alcohol oxidase gene of O. thermomethanolica, is inserted. The terminator is synthesized by chemistry synthesis technique. Mzel restriction site is located at the 5* end and the terminator is connected to the upstream sequence of pUC ori which has anApaU restriction site at the 3' end. This terminator is cut and ligated with pOtNeo2 by using N¾el and^ aLI. The plasmid vector is named pOtNeo3. To make this plasmid more suitable for use with O. thermomethanolica yeast host cells, 359 base-pairs of Ot GAP TT sequence is derived from the gene encoding glyceraldehyde 3- phosphate dehydrogenase enzyme from O. thermomethanolica (GAP) is obtained by chemical synthesis of DNA fragment of 956 base-pairs, 5' end of which consists of the downstream region of PUC ori fragment from the ApdLl restriction site and the 3' end contains a part of neomycin resistant gene with Ncol restriction site. After ligation of this fragment with pOt eo3 plasmid vector that is cut with ApdLl and Ncol, the new plasmid vector is named pOtNeo4.

3. Process of target protein production with gene expression product by methanol induction according to the invention.

This step studies the expression of target gene. The target gene for applying to gene expression product is isolated from prokaryotic or eukaryotic organisms which encodes target proteins and that proteins have potential usage at least for medical, industrial or combination of them. For example, the target gene is an encoded gene in subgroup of lignocellulose or hemicellulose such as xylanase, cellulase, mannanase, beta-glucosidase (β-glucosidase), beta-xylosidase (β- xylosidase), and including encoded gene in subgroup of histidine acid phosphatase such as phytase etc. For optimal condition to transfer plasmids into the host cell, electroporation technique is used, firstly, yeast host cells are prepared to be competent cells (Faber, 1993) while plasmid vectors are cut with restriction endonuclease, Xmnl or Mfel, to obtain a linearized plasmid. This cleavage site is located in the region of the promoter that controls alcohol oxidase production. The 200 nanograms - 8 micrograms of linearized plasmids are mixed with 60 microliter of yeast cells in a cuvette which is soaked in ice for 5 minutes, then the cuvette is taken into an electroporator. The program of the electroporator is set at 1.0 kilovolt, 400 ohm sand 25 microfarads. The 1 milliliter of YPD culture media is added and incubated at 30 degrees Celsius for 1-2 hours without shaking. Then, yeast cells are cultured on YPD agar media containing 100 micrograms per milliliter of neomycin by incubation at 30 degrees Celsius for a few days until the colonies appear on agar plate. The plasmid vector is integrated into chromosomes of yeast host cells by homologous recombination process under the optimal condition.

The method of target protein production by methanol induction using gene expression product according to the invention comprising; a) Construction of recombinant plasmids for expression of target genes by ligating the target genes to the plasmid vector at the appropriate multiple cloning site for the target gene and amplifying these recombinant plasmids in E. coli cells. b) Preparation of recombinant plasmids from a) for transferring and integrating into chromosomes of heat-tolerant yeast O. thermomethanolica by extraction of recombinant plasmid from E. coli and then cut recombinant plasmids with restriction enzymes, Xmnl or Mfel, at the alcohol oxidase promoter site. A sequence of alcohol oxidase promoter is isolated from yeast O. thermomethanolica. As a result, the plasmid is linearized. c) Construction of recombinant yeast O. thermomethanolica for target gene expression by transferring the recombinant plasmid from b) into the heat-tolerant yeast O. thermomethanolica by electroporation. This is achieved by mixing the recombinant plasmid with competent cells of O. thermomethanolica and selecting recombinant yeast cells by culturing on solid culture media containing neomycin antibiotic. d) Production of target proteins by culturing recombinant yeast O. thermomethanolica for target gene expression from c) under condition as follows: recombinant yeasts are cultured in culture media at 25-40 degrees Celsius with aeration. Cells are inoculated into the culture media and incubated at 25-40 degrees Celsius with aeration. Cells are concentrated by centrifugation and resuspended in culture media supplemented with methanol at 1 to 3 percentages. The target protein is then produced by culturing recombinant yeast cells in this media at 25-40 degrees Celsius with aeration and 1 to 3 percentages of methanol is daily added.

The optimum culture condition for the target protein production by culturing recombinant O. thermomethanolica yeast cells is that culturing recombinant yeasts in the YPD culture media at 30 degrees Celsius for 18-24 hours with aeration and then the culture is transferred into BMGY culture media, and cultured for 18-24 hours at 30 degrees Celsius with aeration or until the absorbance at 600 nanometers is 8-12. The cells are then concentrated by centrifugation and resuspended in BMMY culture media containing methanol of 2 percentages with ten times decreased volume. Subsequently, the cells are cultured in the BMMY media at 30 degrees Celsius with aeration for 48 hours with the addition of 2 percentages of methanol each day.

4. Examples that demonstrate the efficacy of product and its method for protein production according to the invention.

In this invention, the genes encoding several enzymes which are phytase, beta-glucosidase and beta-xylosidase are used as model to study the efficiency of the application of the product for gene expression and process of protein production according to this invention for the production of target proteins. The use of the gene expression product according to this invention is achieved by construction of a recombinant plasmid by cutting the plasmid vector, according to this invention, with restriction enzymes and ligating with genes encoding enzymes. The obtained recombinant plasmids are then transferred into yeast host cells by the process of protein production according to this invention. This will result in recombinant yeast cells expressing target enzymes as desired. This is to verify the efficiency of the expression system using yeast host cells.

Example 1 : The study of phytase, beta-glucosidase and beta-xylosidase production from a new type of plasmid vector is controlled under methanol-inducer promoter.

The ability of enzyme production, herein phytase, beta-glucosidase and beta-xylosidase, from the O. thermomethanolica recombinant yeast that contains encoding gene is determined by culturing recombinant yeast in YPD, BMGY and BMMY culture media, respectively, as mentions in the title of the method of target protein production by methanol induction using gene expression products. For BMMY culture media, the 2 percentages of methanol is added during cultured period for both gene expression products. On day 2, samples are collected for enzyme expression analysis as shown in Fig. 6. Clones are selected for expression determination of phytase, beta-glucosidase and beta-xylosidase. The result shows that the expression level of each clone is different. This may be due to the fact that the number of copies of the integrated genes in the chromosome is not equal and the expression level of each enzyme is also unequal depending on the structural characteristics of each enzyme that may affect the export process. However, recombinant yeast cells that contain plasmid vector carrying the encode gene have the ability to secrete proteins extracellularly. The major group of protein is derived from recombinant plasmid while the native proteins are few. This makes the advantages to the next purification step.

Example 2: Comparison of enzyme production between gene expression product in heat-tolerant yeast host cells according to this invention and gene expression product in commercial P. pastoris yeast under methanol induction condition.

This example presents the ability of gene expression product that consists of heat-tolerant yeast and plasmid vector under the operation of Ot AOX promoter according to this invention compares with commercial gene expression product consisting of commercial P. pastoris and carrier plasmid under the operation of traditional AOX1 promoter from P. pastoris by controlling 3 types of enzyme expression, namely, phytase, beta-xylosidase and beta-glucosidase. First, the recombinant yeasts from both products are cultured in YPD, BMGY and BMMY culture media, respectively, as mentions in the title of the method of target protein production by methanol induction using gene expression products. For BMMY culture media, the 2 percentages of methanol is added during cultured period for both gene expression products. On day 2, samples are collected for enzyme- expression analysis as shown in Fig. 8A. The result shows that the expression of phytase from pOtNeo4 plasmid vector in heat-tolerant yeast according to this invention is less than from commercial P. pastoris product. Whereas the expression of beta- glucosidase and beta-xylosidase from pOtNeo4 plasmid vector in heat-tolerant yeast according to this invention is higher than from commercial P. pastoris product at least 2-5 fold. This shows the potential of the expression system containing heat-tolerant yeast and new plasmid vector.

2. Gene expression product for producing target proteins or bio-products from heat- tolerant yeast by non-induction according to the invention

The gene expression product by non-induction comprising:

1) Ogataea thermomethanolica heat-tolerant species as a host cell;

2) Plasmid vectors for target genes expression contain the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) A sequence of promoter, which controls the glyceraldehyde-3- phosphate dehydrogenase production, is isolated from O. thermomethanolica heat-tolerant species. b) An alpha-factor secretion signal for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica. c) A sequence of multiple cloning sites (MCSs) is selected from EcoRl, Kpnl, Sacl, Nsil, Apal, SaR, Pmel, BamHl, Nhel or combination of them. This site is for cloning of target gene encoding proteins into plasmid vector. d) A sequence of transcriptional terminator of alcohol oxidase gene is isolated from O. thermomethanolica. e) A sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters. The TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546. f) A sequence of transcriptional terminator of glyceraldehyde 3-phosphate dehydrogenase gene is isolated from O. thermomethanolica. g) A sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli.

The detail of product components for gene expression by non-induction as follows: 1. Yeast host cell that is a component of gene expression products for producing target proteins or bio-products from heat-tolerant yeast by non-induction according to the invention.

In this invention, O. thermomethanolica heat-tolerant species is used as a host cell. It grows well in temperature range of 30 to 40 degrees Celsius, and is not resistant to neomycin antibiotic. The selected strain for use in this invention is O. thermomethanolica strain BCC16875 or 0. thermomethanolica strain TBRC656, which is the same strain.

2. Plasmid vector that is a component of gene expression products for producing target proteins or bio-products from heat-tolerant yeast by non-induction according to the invention.

Because O. thermomethanolica heat-tolerant species is used as a yeast host cell, and this group of yeast can utilize several types of carbon source such as glucose and sucrose which are efficient carbon sources for yeast cell growth. Therefore, constitutive promoter sequence, which controls glyceraldehyde-3- phosphate dehydrogenase production and is isolated from O. thermomethanolica, is used as a major component for gene expression in the plasmid vector according to this invention.

The plasmid vector which is a component of gene expression product for producing target proteins or bio-products from heat-tolerant yeast by non-induction according to the invention is constructed by cleaving pOtNeo4 plasmid vector containing Ot AOX promoter with restriction enzymes Notl and HindHL Then, the Ot AOX promoter in the pOtNeo4 plasmid vector is replaced by Ot GAP promoter from O. thermomethanolica. The plasmid vector is named pGotNeo4 as shown in Fig. IB.

3. Process of target protein production with gene expression product by non-induction according to the invention.

This step studies the expression of target gene. The target gene for applying to gene expression product are isolated from prokaryotic or eukaryotic organisms which encodes target proteins and that proteins have potential usage at least for medical, industrial or combination of them. For example, the target gene is an encoded gene in subgroup of lignocellulosic or hemicellulosic enzymes such as xylanase, cellulase, mannanase, beta-glucosidase (β-glucosidase), beta- xylosidase (β-xylosidase), and including encoded gene in subgroup of histidine acid phosphatase such as phytase etc. For optimal condition to transfer plasmids into the host cell, electroporation technique is used, firstly, yeast host cells are prepared to be competent cells (Faber, 1993) while plasmid vectors are cut with restriction endonuclease, Notl, to obtain a linearized plasmid. This cleavage site is located in the region of the promoter that controls glyceraldeyde-3 -phosphate dehydrogenase production. The 200 nanograms - 8 micrograms of linearized plasmids are mixed with 60 microliter of yeast cells in a cuvette which is soaked in ice for 5 minutes, then the cuvette is taken into an electroporator. The program of the electroporator is set at 1.0 kilovolt, 400 ohms and 25 microfarads. The 1 milliliter of YPD culture media is added and incubates at 30 degrees Celsius for 1-2 hours without shaking. Then, yeast cells are cultured on YPD agar media containing 100 micrograms per milliliter of neomycin by incubation at 30 degrees Celsius for a few days until the colonies appear on agar plate. The plasmid vector is integrated into chromosomes of yeast host cells by homologous recombination process under the optimal condition. The method of target protein production by non-induction using gene expression product according to the invention comprising; a) Construction of recombinant plasmid for expression of target genes by ligating the target genes to the plasmid vector at the appropriate multiple cloning site for the target gene and amplifying these recombinant plasmids in E. coli cells. b) Preparation of recombinant plasmid from a) for transferring and integrating into chromosomes of heat-tolerant yeast O. thermomethanolica by extraction of recombinant plasmid from E. coli and then cut recombinant plasmids with restriction enzymes at the glyceraldehyde-3 -phosphate dehydrogenase promoter site. A sequence of glyceraldehyde-3 -phosphate dehydrogenase promoter is isolated from O. thermomethanolica. c) Construction of recombinant yeast O. thermomethanolica for target gene expression by transferring the recombinant plasmid from b) into the heat-tolerant yeast O. thermomethanolica by electroporation. This is achieved by mixing the recombinant plasmid with competent cells of O. thermomethanolica and selecting recombinant yeast cells by cwturing on solid culture media containing neomycin antibiotic. d) Production of target proteins by culturing recombinant yeast O. thermomethanolica for target gene expression from c) under culture condition as follows: recombinant yeast cells are cultured in culture media at 25-40 degrees Celsius with aeration. At the appropriate cells optical density, cells are inoculated and re-cultured in the culture media at 25-40 degrees Celsius with aeration. Cells culture media is collected by centrifugation for protein analysis.

The optimal condition for target protein production by culturing recombinant O. thermomethanolica yeast cells is that recombinant yeast cells are cultured in YPD culture media at 30 degrees Celsius for 18 - 24 hours with aeration. Cells were re-cultured in 20 milliliters YPD culture media to obtain cell concentration at 0.01 optical density, at 30 degrees Celsius for 72 hours with aeration. Cells culture media is collected by centrifugation for protein analysis.

In this invention, the encoding enzyme gene such as phytase, beta-glucosidase and beta-xylosidase is used as a model to study the efficacy of gene expression product and its method for protein production. The step of using gene expression product according to this invention is that the recombinant plasmid is constructed by cutting and integrating between plasmid vector and gene encoding enzyme at restriction site using restriction enzymes, and then the recombinant plasmid is transferred into the yeast host cell by the protein production process of this invention. The recombinant yeast cell that can express the target enzyme is generated. This is for testing the efficiency of the expression system using yeast host cell.

Example 1 : The study of phytase, beta-glucosidase and beta-xylosidase production from a new type of plasmid vector is controlled under constitutive promoter. The ability of enzyme production, herein phytase, beta-glucosidase and beta-xylosidase, from the O. thermomethanoUca recombinant yeast that contains encoding gene under the control of constitutive promoter, Ot GAP, is investigated. Recombinant yeast cells are cultured in 5 milliliters of YPD culture media at 30 degrees Celsius for 18 - 24 hours.

Cells are re-cultured in 20 milliliters YPD culture media to obtain cell concentration at 0.01 optical density at 30 degrees Celsius for 72 hours with aeration. Cells culture media is collected by centrifugation for protein analysis as shown in Fig. 7. Clones are selected for expression determination of phytase, beta-glucosidase and beta-xylosidase. The result shows that the expression level of each clone is different. This may be due to the fact that the number of copies of the integrating gene in the chromosome is not equal and the expression level of each enzyme is also unequal depending on the structural characteristics of each enzyme that may affect the export process. However, recombinant yeast cells that contain plasmid vector carrying the encoding gene have the ability to secrete proteins extracellularly and the target enzyme is expressed along with host cell growth. The major group of protein is derived from recombinant plasmid while the native proteins are few. This makes the advantages to the next purify step. Example 2: Comparison of enzyme production between gene expression product in heat-tolerant yeast host cells according to this invention and gene expression product in commercial P. pastoris yeast by non-induction condition.

This example presents the ability of gene expression product that consists of heat tolerant yeast and plasmid vector under the operation of Ot GAP promoter according to this invention compares with commercial gene expression product consisting of commercial P. pastoris and carrier plasmid under the operation of traditional GAP promoter from P. pastoris by controlling 3 types of enzyme expression, namely, phytase, beta-xylosidase and beta-glucosidase. First, the recombinant yeasts from both products are cultured in YPD culture media as mentions in the title of the method of target protein production by non-induction using gene expression product. On day 3, samples are collected for enzyme expression analysis as shown in Fig. 8B. The result shows that the expression level of phytase from pGotNeo4 plasmid vector in heat-tolerant yeast according to this invention is similar to the expression level from commercial P. pastoris product, whereas beta-glucosidase from pGotNeo4 plasmid vector in heat-tolerant yeast according to this invention is expressed in less amount than that from commercial product of the P. pastoris. Beta-xylosidase, on the other hand, is higher than from commercial P. pastoris product.

Best Mode for Carrying out the Invention As mentioned in the topic of Disclosure of Invention.

Claims

1. A gene expression product for producing target proteins or bio-products from heat- tolerant yeast, wherein plasmid vectors for target genes expression by methanol induction and non-induction contain the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) a sequence of promoter is isolated from O. thermomethanolica heat-tolerant species; b) an alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica; c) a sequence of multiple cloning sites (MCSs) is selected from EcoKL, Kpnl, Sad, Nsil, Apal, Sail, Pmel, BamHl, Nhel or combination of them; d) a sequence of transcriptional terminator of alcohol oxidase gene is isolated from O. thermomethanolica; e) a sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters; the TEF1 promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546; f) a sequence of transcriptional terminator of glyceraldehyde 3-phosphate dehydrogenase gene is isolated from O. thermomethanolica; g) a sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli.

2. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 1, wherein the sequence of promoter which is isolated from O. thermomethanolica heat-tolerant species is the promoter that controls the alcohol oxidase production or the glyceraldehyde-3 -phosphate dehydrogenase production.

3. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 1 or 2, wherein plasmid vectors for target genes expression by methanol induction contain the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) a sequence of promoter, which controls the alcohol oxidase production, is isolated from O. thermomethanolica heat-tolerant species; b) an alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica; c) a sequence of multiple cloning sites (MCSs) is selected from EcoRl, Kpnl, Sacl, Nsil, Apdl, Sail, Pmel, BamH , Nhel or combination of them; d) a sequence of transcriptional terminator of alcohol oxidase gene is isolated from O. thermomethanolica; e) a sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters; the TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546; f) a sequence of transcriptional terminator of glyceraldehyde-3 -phosphate dehydrogenase gene is isolated from O. thermomethanolica; g) a sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli.

4. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 3, wherein gene expression product for producing target proteins or bio- products from heat-tolerant yeast by methanol induction comprising:

1) Ogataea thermomethanolica heat-tolerant species as host cell;

2) plasmid vectors for target genes expression by methanol induction contain the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) a sequence of promoter, which controls the alcohol oxidase production, is isolated from O. thermomethanolica heat-tolerant species; b) an alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica; c) a sequence of multiple cloning sites (MCSs) is selected from EcoRL, Kpnl, Sacl, Nsil, Apal, SaR, Pmel, Bam l, Nhel or combination of them; d) a sequence of transcription terminator of alcohol oxidase gene is isolated from O. thermomethanolica; e) a sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters; the TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546; f) a sequence of transcriptional terminator of glyceraldehyde-3-phosphate dehydrogenase gene is isolated from O. thermomethanolica; g) a sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli.

5. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 1 or 2, wherein plasmid vectors for target genes expression by non- induction contain the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) a sequence of promoter, which controls the glyceraldehyde-3 -phosphate dehydrogenase production, is isolated from O. thermomethanolica heat-tolerant species; b) an alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica; c) a sequence of multiple cloning sites (MCSs) is selected from EcoBI, KpnI, Sacl, Nsil, Apal, Sail, Pmel, BamHl, Nhel or combination of them; d) a sequence of transcriptional terminator of alcohol oxidase gene is isolated from O. thermomethanolica; e) a sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters; the TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546; f) a sequence of transcriptional terminator of glyceraldehyde-3-phosphate dehydrogenase gene is isolated from O. thermomethanolica; g) a sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli.

6. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 5, wherein gene expression product for producing target proteins or bio- products from heat-tolerant yeast by non-induction comprising:

1) Ogataea thermomethanolica heat-tolerant species as a host cell;

2) plasmid vectors for target genes expression by non-induction contain the sequence component for gene expression in the direction from the 5' end to the 3' end as follows: a) a sequence of promoter, which controls the glyceraldehyde-3 -phosphate dehydrogenase production, is isolated from O. thermomethanolica heat-tolerant species; b) an alpha-factor secretion signal sequence for directing secreted expression of the recombinant protein is isolated from O. thermomethanolica; c) a sequence of multiple cloning sites (MCSs) is selected from EcoRI, Kpnl, Sacl, Nsil, Apal, Sali, Pmel, BamHi, Nhel or combination of them; d) a sequence of transcription terminator of alcohol oxidase gene is isolated from O. thermomethanolica; e) a sequence of neomycin resistance gene for recombinant plasmid selection is controlled by two linked promoters; the TEFl promoter is isolated from O. thermomethanolica and L-ldh promoter is isolated from L. plantarum BCC9546; f) a sequence of transcriptional terminator of glyceraldehyde-3-phosphate dehydrogenase gene is isolated from O. thermomethanolica; g) a sequence of pUC origin of replication (pUC ori) for plasmid replication in E. coli.

7. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 4 or 6, wherein the host cell, O. thermomethanolica heat-tolerant species, grows well at 30 to 40 degrees Celsius and is not resistant to neomycin antibiotic.

8. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 4 or 6 or 7, wherein the strain of host cell, O. thermomethanolica heat- tolerant species, is O. thermomethanolica strain TBRC656.

9. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to any of claims 1-6, wherein the target gene for applying to gene expression product is isolated from prokaryote or eukaryote.

10. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 9, wherein the target gene for applying to gene expression product is the gene which encodes potential protein for using at least in medical, industrial or combination of them.

11. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to any of claims 1 - 10, wherein the target gene for applying to gene expression product is the gene which encodes enzyme from at least one of lignocellulosic, hemicellulosic enzymes, histidine acid phosphatase or combination of them.

12. The gene expression product for producing target proteins or bio-products from heat-tolerant yeast according to claim 11, wherein the gene that encodes enzyme from at least one of lignocellulosic, hemicellulosic enzymes or histidine acid phosphatase is selected from genes that encode enzymes from at least one of beta-glucosidase, beta-cellulase, phytase or combination of them.

13. A method of target protein production by methanol induction using gene expression product according to any of claims 1-12, comprising: a) construction of recombinant plasmids for expression of target genes by ligating the target genes to the plasmid vector at the appropriate multiple cloning site for the target gene and amplifying these recombinant plasmids in E. coli cells; b) preparation of recombinant plasmids from a) for transferring and integrating into chromosomes of heat-tolerant yeast O. thermomethanolica by extraction of recombinant plasmids from E. coli and then cut recombinant plasmids with restriction enzymes at the alcohol oxidase promoter site; a sequence of alcohol oxidase promoter is isolated from O. thermomethanolica; c) construction of recombinant yeast O. thermomethanolica for target gene expression by transferring the recombinant plasmid from b) into the heat-tolerant yeast O. thermomethanolica by electroporation. This is achieved by mixing the recombinant plasmid with competent cells of O. thermomethanolica and selecting recombinant yeast cells by culturing on solid culture media containing neomycin antibiotic; d) production of target proteins by culturing recombinant yeast O. thermomethanolica for target gene expression from c) under condition as follows: recombinant yeasts are cultured in culture media at 25-40 degrees Celsius with aeration; cells are inoculated into the culture media and incubated at 25-40 degrees Celsius with aeration; cells are concentrated by centrifugation and resuspended in culture media supplemented with methanol at 1 to 3 percentages; the target protein is then produced by culturing recombinant yeast cells in this media at 25-40 degrees Celsius with aeration and 1 to 3 percentages of methanol is daily added.

14. The method of target protein production by methanol induction using gene expression product according to claim 13, wherein the optimal condition for target protein production by culturing recombinant O. thermomethanolica yeast cells is that culturing the recombinant yeasts in the YPD culture media at 30 degrees Celsius for 18-24 hours with aeration and then the culture is transferred into BMGY culture media, and cultured for 18-24 hours at 30 degrees Celsius with aeration or until the absorbance at 600 nanometers is 8-12; the cells are then concentrated by centrifugation and resuspended in BMMY culture media containing methanol of 2 percentages with ten times decreased volume; subsequently, the cells are cultured in the BMMY media at 30 degrees Celsius with aeration for 48 hours with the addition of 2 percentages of methanol each day.

15. The method of target protein production by non-induction using gene expression product, according to claims 1-12, comprising: a) construction of recombinant plasmids for expression of target genes by ligating the target genes to the plasmid vector at the appropriate multiple cloning site for the target gene and amplifying these recombinant plasmids in E. coli cells; b) preparation of recombinant plasmid from a) for transferring and integrating into chromosomes of heat-tolerant yeast O. thermomethanolica by extraction of recombinant plasmid from E. coli and then cut recombinant plasmids with restriction enzymes at the glyceraldehyde-3 -phosphate dehydrogenase promoter site; a sequence of glyceraldehyde-3 -phosphate dehydrogenase promoter is isolated from O. thermomethanolica; c) construction of recombinant yeast O. thermomethanolica for target gene expression by transferring the recombinant plasmid from b) into the heat-tolerant yeast O. thermomethanolica by electroporation. This is achieved by mixing the recombinant plasmid with competent cells of O. thermomethanolica and selecting recombinant yeast cells by culturing on solid culture media containing neomycin antibiotic; d) production of target proteins by culturing recombinant yeast O. thermomethanolica yeast cells for target gene expression from c) under condition as follows: recombinant yeast cells are cultured in culture media at 25-40 degrees Celsius with aeration; at the appropriate cells optical density, cells are inoculated and re-cultured in the culture media at 25-40 degrees Celsius with aeration; cells culture media is collected by centrifugation for protein analysis.

16. The method of target protein production by non-induction using gene expression product, according to claim 15, wherein the optimal condition for target protein production by culturing recombinant O. thermomethanolica yeast cells is that recombinant yeast cells are cultured in YPD culture media at 30 degrees Celsius for 18 - 24 hours with aeration; and cells were re- cultured in 20 milliliters YPD culture media to obtain cell concentration at 0.01 optical density at 30 degrees Celsius for 72 hours with aeration. Cells culture media is collected by centrifugation for protein analysis.