Patent Publication Number: US-11643663-B2

Title: Determination of regulatory DNA regions of alcohol dehydrogenase 3 (ADH3) promoter and design of synthetic promoters for recombinant protein production

Description:
INCORPORATION BY REFERENCE 
     The file entitled “17071-27 Sequence Listing.txt” is hereby incorporated by reference into the present application. This ASCII text file was created 2 Sep. 2021 to correct formatting. This file is 15,969 bytes (−16 KB) in size and contains no new matter. 
     TECHNICAL FIELD RELATED TO THE INVENTION 
     This invention relates to DNA regions responsible for the regulation of the alcohol dehydrogenase 3 (ADH3) promoter of  Pichia pastoris  ( Komagatella pastoris ), vectors and expression cassettes containing these DNA regions, and host cells comprising these vectors and expression cassettes. It also includes mutant ADH3 promoters that operate at different strengths that can be used in the production of recombinant protein. 
     STATE OF THE ART 
     The first choice in recombinant protein production is usually a prokaryotic expression system because it is easy and cheap. However, during the production of eukaryotic proteins in prokaryotic systems, problems may be encountered, such as the protein being unstable or not performing its biological activity. The intracellular environment of yeasts is more suitable for the production of eukaryotic proteins than prokaryotic systems. Yeasts as eukaryotic expression systems has ability to perform post-translational biochemical reactions (such as disulfide bond formation, glycosylation), which are essential for eukaryotic proteins. 
       Saccharomyces cerevisiae  is the most widely used eukaryotic host due to its extensive knowledge of genetics and physiology. However, there are some disadvantages of using  S. cerevisiae  in recombinant protein production such as loss of plasmid in large-scale production, hyperglycosylation and low yield. Recently, as an alternative to  S. cerevisiae , a methylotrophic yeast  Pichia pastoris  ( Komagataella pastoris, K. phaffi, K. pseudopastoris ) was developed. In literature, there are different sources which  P. pastoris  called as  Komagataella pastoris, K. phaffi, K. pseudopastoris . Here, it was referred to as  P. pastoris. P. pastoris  yeast as a eukaryotic microorganism has the advantages of  S. cerevisiae  in terms of molecular and genetic manipulations, but it is a more efficient host system than  S. cerevisiae. P. pastoris  is an excellent host for the production of recombinant proteins, particularly in the industrial field. 
     The most commonly used promoter for recombinant protein production with  P. pastoris  is methanol-inducible AOX1 promoter.  P. pastoris , a methylotrophic yeast, requires high levels of enzymes such as alcohol oxidase during its development in methanol media. In the  P. pastoris  genome, the alcohol oxidase gene is present in two copies: AOX1 and AOX2. While AOX1 is responsible for 85% of alcohol oxidase activity, AOX2 is responsible for 15%. The highest expression level in recombinant protein production with  P. pastoris  (22 g/L in intracellular production, up to 15 g/L in extracellular production) was achieved with P AOX1 . In addition, a constitutive P GAP  (glyceraldehyde 3-phosphate dehydrogenase) is also commonly used promoter in  P. pastoris . However, its use is not as common as P AOX1  as it is not suitable for the production of foreign proteins which are toxic to the cell. 
     Alternative  P. pastoris  promoters and expression levels are summarized in Table 1. The approximate expression levels of these promoters were compared with the P GAP  and P AOX1  expression levels. The expression levels measured in different culture conditions are given in intervals. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Alternative  P. pastoris  promoters and expression 
               
               
                 levels (Yogi and Glieder, 2013) 
               
            
           
           
               
               
               
               
            
               
                 Gene 
                   
                   
                   
               
               
                 name 
                 Gene product 
                 Regulation 
                 Expression level 
               
               
                   
               
               
                 AOX1 
                 Alcohol oxidase 1 
                 Induced by 
                 Strong 
               
               
                   
                   
                 methanol 
                 (naturally 5% of mRNA 
               
               
                   
                   
                   
                 and 30% of total protein) 
               
               
                 GAP 
                 Glyceraldehyde 
                 Constitutive 
                 Strong 
               
               
                   
                 3-phosphate 
                   
                 (similar to P AOX1 ) 
               
               
                   
                 dehydrogenase 
               
               
                 AOX2 
                 Alcohol oxidase 2 
                 Induced by 
                 5-10% of P AOX1   
               
               
                   
                   
                 methanol 
               
               
                 DAS 
                 Dihydroxyacetone 
                 Induced by 
                 Strong 
               
               
                   
                 synthase) 
                 methanol 
                 (similar to P AOX1 ) 
               
               
                 ENO1 
                 Enolase 
                 Constitutive 
                 20-70% of P GAP   
               
               
                 FLD1 
                 Formaldehyde 
                 Induced by 
                 Strong 
               
               
                   
                 dehydrogenase 
                 methanol and 
                 (similar to P AOX1 ) 
               
               
                   
                   
                 methylamine 
               
               
                 GPM1 
                 Phosphoglycerate 
                 Constitutive 
                 15-40% of P GAP   
               
               
                   
                 mutase 
               
               
                 PET9 
                 ADP/ATP carrier 
                 Constitutive 
                 10-1700% of P GAP   
               
               
                   
                 of the inner 
               
               
                   
                 mitochondrial 
               
               
                   
                 membrane 
               
               
                 PEX8 
                 Peroxisomal 
                 Induced by 
                 Weak 
               
               
                   
                 matrix protein 
                 methanol or 
               
               
                   
                   
                 oleate 
               
               
                 TEF1 
                 Translation 
                 Constitutive 
                 Strong 
               
               
                   
                 elongation 
                   
                 (similar to P GAP ) 
               
               
                   
                 factor 1 alpha 
               
               
                 THI11 
                 Protein involved 
                 Completely 
                 70% of P GAP   
               
               
                   
                 in thiamine 
                 repressed by 
                 on medium 
               
               
                   
                 biosynthesis 
                 thiamine 
                 lacking thiamin 
               
               
                   
               
            
           
         
       
     
     Studies on using  P. pastoris  expression system more effectively and efficiently are ongoing. Genetic modifications, in natural promoters and development of existing promoters for the purpose of use are among these studies. Thus, expression of a foreign protein can be increased or decreased as desired using the related promoters. 
     Patents related to the regulation of the AOX1 promoter, which is commonly used in the production of recombinant protein with  P. pastoris  are available. Refer. U.S. Pat. Nos. 6,699,691 and 9,012,175. 
     The subject of this invention is  P. pastoris  ADH3 promoter. Although there is a patent on protein expression with this promoter, sec. U.S. Pat. No. 8,222,386, there is no experimental data on DNA regions involved in the regulation of the promoter and the shortest DNA sequence in which ADH3 promoter activity is maintained at 100%. In addition, the patent mentioned does not provide the absolute expression level of the ADH3 promoter. In our previous studies, recombinant protein production in  P. pastoris  with ADH3 promoter was compared with the most commonly used  P. pastoris  promoters AOX1 and GAP. The results showed that in the production of recombinant proteins under fermenter conditions, the specific productivity of the ADH3 promoter was 1.3 times the AOX1 promoter (Karaoglan et al. 2016a). 
     There ae differences in the nomenclature of ADH3 gene in the literature. Genome sequences of  P. pastoris  GS115 and  P. pastoris  DSMZ 70382 strains were recently discovered and one gene encoding alcohol dehydrogenase enzyme was identified (De Schutter et al. 2009; Mattanovich et al. 2009). The same gene sequence was referred to as the ADH1 gene in the aforementioned patent (U.S. Pat. No. 8,222,386) and the promoter region responsible for the regulation of this gene in the same patent was referred to as ADH1 promoter (Cregg and Tolstorukov 2008), and, referred to as ADH2 in second genome study (DSMZ 70382 strain) (Mattanovich et al. 2009). However, in the NCBI database, this gene was identified and named ADH3 in  P. pastoris  (strain GS115) because of the similarity to ADH3 amino acid sequence of  S. cerevisiae  (De Schutter et al. 2009). The ADH3 gene has been characterized by our previous studies and has been shown to be the only gene responsible for ethanol consumption in  P. pastoris  ethanol metabolism (Karaoglan et al. 2016b). All the genes mentioned above have the same gene sequence. In our previous work and in the present invention, it has been referred to as the ADH3 promoter based on the database naming. 
     PURPOSE OF THE INVENTION 
     The invention relates to  P. pastoris  ADH3 promoter. The main purpose of the invention is to improve the properties of  P. pastoris  ADH3 promoter. In this way, protein production can be performed more efficiently. In order for the invention to fulfill this purpose, it is necessary to determine the regions (e.g., regulatory regions and transcription factor binding sites) that play a role in the regulation on the ADH3 promoter (SEQ ID NO: 1). The other purpose depending on the determination of regulator regions is to provide tools for the creation of new synthetic promoters. 
    
    
     
       DESCRIPTION OF FIGURES 
       The method and advantages of the invention will be better understood by means of the figures and their descriptions. 
         FIG.  1   : The construction steps of expression vectors (cassettes) pADH3ZαA-XylB/HIS4 (A. schematic illustration of construction steps with plasmids, B. A1 plasmid, C. A2-1 plasmid, D. A2-2 plasmid, E. A3 plasmid, F. B1 plasmid, G. B2 plasmid, H. C plasmid) 
         FIG.  2   : Schematic representation of ADH3 promoter regions of different lengths 
         FIG.  3   : Schematic representation of the vector pPICZαA/HIS4-XylB. 
         FIG.  4   : Schematic representation of the vectors obtained by replacing the AOX1 promoters with the ADH3 promoters in the plasmid pPICZαA/HIS4-XylB. 
         FIG.  5   : Schematic representation of the deletion analyzes of the ADH3 promoter. It shows the deleted regions and promoter activities. XylB gene encoding xylanase was used as a reporter gene and promoter activities were determined by xylanase activity. 
         FIG.  6   : Graphical representation of the promoter activities obtained by 5′ end-deletion analysis of the ADH3 promoter. 
         FIG.  7   : Graphical representation of the promoter activities obtained by internal deletion analysis of the ADH3 promoter. 
         FIG.  8   : Schematic representation of synthetic promoters. It shows the regions that make up the synthetic promoters, the endonuclease recognition regions used for the binding of the regions and the promoter activities obtained. XylB gene encoding xylanase was used as a reporter gene and promoter activities were determined by xylanase activity. 
     
    
    
     DESCRIPTION OF THE REFERENCES 
     A1: First construction step of expression vector pPICZαA-XylB/HIS4 with A1 plasmid 
     A2-1: Second construction step of expression vector pPICZαA-XylB/HIS4 with A2-1 plasmid 
     A2-2: Second construction step of expression vector pPICZαA-XylB/HIS4 with A2-2 plasmid 
     A3: Third construction step of expression vector pPICZαA-XylB/HIS4 with A3 plasmid 
     B1: First construction step of expression vector pADH3ZαA with B1 plasmid 
     B2: Second construction step of expression vector pADH3ZαA with B2 plasmid 
     C: Expression vector pADH3ZαA-XylB/HIS4 as C plasmid 
     DESCRIPTION OF THE INVENTION 
     The invention relates to the method of expression of a recombinant protein, peptide or functional nucleic acid in a cell, determination of DNA regions responsible for regulation of alcohol dehydrogenase 3 (ADH3) promoter and design of synthetic promoters for recombinant protein production. 
     Within the scope of the invention, ADH3 promoter region was determined as the shortest DNA sequence in which ADH3 promoter activity was maintained at 100% and 5′ end- and internal deletion analyzes were performed on this region determined. The results showed the effects of these regions on promoter activity. Also, synthetic promoters were constructed using the DNA regions known to be effective on promoter activity. It is also possible to form new synthetic promoters of different strengths using the data presented in the present invention. 
     The general method of expression of a recombinant protein, peptide or functional nucleic acid in the cell is as follows; determination of ADH3 promoter to be selected, operably linking promoter to nucleic acid molecule encoding of a protein, peptide or functional nucleic acid and transforming the host cell with vector or the nucleic acid molecule, growth of the transformed host cell under suitable culture conditions, inducing expression of this protein, peptide or functional nucleic acid, and isolation of this protein, peptide or functional nucleic acid. 
     In the following description for the method according to the invention, the (negative) numbers shown in brackets indicate their position relative to the translation initiation codon of the promoter. 
     1. Determination of the ADH3 Promoter 
     In order to determine the regions involved in the regulation of the ADH3 promoter (e.g., regulatory regions and transcription factor binding sites), it is first necessary to determine the promoter region of the ADH3.
         To determine the ADH3 promoter, the 2000 bp DNA region at the 5′ end of the ADH3 gene was examined and divided into four regions.   The shortest DNA region in which ADH3 promoter activity maintained at 100% was determined.   In order to identify the promoter region precisely, 5′ end-deletion analyzes were performed on the shortest DNA region determined in the previous stage.   The results of 5′ end-deletion analysis, the increases and decreases observed in the promoter activity, can provide clues about the regulatory regions.       

     In the first step of the method for obtaining the mutant promoters of the invention, to identify the ADH3 promoter; The 2000 bp DNA region at the 5 ′end of the ADH3 gene was examined as the regions of 2000 (Nucleotide −2000 to 0), 1500 (Nucleotide −1500 to 0), 1000 (Nucleotides −1000 to 0) and 500 (Nucleotide −500 to 0) bp. The shortest DNA region in which the activity of the ADH3 promoter was maintained at 100% was determined to be 1000 bp (Nucleotide −1000 to 0). Then, in order to identify the promoter region precisely, 5′ end-deletion analysis was applied to 1000 bp promoter region and it was serially truncated 100 bp from the 5′ upstream region. 
     5′ end-deletion analysis: To determine the exact length of the ADH3 promoter, the 1000 bp promoter was serially truncated 100 bp from the 5′ end. The promoter regions thus obtained were the DNA regions between −900 to 0, −800 to 0, −700 to 0, −600 to 0, −25 to 500 to 0, −400 to 0, −300 to 0, −200 to 0, −100 to 0 nucleotides. Among the obtained promoters, the shortest DNA region in which activity was unchanged, i.e. the exact promoter region, was determined to be 900 bp (Nucleotide −900 to 0) and this region was identified as ADH3 promoter (SEQ ID NO:1). The promoter activities obtained by 5′ end-deletion analysis were given graphically in  FIG.  6   . 
     2. Determination of Regulatory DNA Regions in the ADH3 Promoter 
     To more accurately identify the DNA regions responsible for regulation in the ADH3 promoter, internal deletion analysis was performed on 900 bp promoter (Sequence ID NO: 1). 
     The regions deleted were the nucleotides between 0 to 99 (−900 to −801), 77 to 176 (−823 to −724), 154 to 253 (−746 to −647), 231 to 330 (−669 to −570), 308 to 407 (−592 to −493), 385 to 484 (−515 to −416), 462 to 561 (−438 to −339), 539 to 638 (−361 to −262), 616 to 715 (−284 to −185), 693 to 792 (−207 to −108), 770 to 814 (−130 to −86) and 200 to 500 (−700 to −400). 
     The promoter activities obtained by internal deletion analysis were given graphically in  FIG.  7   . 
     According to the present invention, the mutations between the nucleotides 0 to 99 (−900 to −801), 462 to 561 (−438 to −339), 539 to 638 (−361 to −262), 616 to 715 (−284 to −185), 693 to 792 (−207 to −108) and 770 to 814 (−130 to −86) on SEQ ID NO:1 were determined to be effective in the regulation of the ADH3 promoter in terms of positive or negative regulation under the ethanol-induced conditions. 
     Also, it was determined that the mutations between 77 to 176 (−823 to −724), 154 to 253 (−746 to −647), 231 to 330 (−669 to −570), 308 to 407 (−592 to −493), 385 to 484 (−515 to −416) and 200 to 500 (−700 to −400) nucleotides on SEQ ID NO: 1 and the combinations of these mutations had no significant effect on the promoter activity. 
     The protein expression yield of the synthetic promoter sequences may vary depending on the number of expression cassettes integrated into the host cell genome. In particular, there was an increase in promoter activity in strains containing more than one copy expression cassette. The clone in which the expression cassette, containing the DNA region from 500 to 900 (−400 to 0) nucleotides on ADH3 promoter (SEQ ID NO:1) as a promoter, was integrated in 2 copies, showed an increase in protein production relative to the single copy clone. 
     3. Design of Synthetic Promoters Using the Promoter Regions that Affect the Strength of the ADH3 Promoter 
     The data on promoter regions that affect the strength of the ADH3 promoter can be used to design promoters with different characteristics. The promoters obtained according to the preferred embodiment of the present invention are used in a yeast cell, preferably a methylotrophic yeast cell. These methylotrophic yeast cells can be  Pichia , especially  P. pastoris, Candida, Hansenula  and  Torulopsis  cells. As in an example, previous studies have shown that the oxygen-regulated ADH promoter of  P. stipitis  was regulated in the same manner in  P. pastoris  yeast (Chien and Lee, 2005). 
     3a. Construction of Expression Vectors (Cassettes) 
     The construction steps of expression cassettes were shown in  FIG.  1   . 
     The construction steps of expression vector pPICZαA-XylB/HIS4 were schematized by line A (A1, A2-1, A2-2 and A3). 
     The construction steps of expression vector pADH3ZαA were schematized by line B (B1 and B2). 
     The expression vector obtained from line A, pPICZαA-XylB/HIS4 and the expression vector obtained from line B, pADH3ZαA were combined and the expression vector pADH3ZαA-XylB/HIS4 schematized by line C was obtained. 
     The native ADH3 promoter regions of different lengths were amplified by PCR using the forward primer containing BamHI restriction site and the reverse primer containing AsuII restriction. PCR analyzes were performed using KOD Hot Start DNA Polymerase Kit according to the manufacturer&#39;s instructions. Mutant ADH3 promoter regions were obtained using overlap PCR method. The primers used in PCR reactions and the ADH3 promoter regions obtained were given in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Primer list (Macrogen Inc., Seoul, South Korea) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Melting 
               
               
                   
                   
                   
                 Temperature 
               
               
                   
                 SEQ ID No: 
                 Name 
                 [° C.] 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 2 
                 1000p-F 
                 75.0 
               
               
                   
                 3 
                 900p-F 
                 74.7 
               
               
                   
                 4 
                 800p-F 
                 74.2 
               
               
                   
                 5 
                 700p-F 
                 66.2 
               
               
                   
                 6 
                 600p-F 
                 69.0 
               
               
                   
                 7 
                 500p-F 
                 72.0 
               
               
                   
                 8 
                 400p-F 
                 69.0 
               
               
                   
                 9 
                 300p-F 
                 67.6 
               
               
                   
                 10 
                 200p-F 
                 70.4 
               
               
                   
                 11 
                 100p-F 
                 66.2 
               
               
                   
                 12 
                 ADH3-R 
                 63.0 
               
               
                   
                 13 
                 ADH3F -801 
                 74.4 
               
               
                   
                 14 
                 ADH3R -823 
                 75.5 
               
               
                   
                 15 
                 ADH3F -724 
                 75.5 
               
               
                   
                 16 
                 ADH3R -746 
                 76.6 
               
               
                   
                 17 
                 ADH3F -647 
                 76.6 
               
               
                   
                 18 
                 ADH3R -669 
                 76.6 
               
               
                   
                 19 
                 ADH3F -570 
                 76.6 
               
               
                   
                 20 
                 ADH3R -592 
                 69.8 
               
               
                   
                 21 
                 ADH3F -493 
                 69.8 
               
               
                   
                 22 
                 ADH3R -515 
                 74.4 
               
               
                   
                 23 
                 ADH3F -416 
                 74.4 
               
               
                   
                 24 
                 ADH3R -438 
                 75.5 
               
               
                   
                 25 
                 ADH3F -339 
                 75.5 
               
               
                   
                 26 
                 ADH3R -361 
                 76.6 
               
               
                   
                 27 
                 ADH3F -262 
                 76.6 
               
               
                   
                 28 
                 ADH3R -284 
                 76.6 
               
               
                   
                 29 
                 ADH3F -185 
                 76.6 
               
               
                   
                 30 
                 ADH3R -207 
                 73.2 
               
               
                   
                 31 
                 ADH3F -108 
                 73.2 
               
               
                   
                 32 
                 ADH3R -130 
                 74.4 
               
               
                   
                 33 
                 ADH3F -86 
                 74.4 
               
               
                   
                 34 
                 ADH3R -700 
                 72.1 
               
               
                   
                 35 
                 ADH3F -400 
                 72.1 
               
               
                   
                   
               
            
           
         
       
         
         
           
             The construction steps (A1, A2-1, A2-2 and A3) of expression vector pPICZαA-XylB/HIS4 given in line A; 
           
         
       
    
     Plasmid A1 shown in  FIG.  1   , the commercial pPICZαA, was ligated to the reporter XylB gene from the XhoI-XbaI cloning site to obtain A2-1 and was named pPICZαA-XylB. 
     Plasmid A2-2, commercial pAO815, was used to obtain the HIS4 gene used as marker. The HIS4 gene obtained by cutting with BglII-BamHI was ligated to the pPICZαA-XylB which was linearized with BamHI to obtain the A3 plasmid and was named pPICZαA-XylB/HIS4.
         The construction steps (B1 and B2) of expression vector pADH3ZαA given in line B;       

     B1, the commercial pPICZαA, was digested with BglII-AsuII to excise the AOX1 promoter. The new promoterless construct was ligated to the PCR products of different ADH3 promoters digested with BamHI-AsuII and B2 was obtained. The plasmid B2 is named pADH3ZαA.
         Ligation of pPICZαA-XylB/HIS4 obtained from line A and pADH3ZαA obtained from line B to obtain pADH3ZαA-XylB/HIS4 shown in line C       

     pPICZαA-XylB/HIS4 (A3) and pADH3ZαA (B2) plasmids were ligated from the XhoI-SmaI restriction site. The ligated fragments are the portion containing ADH3 promoter in pADH3ZαA and the portion containing the XylB and HIS4 genes in pPICZαA-XylB/HIS4 plasmids that were obtained by XhoI-SmaI digestion reactions of both plasmids. The final plasmid was named pADH3ZαA-XylB/HIS4. 
     3b. Transformation (Integration) of Expression Vectors into Host Cell Genome 
     All ADH3 expression plasmids of the invention were generated by the same strategy. Plasmids were linearized with Kpn2I and integrated into the host cell genome from the his4 locus.  P. pastoris  GS115 was used as the host cell. 
     The integrated host cells were grown in appropriate culture conditions to produce xylanase enzyme (reporter protein) under the control of promoters constructed within the scope of the invention. 
     It is possible to carry out different protein productions in different yeast cells using mutant ADH3 promoters and novel mutant promoters which can be formed as addition, deletion, substitution and inversion of the promoter regions of ADH3. 
     An example of the use of the invention in methylotrophic yeast cells is given in Example 1 for use in the  P. pastoris  cell. 
     Example 1 
     Material and Methods 
     Strains, Plasmids and Media 
       P. pastoris  strains used in studies were X33 (Wild-type) and GS115 (his4, Invitrogen). The media used for  P. pastoris : YPD (1% yeast extract, 2% peptone, 2% glucose), MG (1.34% YNB, 4×10 −5 % biotin and 2% glycerol) and ME (1.34% YNB, 4×10 −5 %). biotin and 1% ethanol), BMGY (2% peptone, 1% yeast extract, 1.34% YNB, 4×10 −5 % biotin and 2% glycerol) and BMEY (2% peptone, 1% yeast extract, 1.34% YNB, 4×10% −5  biotin and 1% ethanol). 
     As the expression vector for the recombinant protein production, pADH3ZαA-XylB/HIS4, the plasmid containing the native and mutant ADH3 promoters, constructed in accordance with the invention was used.  Aspergillus niger  xylanase gene (XylB) was used as the reporter gene.  Escherichia coli  XL1-Blue strain was used for cloning and propagation of plasmids. LB medium (0.5% yeast extract, 1% peptone and 1% NaCl) was prepared with appropriate antibiotic additions to grow  E. coli  cells. 
     General Molecular Biology Techniques 
     The restriction endonucleases and their buffer solutions were obtained from Fermentas (MD, USA). DNAMAN 7.0 (Lynnon Corporation) was used for all primer designs and DNA analyzes. Primers were obtained from Macrogen Inc. (Seoul, South Korea). The National Biotechnology Information Center Network Service (Bethesda, Md., USA; http://www.ncbi.nlm.nlh.gov) was used for screening of gene and protein sequences. Molecular cloning techniques were performed in accordance with Sambrook and Russel (2001) and kit protocols. Southern blot method was used for the confirmation of the integration of the expression cassette into the genome and the determination of clones containing a single copy expression cassette. In the Southern blot analysis, 527 bp HIS4 fragment which was amplified by PCR from plasmid pPIC3.5K and labeled with digoxigenin (DIG) was used as probe. 
     Determination of Xylanase Enzyme Activity 
     Xylanase activity was determined by measuring the reducing sugar released from the xylan (Miller 1959). The supernatant sample (0.1 mL) was added to 0.9 mL of 0.05 M sodium citric acid buffer (pH 5.00) containing 1% (w/v) beechwood xylan. The reaction mixture was incubated at 50° C. for 5 minutes. At the end of the incubation period, 100 μl of the reaction mixture was added into the 900 μl of DNSA to stop the reaction. The mixture was incubated at boiling water bath for 5 minutes and after cooling, absorbance values were recorded spectrophotometrically at a wavelength of 540 nm. The amount of reducing sugar was determined by the dinitrosalicylic acid (DNSA) method. The blank samples were prepared by stopping the reaction with DNSA solution immediately after the enzyme was added to the substrate. One unit of xylanase activity was defined as the amount of enzyme needed to produce 1 μmol of reducing sugar (equivalent to glucose) under experimental conditions at 1 minute. The standard curve was plotted with 1-10 μmol xylose. 
     Results 
     Determination of ADH3 Promoter Region 
     Expression vectors containing the DNA regions of the 2000, 1500, 1000 and 500 bp in the 5′ upstream portion of the ADH3 gene as promoters were integrated into the genome of the  P. pastoris  GS115 strain from his4 locus. Transformants containing a single copy expression cassette were selected and their copy number confirmed by Southern blot analysis (result not shown here). The transformants were inoculated into the medium containing glycerol and grown at 28° C. on a shaker for about 16 hours. The grown cells were transferred to the ethanol medium at the equal optical densities, and the supernatants were collected at the 8th hour of incubation. Activities of different length promoters were determined indirectly by measuring xylanase activity in supernatants. The results were determined relatively by normalizing the promoter activities to that of 2000 bp promoter defined as 100%. There was no difference in the tendency of promoter activities between minimal and rich media. The shortest DNA region in which the ADH3 promoter activity maintained unchanged was determined to be 1000 bp between the promoters 2000, 1500, 1000 and 500 bp (data not shown). 
     For a more accurate analysis of the promoter region, 1000 bp DNA region of the ADH3 promoter was serially truncated 100 bp from the 5′ end and deletion analysis was continued to 100 bp DNA region. The results showed that the shortest DNA region in which the ADH3 promoter activity was 100% continued to be 900 bp and the ADH3 promoter region for further studies was determined as 900 bp (SEQ ID No: 1). The deletion of the region of −900 to −800 on the SEQ ID No: 1 caused a 30% reduction in promoter activity. This result can be expressed as the maintenance of 70% of the promoter activity with 800 bp promoter (Nucleotides −800 to 0). The promoter activity was maintained at about 70% in the DNA regions of 700 (nucleotide −700 to 0), 600 (nucleotide −600 to 0), 500 (nucleotide −500 to 0) and 400 (nucleotide −400 to 0). This means that 5′ end-deletion analysis between −800 and −400 did not have an effect of increasing or decreasing the promoter activity. The promoter activity was approximately 130% with a 300 bp promoter (Nucleotide −300 to 0), and approximately 95% with a 200 bp promoter (Nucleotide −200 to 0) and about 6% with a 100 bp promoter (Nucleotide −100 to 0). Deletion of the repressor region results in an increase in promoter activity, while deletion of the activator region causes a decrease in promoter activity. The results showed that the promoter activity is 70% with 400 bp promoter and 130% with 300 bp promoter showed that there may have been a repressor region between −400 and −300 nucleotides. Promoter activity of the 200 bp region (Nucleotide −200 to 0) was obtained 95%, but this was a significant decrease in activity relative to the 300 bp promoter. This indicated that the activator sites could be located in a region between the nucleotides −300 and −200. Increases and decreases in promoter activity during 5′ end-deletion can give some idea about regulator regions, but internal deletion analysis is required to obtain more accurate results. 
     Mutant ADH3 Promoters 
     Internal deletion analyzes (SEQ ID No: 1) were performed by overlap PCR method. The resulting mutant ADH3 promoters were ligated to expression vectors. The linearized plasmids were then integrated into the  P. pastoris  GS115 his4 locus. Transformants containing a single copy expression cassette were selected and their copy number was confirmed by Southern blot analysis (data not shown). 
     In the expression studies, the transformants were inoculated into the medium containing glycerol and grown at 28° C. for about 16 hours in the shaker. The grown cells were transferred to medium containing ethanol with equal optical density. The supernatants were collected at the 8th hour of incubation. Activities of the mutant ADH3 promoters were determined by measuring xylanase activity in the supernatants. The results were determined relatively by normalizing the promoter activities to that of 900 bp promoter defined as 100%. 
     Deleted regions in ADH3 promoter structures was given in Table 3. Deleted regions: Nucleotides 0 to 99 (−900 to −801), 77 to 176 (−823 to −724), 154 to 253 (−746 to −647), 231 to 330 (−669 to −570), 308 to 407 (−592 to −493), 385 to 484 (−515 to −416), 462 to 561 (−438 to −339), 539 to 638 (−361 to −262), 616 to 715 (−284 to −185), 693 to 792 (−207 to −108), 770 to 814 (−130 to −86) and 200 to 500 (−700 to −400). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Deleted sequences inADH3 promoter structures 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Position* 
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Construct 
                 5′ 
                 3′ 
                 SEQ ID No: 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 ΔA 
                 −900 
                 −801 
                 36 
               
               
                   
                 ΔB 
                 −823 
                 −724 
                 37 
               
               
                   
                 ΔC 
                 −746 
                 −647 
                 38 
               
               
                   
                 ΔD 
                 −669 
                 −570 
                 39 
               
               
                   
                 ΔE 
                 −592 
                 −493 
                 40 
               
               
                   
                 ΔF 
                 −515 
                 −416 
                 41 
               
               
                   
                 ΔG 
                 −438 
                 −339 
                 42 
               
               
                   
                 ΔH 
                 −361 
                 −262 
                 43 
               
               
                   
                 ΔI 
                 −284 
                 −185 
                 44 
               
               
                   
                 ΔJ 
                 −207 
                 −108 
                 45 
               
               
                   
                 ΔK 
                 −130 
                 −86 
                 46 
               
               
                   
                 Δ700-400 
                 −700 
                 −400 
                 47 
               
               
                   
                   
               
               
                   
                 *The positions were given according to SEQ ID No: 1 
               
            
           
         
       
     
     According to the present invention, mutations between the nucleotides from 0 to 99 (−900 to −801), 462 to 561 (−438 to −339), 539 to 638 (−361 to −262), 616 to 715 (−284 to −185), 693 to 792 (−207 to −108) and 770 to 814 (−130 to −86) on SEQ ID NO: 1 have been found to be effective in the regulation of the ADH3 promoter activity. 
     The results showed that the deletion between the 0 to 99 (−900 to −801) nucleotides resulted in a decrease of about 30% in the promoter activity, while the deletion between the nucleotides 462 to 561 (−438 to −339) was about 20% in the promoter activity; deletion of the region 616 to 715 (−284 to −185) is about 70%; deletion of the region 693 to 792 (−207 to −108) is about 70%; deletion of the region 770 to 814 (−130 to −86) resulted in a decrease of about 30%. These regions, which cause a decrease in the promotor activity as a result of internal deletion analyzes, are the regions responsible for the positive regulation in the promoter activity (UAS, Upstream Activator Site). 
     Internal deletion between 539 to 638 (−361 to −262) nucleotides on SEQ ID NO: 1 caused a 63% increase in ADH3 promoter activity. This region in which deletion analysis results in increased promoter activity, is a region responsible for negative regulation in promoter activity (URS, Upstream Repressor Site). 
     Comparison of internal deletion analysis and 5′ end-deletion analysis showed that internal deletion between 539 and 638 (−361 to −262) nucleotides resulted in a 63% increase in ADH3 promoter activity and 5′ end-deletion analysis between −400 and −300 in the resulted in a 60% increase in the promoter activity, from about 70% to 130%. 
     No significant difference in activity was observed between 5′ end-deletion analyzes between the 700 (−700 to 0), 600 (−600 to 0), 500 (−500 to 0), and 400 (−400 to 0) bp ADH3 promoters. The results indicated that there were no regulatory sequences in these regions responsible from promoter activity. Also, in internal deletion assays, when the DNA region between 200 and 500 (−700 to −400) in SEQ ID NO: 1 was deleted, the promoter activity was maintained at 94%. It has also been identified that mutations between the nucleotides 77 to 176 (−823 to −724), 154 to 253 (−746 to −647), 231 to 330 (−669 to −570), 308 to 407 (−592 to −493), 385 to 484 (−515 to −416) do not have a significant effect on promoter activity. 
     The present invention provides important data on the positive or negative regulatory regions on the ADH3 promoter. This data obtained by 5′ end- and internal deletion analysis has been summarized in  FIG.  5   . Using this data, it is possible to obtain ADH3 promoters of different strengths. 
     Construction of Synthetic ADH3 Promoters 
     The results of the deletion assays identified nucleotides from 0 to 99 (−900 to −801) as UAS1; nucleotides 616 to 792 (−284 to −108) as UAS2; nucleotides 539 to 638 (−361 to −262) as URS1 on SEQ NO:1. Five different synthetic promoters were constructed using activator (UAS1 and UAS2) regions defined. All synthetic promoters created had a region of nucleotide 792 to 900 (−108 to 0) containing the ADH3 promoter TATA box. The ADH3-SNT5 promoter further comprises the fragment comprising the nucleotides from 100 to 538 (−800 to −362). 
     Enzyme recognition sites were used for combining the regions used in the construction of synthetic promoters. The regions included in the synthetic promoters and the endonuclease enzyme recognition sites used to combine these regions are shown schematically in  FIG.  8   . 
     The regions used in the construction of the synthetic promoters were amplified by PCR method and the PCR products were digested with the enzymes of recognition regions shown in  FIG.  8   . The fragments were ligated from these regions using the ligase enzyme. The DNA sequences of the synthetic promoters constructed were given in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 The DNA sequences of the synthetic promoters 
               
            
           
           
               
               
               
            
               
                   
                 Promoter 
                 SEQ ID No: 
               
               
                   
                   
               
               
                   
                 ADH3-SNT1 
                 48 
               
               
                   
                 ADH3-SNT2 
                 49 
               
               
                   
                 ADH3-SNT3 
                 50 
               
               
                   
                 ADH3-SNT4 
                 51 
               
               
                   
                 ADH3-SNT5 
                 52 
               
               
                   
                   
               
            
           
         
       
     
     The resulting synthetic promoters were ligated to the expression vector by the strategy used in the deletion analysis ( FIG.  1   ). The linearized plasmids are integrated into the  P. pastoris  GS115 his4 locus. 
     In the protein expression studies, the transformants were cultured in glycerol-containing medium at 28° C. for about 16 hours in shaking incubator. The grown cells were transferred to the ethanol medium in equal their optical densities and the supernatants were collected at the 8th hour of incubation. The activity of synthetic promoters was determined by measuring xylanase activity in supernatants. The results were calculated relatively by normalizing the promoter activities to that of 900 bp promoter defined as 100%. 
     The results showed that the promoter activities of ADH3-SNT1, ADH3-SNT2, ADH3-SNT3, ADH3-SNT4 and ADH3-SNT5 were 164%, 167%, 173%, 197% and 201%, respectively. 
     REFERENCES 
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         U.S. Pat. No. 6,699,691 B2 March 2004 Inan et al. 
         U.S. Pat. No. 9,012,175 B2 April 2015 Hartner et al. 
         U.S. Pat. No. 8,222,386 B2 July 2012 Cregg et al. 
       
    
     Others 
     
         
         Chien, L. J., Lee, C. K. 2005. “Expression of bacterial hemoglobin in the yeast,  Pichia pastoris , with a low O2-induced promoter”, Biotechnology Letters, 27 (19), 1491-1497. 
         De Schutter, K., Lin Y. C., Tiels P., Van Hecke A., Glinka S., Weber-Lenhmann J., Rouze P., Peer Y., Van D. E. and Callewaert N. 2009. “Genome sequence of the recombinant protein production host  Pichia pastoris ”, Nature Biotechnology, 27, 6, 561-566. 
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