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WO2010134096A2 - Souche de levure exprimant le cytochrome p450 réductase humain modifié et/ou un gène du cytochrome p450 - Google Patents

Souche de levure exprimant le cytochrome p450 réductase humain modifié et/ou un gène du cytochrome p450 Download PDF

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Publication number
WO2010134096A2
WO2010134096A2 PCT/IN2010/000322 IN2010000322W WO2010134096A2 WO 2010134096 A2 WO2010134096 A2 WO 2010134096A2 IN 2010000322 W IN2010000322 W IN 2010000322W WO 2010134096 A2 WO2010134096 A2 WO 2010134096A2
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seq
cytochrome
yeast
yeast strain
protease
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WO2010134096A3 (fr
Inventor
Rajeev Soni
Prabuddha Kumar Kundu
Kajal Arora
Purna Chandra Rao
Kunal Bhalla
Sunil Kumar Suman
Vipul Kumar
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PREMAS BIOTECH PVT Ltd
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PREMAS BIOTECH PVT Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90209—Oxidoreductases (1.) acting on NADH or NADPH (1.6), e.g. those with a heme protein as acceptor (1.6.2) (general), Cytochrome-b5 reductase (1.6.2.2) or NADPH-cytochrome P450 reductase (1.6.2.4)

Definitions

  • the present invention relates to a genetically modified yeast strain comprising integrated modified human cytochrome P450 reductase and/or a plasmid comprising modified human cytochrome P450, wherein the strain is useful for heterologous over-expression of the human recombinant cytochrome P450 (s).
  • cytochrome P450 BACKGROUND OF THE INVENTION
  • Cytochrome P450 is a superfamily of haemoproteins that catalyse the oxidation of a wide variety of endogenous and xenobiotic chemicals, including therapeutic drugs and carcinogens.
  • P450s have been found in all tissues but are localized mainly in the liver. P450- dependent metabolism requires two protein components, P450 and NADPH-P450 oxidoreductase. Both enzymes are embedded in the membrane of endoplasmic reticulum, and cytochrome P450 reductase shuttles electrons from NADPH to P450 for its functional activity.
  • NADPH + H + + O 2 + RH NADP + + H 2 O + R-OH
  • P450s The role of P450s in drug metabolism has made the P450 enzyme system an important tool in drug development process. Although most of the interest in the pharmaceutical industry has focused on the role of cytochrome P450s in drug development, these enzymes also offer potential in the discovery not only of drugs, but also of other useful chemicals.
  • Microsomal preparations from liver or cellular systems such as liver slices and human hepatocytes have been extensively used and provide the advantage of a maintained cellular integrity containing other enzymes, cofactors, transporters etc., contributing to the activity as well. These systems, however, are not useful for generating large amount of cytochrome P450.
  • in vivo experiments using animal models have been replaced by in vitro studies using human enzymes from different sources. Recombinant expression methods for the production of large quantity of these enzymes alone or in combination have proven useful for applications such as drug metabolism and drug-drug interactions.
  • recombinant expression systems are available today to produce large quantities of recombinant proteins. Whether it is a prokaryotic system, viral system, systems mimicking eukaryotic expression systems like yeast, insect or mammalian cell line expression system, using the right expression system for a particular application is the key to success. Often, model organisms are chosen on the basis that they are amenable to experimental manipulation. This usually includes characteristics such as size, generation time, cell culture complexity, accessibility, genetic manipulation, conservation of mechanisms, expression level, extracellular expression, post-translational modification & processing, scale-up and potential economic benefit.
  • heterologous gene Another important factor required for desired expression of a heterologous gene is the choice of correct host system as explained above. Mammalian cells as hosts for heterologous expression is best for the expression of human genes but unfortunately this is associated with low yields, high cost and long generation times. On the other hand prokaryotic system, although results in high protein yields in considerably less time but lacks the post-translational modifications and processing required for most human proteins.
  • Yeast has been the best choice of a eukaryotic system as being a single-celled eukaryote, has proven to be more like human cells in its molecular structure and function than anyone imagined. The cell cycle in yeast is very similar to the cell cycle in humans, and regulated by homologous proteins. It is less expensive to scale up when compared over other more complex systems like insects and mammalian cells that exhibit slow growth. Additional features include high expression and the ability to perform post translational modifications as compared to E. coli based systems.
  • Vacuoles or lysosomal compartment are known to be the major site for protein degradation/turnover in cells, and are populated by vacuolar proteases and several other luminal hydrolases and factors that contribute to protein degradation/turnover. Proteolysis can occur during expression or during the first stages of purification. Multiple research publications and literature reviews have demonstrated that the protein degradation can be decreased by deleting the major protease producing genes. "Proteases" refer to a group of enzymes whose catalytic function is to breakdown proteins, by hydrolysis of the peptide bonds. They are also called proteolytic enzymes or proteinases. Consequently, production of many proteins, particularly heterologous expressed proteins by use of strains that are deficient in proteases can significantly improve overall yields (Martin A. G.
  • cytochrome P450s have been established in several organisms. Saccharomyces cerevisiae was used as the first host for the heterologous expression of mammalian cytochrome P450 (Oeda K, Sakaki T, Ohkawa H., DNA. 1985 Jun; 4(3): 203-10). Although various P450 isozymes have been successfully expressed in S. cerevisiae, their expression levels fluctuated (Gonzalez FJ, Korzekwa KR, Annu Rev Pharmacol Toxicol. 1995; 35: 369-90. Review and US Patents namely US 5635369 and US 6579693) and some P450 isozymes, for unknown reasons, were not expressed in S. cerevisiae.
  • Bellamine, et al. (US patent 6579693) describes simultaneous replacement of both the endogenous yeast NADPH-cytochrome P450 reductase and the endogenous yeast cytochrome b5 with their human homologues to obtain a genetically modified yeast strain expressing human NADPH-cytochrome P450 reductase and cytochrome b5.
  • the modified yeast strain described by Bellamin et al comprises a plasmid carrying a nucleotide sequence coding for human cytochrome P450.
  • an additional feature that needs to be considered is the optimization of codon usage of the host organism by modifying the DNA sequence to be expressed, thereby maintaining the amino acid integrity and functionality of the expressed protein.
  • codon biasing alone, of any sequence as per the desired host may not be sufficient for the good protein expression.
  • Further optimization of the gene sequence along with codon biasing is critical for enhanced expression of the desired protein.
  • Another feature of utmost importance for the efficient protein expression is the Kozak consensus sequence which plays a major role in the initiation of translation process in eukaryotes in addition to other factors.
  • Pompon, et al. (US patent 5635369) describes a diploid yeast strain having integrated NADPH-cytochrome P450 reductase and cytochrome b5 genes and a plasmid carrying a cassette for expression of the heterologous cytochrome P450 gene.
  • the yeast strain co-expresses the human NADPH reductase and cytochrome b5 along with cytochrome P450.
  • the present invention relates to development of a protease-deficient haploid yeast strain by respective disruption of major protease encoding genes of S. cerevisiae and further modification of yeast genome by integration of yeast sequence biased human cytochrome P450 reductase gene to be used for high level expression of catalytically active cytochrome P450s.
  • One aspect of the present invention relates to a modified DNA sequence selected from a group consisting of nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified sequence codes for human cytochrome P450 reductase.
  • Another aspect of the present invention relates to a modified DNA sequence selected from a group consisting of nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, wherein the modified DNA sequence codes for cytochrome b5.
  • Another aspect of the present invention relates to a recombinant DNA molecule comprising the polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 41.
  • Another aspect of the present invention relates to a yeast strain comprising the modified
  • yeast strain comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for a cytochrome P450, wherein the yeast strain is a protease A and protease B deficient strain.
  • Another aspect of the present invention relates to a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising integrated modified DNA sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified DNA sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5; contacting the substrate with the microsomal fraction from the said yeast strain; and analyzing metabolites of the substrate.
  • Yet another aspect of the present invention relates to a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising integrated modified DNA sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified DNA sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
  • Yet another aspect of the present invention relates to a process of producing cytochrome P450 using protease A and protease B deficient strain comprising an integrated modified DNA sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified DNA sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, wherein the process comprises growing the yeast strain by using the conventional method to obtain cytochrome P450.
  • Yet another aspect of the present invention relates to use of the yeast strain comprising the modified DNA sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 for substrate analysis.
  • Still yet another aspect of the present invention relates to a modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: , 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence codes for cytochrome P450.
  • Still yet another aspect of the present invention relates to a recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence codes for cytochrome P450, wherein the modified DNA sequence is operably linked to a promoter sequence.
  • yeast strain comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the yeast strain is a protease A and protease B deficient strain.
  • Another aspect of the present invention relates to a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising the modified DNA sequence coding for cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 and a polynucleotide sequence coding for cytochrome b5; contacting the substrate with the microsomal fraction
  • Yet another aspect of the present invention relates to a process of producing cytochrome P450, the process comprising growing protease A and protease B deficient yeast strain comprising the modified DNA sequence coding for cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 by using a conventional method to obtain cytochrome P450.
  • yeast strain comprising the modified DNA sequence coding for cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 for substrate analysis.
  • Figure 2 shows construction of PYPD strain with stable integration of modified human cytochrome P450 reductase for expression of modified human CYP3A4 under the galactose promoter
  • Figure 3 shows construction of PYPD strain with stable integration of modified human cytochrome P450 reductase for expression of modified human CYP3A4 under the galactose promoter in the presence of modified human cytochrome b5
  • Figure 4 shows construction of PYPD strain with stable integration of modified cytochrome P450 reductase for expression of modified human CYP3A4 under the alcohol dehydrogenase promoter
  • Figure 5 shows construction of PYPD strain with stable integration of modified human cytochrome P450 reductase for expression of modified human CYP3A4 under the alcohol dehydrogenase promoter in the presence of modified human cytochrome b5
  • the present invention discloses an exemplary sequence listing containing both the nucleotide and amino acid sequence of human cytochrome P450 reductase gene wild type and modified and cytochrome b5.
  • SEQ ID NO: 1 shows nucleotide sequence of human CYP 1A2 (Wild type)
  • SEQ ID NO: 2 shows modified and optimized nucleotide sequence of human CYP1A2 as set forth in SEQ ID NO: 1
  • SEQ ID NO: 3 shows partially modified and partially optimized sequence of nucleotide sequence of human CYP 1A2 as set forth in SEQ ID NO: 1
  • SEQ ID NO: 4 shows partially modified and partially optimized nucleotide sequence of human CYP I A2 as set forth in SEQ ID NO: 1
  • SEQ ID NO: 5 shows nucleotide sequence of human CYP2B6 (It is a mutated sequence having natural mutation at 3 positions, wherein thymine is substituted with cytosine at position 437, cytosine at position 499 and adenine at position 501 are substituted with guanine and cytosine respectively; and thymine at positions 712 and 713 are substituted with cytosine).
  • SEQ ID NO: 6 shows modified and optimized nucleotide sequence of human CYP2B6 as set forth in SEQ ID NO: 5
  • SEQ ID NO: 7 shows partially modified and partially optimized nucleotide sequence of human CYP2B6 as set forth in SEQ ID NO: 5
  • SEQ ID NO: 8 shows partially modified and partially optimized nucleotide sequence of human CYP2B6 as set forth in SEQ ID NO: 5
  • SEQ ID NO: 9 shows nucleotide sequence of human CYP2C8 (Wild type)
  • SEQ ID NO: 10 shows modified and optimized nucleotide sequence of human CYP2C8 as set forth in SEQ ID NO: 9
  • SEQ ID NO: 11 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 12 shows partially modified and partially optimized nucleotide sequence of human CYP2C8 as set forth in SEQ ID NO: 9
  • SEQ ID NO: 13 shows nucleotide sequence of human CYP2C9 (Wild type with additional TCT after ATG)
  • SEQ ID NO: 14 shows modified and optimized nucleotide sequence of human CYP2C9 as set forth in SEQ ID NO: 13
  • SEQ ID NO: 15 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 16 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 17 shows nucleotide sequence of human CYP2C19 (Wild type with additional TCT after ATG)
  • SEQ ID NO: 18 shows modified and optimized nucleotide sequence of human CYP2C19 as set forth in SEQ ID NO: 17
  • SEQ ID NO: 19 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 20 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 21 shows nucleotide sequence of human CYP2D6 (Wild type)
  • SEQ ID NO: 22 shows modified and optimized nucleotide sequence of human CYP2D6 as set forth in SEQ ID NO: 21
  • SEQ ID NO: 23 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 24 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 25 shows nucleotide sequence of human CYP2E1 (Wild type with additional TCT after ATG)
  • SEQ ID NO: 26 shows modified and optimized nucleotide sequence of human CYP2E1 as set forth in SEQ ID NO: 25
  • SEQ ID NO: 27 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 28 shows partially modified and partially optimized nucleotide sequence of human CYP2E1 as set forth in SEQ ID NO: 25
  • SEQ ID NO: 29 shows nucleotide sequence of human CYP3A4 (Wild type)
  • SEQ ID NO: 30 shows modified and optimized nucleotide sequence of human CYP3A4 as set forth in SEQ ID NO: 29
  • SEQ ID NO: 31 shows partially modified and partially optimized nucleotide sequence of human CYP3A4 as set forth in SEQ ID NO: 29
  • SEQ ID NO: 32 shows partially modified and partially optimized nucleotide sequence of human
  • SEQ ID NO: 33 shows nucleotide sequence of human cytochrome P450 reductase (Wild type)
  • SEQ ID NO: 34 shows modified and optimized nucleotide sequence of human cytochrome P450 reductase as set forth in SEQ ID NO: 33
  • SEQ ID NO: 35 shows partially modified and partially optimized nucleotide sequence of human cytochrome P450 reductase as set forth in SEQ ID NO: 33
  • SEQ ID NO: 36 shows partially modified and partially optimized nucleotide sequence of human cytochrome P450 reductase as set forth in SEQ ID NO: 33
  • SEQ ID NO: 37 shows nucleotide sequence of human cytochrome b5 (It is a mutated sequence having mutation at position 287 wherein cytosine is substituted with thymine).
  • SEQ ID NO: 38 shows modified and optimized nucleotide sequence of human cytochrome b5 as set forth in SEQ ID NO: 37
  • SEQ ID NO: 39 shows partially modified and partially optimized nucleotide sequence of human . cytochrome b5 as set forth in SEQ ID NO: 37
  • SEQ ID NO: 40 shows partially modified and partially optimized nucleotide sequence of human cytochrome b5 as set forth in SEQ ID NO: 37
  • SEQ ID NO: 41 shows recombinant polynucleotide sequence comprising modified and optimized nucleotide sequence of human cytochrome b5 as set forth in SEQ ID NO: 38 under the control of GALl promoter along with CYCl terminator
  • SEQ ID NO: 42 shows amino acid sequence of human CYP 1A2 protein
  • SEQ ID NO: 43 shows amino acid sequence of human CYP2B6 protein
  • SEQ ID NO: 44 shows amino acid sequence of human CYP2C8 protein
  • SEQ ID NO: 45 shows amino acid sequence of human CYP2C9 protein
  • 46 shows amino acid sequence of human CYP2C19 protein
  • SEQ ID NO: 47 shows amino acid sequence of human CYP2D6 protein
  • SEQ ID NO: 48 shows amino acid sequence of human CYP2E1 protein
  • SEQ ID NO: 49 shows amino acid sequence of human CYP3A4 protein
  • SEQ ID NO: 50 shows amino acid sequence of human cytochrome P450 reductase
  • SEQ ID NO: 51 shows amino acid sequence of human cytochrome b5
  • DNA DNA
  • polynucleotide and “nucleotide” used herein can be used interchangeably.
  • protease deficient yeast strain refers to the genetically engineered yeast strain with deletion of protease A and protease B coding genes.
  • modified refers to modification (s) carried out in the DNA sequences coding for human cytochrome P450 reductase, human cytochrome P450 or human cytochrome b5, wherein the sequence was modified using codon usage of the yeast for higher expression of the protein.
  • optimal refers to multi-parameter gene optimization of yeast codon biased sequence for critical elements like GC content, cryptic splice sites, premature poly (A), direct repeats, RNA secondary structures, killer sequences and internal RBS binding sites whose presence may lead to poor yield of heterologous proteins in an in-vitro system.
  • the present invention relates to development of a protease-deficient haploid yeast strain by respective disruption of major protease encoding genes of S. cerevisiae and further modification of yeast genome by integration of yeast sequence biased human cytochrome P450 reductase for high level expression of catalytically active cytochrome P450s.
  • the present invention particularly relates to a genetically engineered protease A and B deficient yeast strain comprising integrated yeast sequence biased human cytochrome P450 reductase for heterologous over-expression of the human recombinant cytochrome P450.
  • the yeast strain disclosed in the present invention is useful for heterologous over-expression of yeast sequence biased catalytically active cytochrome P450s with higher specific activity over wild type sequence expressed cytochrome P450.
  • cDNA encoding human cytochrome P450 (s) was co-expressed with their regulatory partners such as human cytochrome P450 reductase and/or human cytochrome b5 in a genetically engineered protease deficient haploid Saccharomyces cerevisiae strain.
  • the yeast strain (s) disclosed in the present invention possesses complete endogenous system of regulatory enzymes. These strains were used for assessing the activity of human cytochrome P450 (s). It was observed that the human cytochrome P450 coded by the wild type sequence resulted in no significant activity as compared to human cytochrome P450 encoded by the modified DNA sequence.
  • the yeast expression system disclosed in the present invention combines the over- expression of yeast sequence biased human cytochrome P450 along with their regulatory partners under galactose inducible promoter in a protease deficient yeast strain.
  • the yeast strain disclosed in the present invention has resulted in significantly higher yields with better specific activity (Table 1).
  • the present invention provides a solution to the problem associated with production of recombinant human cytochrome P450 and its specific activity by providing a genetically engineered protease deficient haploid yeast strain comprising integrated yeast sequence biased human cytochrome P450 reductase and yeast sequence biased human cytochrome P450 to express the catalytically active human cytochrome P450 (s).
  • Advantages of the yeast strain (s) disclosed in the present invention are high overall protein yield, higher specific activity, cost effective and economical process of production of human cytochrome P450.
  • the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 33 coding for human cytochrome P450 reductase having amino acid sequence as set forth in SEQ ID NO: 50. In another embodiment the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 1, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 42.
  • the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 5, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 43.
  • the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 9, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 44.
  • the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 13, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 45.
  • the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 17, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 46. In another embodiment the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 21, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 47.
  • the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 25, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 48. In another embodiment the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 29, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO: 49.
  • the present invention provides a polynucleotide sequence as set forth in SEQ ID NO: 37 coding for human cytochrome b5 having amino acid sequence as set forth in SEQ ID NO: 51
  • a recombinant polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 41, wherein the recombinant polynucleotide sequence consists of human yeast sequence biased polynucleotide sequence as set forth in SEQ ID NO: 38 coding for cytochrome b5.
  • a plasmid comprising a modified polynucleotide sequence coding for cytochrome P450.
  • a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 coding for cytochrome P450.
  • a plasmid comprising a modified polynucleotide sequence coding for cytochrome P450 and a modified polynucleotide sequence coding for cytochrome b5.
  • a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5.
  • a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID, NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5 having nucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.
  • a plasmid comprising a modified polynucleotide sequence coding for cytochrome P450 and a modified polynucleotide sequence coding for cytochrome b5 having nucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.
  • a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome, wherein the yeast strain is protease A and protease B deficient strain.
  • yeast strain comprising the yeast sequence biased polynucleotide sequence coding for human cytochrome P450 reductase integrated in yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, wherein the yeast strain is protease A and protease B deficient strain.
  • a yeast strain comprising the integrated yeast sequence biased polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 coding for human cytochrome P450 reductase, wherein the polynucleotide sequence is integrated in the yeast genome and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, wherein the yeast strain is protease A and protease B deficient strain.
  • a yeast strain comprising the yeast sequence biased polynucleotide sequence coding for human cytochrome P450 reductase integrated in yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5, wherein the yeast strain is protease A and protease B deficient strain.
  • a yeast strain comprising the integrated yeast sequence biased polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 coding for human cytochrome P450 reductase and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5.
  • a yeast strain comprising the integrated yeast sequence biased polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 coding for human cytochrome P450 reductase and a plasmid comprising a polynucleotide sequence coding for human cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30,
  • a yeast strain comprising the integrated yeast sequence biased polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 coding for human cytochrome P450 reductase and a plasmid comprising a polynucleotide sequence coding for human cytochrome P45.0 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
  • Another embodiment - of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome, the process comprises growing the yeast strain by using conventional methods.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated in the yeast genome.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises yeast sequence biased human cytochrome P450 reductase integrated in yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO; 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises yeast sequence biased human cytochrome P450 reductase integrated in yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises yeast sequence biased human cytochrome P450 reductase integrated in yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 coding for cytochrome b5.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 coding for cytochrome b5.
  • the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 is integrated in the yeast genome, and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, S
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using protease A and protease B deficient yeast strain, the process comprises growing the yeast strain by using conventional methods, wherein the yeast strain comprises the yeast sequence biased human cytochrome P450 reductase, and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 coding for
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing the yeast strain as disclosed in the present invention, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 coding for cytochrome P450, contacting the substrate with the microsomal fraction of SEQ ID
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 coding for cytochrome P450 and a polynucleotide sequence
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 coding for cytochrome b5; contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 coding for cytochrome b5, contacting the substrate with the microsomal fraction of the said yeast strain and analyzing the metabolites of the substrate; wherein the yeast strain is protease A and protease B deficient strain.
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 coding for cytochrome P450 and a polynucleotide sequence having nucleo
  • the present invention provides a process of determining the metabolite of a substrate, the process comprises providing a yeast strain comprising the yeast sequence biased human cytochrome P450 reductase integrated in the yeast genome having nucleotide sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
  • the present invention provides use of the yeast strain(s) as disclosed in the present invention for analysis of a substrate.
  • the yeast strain comprising the integrated modified human cytochrome P450 reductase, and a plasmid comprising the modified human cytochrome P450 and modified human cytochrome b5 show highest activity of cytochrome P450 as compared to i) the yeast strain comprising wild type human cytochrome P450 along with the endogenous cytochrome P450 reductase and ii) the yeast strain comprising wild type human cytochrome P450 along with the wild type human cytochrome P450 reductase.
  • a modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified sequence codes for human cytochrome P450 reductase.
  • a recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified sequence is operably linked to a promoter sequence.
  • the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified sequence is operably linked to a promoter sequence, wherein the promoter is ADH 2 or GALl.
  • a modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, wherein the modified DNA sequence codes for cytochrome b5.
  • a recombinant DNA molecule comprising the modified DNA sequence coding for cytochrome b5 having polynucleotide sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, wherein the modified sequence is operably linked to a promoter sequence.
  • One embodiment of the present invention provides a recombinant DNA molecule comprising the polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 41.
  • Another embodiment of the present invention provides a recombinant DNA molecule comprising the modified DNA sequence coding for cytochrome b5 having polynucleotide sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, wherein the modified sequence is operably linked to a promoter sequence, wherein the promoter is ADH 2 or GAL 1.
  • the recombinant DNA molecule as disclosed in the present invention further comprises a
  • a yeast strain comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the yeast strain is a protease A and protease B deficient strain.
  • a yeast strain comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for a cytochrome P450, wherein the yeast strain is a protease A and protease B deficient strain.
  • a yeast strain comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified DNA sequence is integrated in the genome of the yeast strain.
  • One aspect of the present invention relates to a yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the yeast strain is a protease A and protease B deficient strain.
  • yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36
  • yeast strain is a protease A and protease B deficient strain.
  • a yeast strain comprising the recombinant DNA molecule comprising the nucleotide sequence as set forth in SEQ ID NO: 41, wherein the recombinant DNA molecule is integrated in the genome of said yeast strain.
  • yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for a cytochrome P450, wherein the plasmid further comprises a polynucleotide sequence coding for cytochrome b5, wherein the yeast strain is a protease A and protease B deficient strain.
  • yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ
  • a plasmid comprising a polynucleotide sequence coding for a cytochrome P450, having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3,
  • SEQ ID NO: 4 SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:
  • SEQ ID NO: 12 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:
  • yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, wherein the plasmid further comprises a polynucleotide sequence coding for cytochrome b5, wherein the polynucleotide sequence of cytochrome b5 is selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40, wherein the yeast strain is a protease A and protease B deficient strain.
  • One embodiment of the present invention provides a yeast strain having MTCC accession number MTCC 5476 deposited at IMTECH, Chandigarh, India
  • One embodiment of the present invention provides a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising integrated modified DNA sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified DNA sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5; contacting the substrate with the microsomal fraction of the said yeast strain; and analyzing metabolites of the substrate.
  • Another embodiment of the present invention provides a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising integrated modified DNA sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified DNA sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain comprising integrated modified DNA sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, wherein the modified DNA sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, wherein the process comprises growing the yeast strain by using the conventional method to obtain human cytochrome P450.
  • Another embodiment of the present invention provides a process of producing cytochrome P450 using a protease A and protease B deficient yeast strain comprising integrated modified DNA sequence selected from a group consisting of SEQ ID NO: 34, SEQ ID NO: 35.
  • the modified DNA sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450 selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32., wherein the process comprises growing said yeast strain by using the conventional method to obtain human cytochrome P450; Another embodiment of the present invention provides a process of producing cytochrome P450 using
  • yeast strain(s) comprising the modified DNA sequence selected from a group consisting of nucleotide sequence as set forth in SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36 and a plasmid comprising polynucleotide sequence coding for cytochrome P450 for substrate analysis, wherein the modified DNA sequence codes for human cytochrome P450 reductase.
  • a recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence codes for cytochrome P450, wherein the modified sequence is operably linked to a promoter sequence.
  • the present invention provides a recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence codes for cytochrome P450, wherein the modified sequence is operably linked to a promoter sequence, wherein the recombinant DNA molecule further comprises a Kozak sequence.
  • the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence codes for cytochrome P450, wherein the modified sequence is operably linked to a promoter sequence, wherein the promoter is ADH2 or GALl.
  • the present invention also provides a yeast strain comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the yeast strain is a protease A and protease B deficient strain.
  • One embodiment of the present invention provides a yeast strain comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence is integrated in the genome of said yeast strain.
  • a yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence codes for cytochrome P450, wherein the modified sequence is operably linked to a promoter sequence, wherein the yeast strain is a protease A and protease B deficient strain.
  • a yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, wherein the modified DNA sequence codes for cytochrome P450, wherein the modified sequence is operably linked to a promoter sequence, wherein the yeast strain is a protease A and protease B deficient strain, wherein the re
  • a yeast strain comprising the recombinant DNA molecule comprising the modified DNA sequence selected from a group consisting of the nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:
  • the modified DNA sequence codes for cytochrome P450, wherein the modified sequence is operably linked to a promoter sequence, wherein the yeast strain is a protease A and protease B deficient strain, wherein the recombinant DNA further comprises a polynucleotide sequence coding for cytochrome b5, wherein the nucleotide sequence coding for cytochrome b5 is selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.
  • One embodiment of the present invention provides a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising the modified DNA sequence coding for cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:
  • SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 and a polynucleotide sequence coding for cytochrome b5; contacting the substrate with the microsomal fraction of the said yeast strain; and analyzing the metabolites of the substrate.
  • One embodiment of the present invention provides a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising the modified DNA sequence coding for cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 and a polynucleotide sequence coding for cytochrome b5; contacting the substrate with the microsomal fraction of the said
  • One embodiment of the present invention provides a process of determining metabolite of a substrate, the process comprising providing a protease A and protease B deficient yeast strain comprising the modified DNA sequence coding for cytochrome P450 having nucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 and a polynucleotide sequence coding for cytochrome b5 selected from the group consisting of SEQ ID NO: 38,
  • Another embodiment of the present invention provides a process of producing cytochrome P450, the process comprising growing protease A and protease B deficient yeast strain comprising the modified DNA sequence having polynucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 by using the conventional method to obtain cytochrome P450;
  • yeast strain comprising the modified DNA sequence having polynucleotide sequence selected from a group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 for substrate analysis.
  • Development of protease deficient yeast strain The protease deficient strain was constructed by site specific homologous recombination.
  • the yeast strain YPH500 of the Saccharomyces species of genotype UKAWHL (LGC Promochem India Pvt. Ltd; ATCC accession no: 204680) was used for the disruption of protease genes.
  • This is a haploid strain of mating type alpha, wherein the genes to be disrupted for making protease deficient strain are protease A and protease B coding genes.
  • the strategy for the construction of protease deficient strain involves the revival of selected mutated amino acid markers at the desired position in the yeast genome without compromising the existing auxotrophic yeast genotype. This is performed by inserting selected amino acid auxotrophic markers into yeast genome through site specific homologous recombination.
  • protease gene was disrupted first in YPH500 strain with genotype UKAWHL to produce Protease B coding gene disrupted recombinant strain of genotype UKAHL named as PYBPD, by the revival of tryptophan auxotrophic marker.
  • the recombinant strain PYBPD was selected on SD UKAHL media agar plates.
  • Protease A coding gene was disrupted in PYBPD strain with genotype
  • UKAHL This resulted in insertion and thereby revival of Histidine auxotrophic marker with new genotype UKAL from UKAHL.
  • the recombinant strain is named as PYPD and was selected on UKAL, SD media agar plates.
  • protease A and protease B coding gene deficient haploid strain PYPD
  • the above two sequential steps resulted in protease A and protease B coding gene deficient haploid strain (PYPD) of mating type alpha with genotype UKAL.
  • protease deficient strain with integrated human cytochrome P450 reductase gene was carried out by the integration of plasmid pYRIl 13KRN comprising of human cytochrome P450 reductase, auxotrophic LEU2 marker along with other necessary components in PYPD strain by site specific recombination at LEU2 locus, resulting in the revival of Leucine auxotrophic marker in recombinant strain generating the genotype UKA from UKAL.
  • Figure 1 illustrates development of modified protease A and protease B deficient yeast strain comprising optimized and fully yeast biased human cytochrome P450 reductase having nucleotide sequence as set forth in SEQ ID NO: 34 integrated at LEU2 locus of the yeast genome.
  • the modified yeast strain is designated as PYPDl 13KRN.
  • the integrated cytochrome P450 reductase gene is under the control of GALl promoter along with CYCl terminator.
  • protease deficient yeast strain comprising integrated modified human cytochrome P450 reductase gene and an episomal cytochrome P450(s) and/or cytochrome b5 under the control of GALl promoter along with CYCl terminator Construction of yeast episomal vector comprising cytochrome P450 3A4
  • the yeast episomal vector, pYRE113K34 (6870 bp) comprising of human CYP3A4 (SEQ ID NO: 30) was prepared as follows.
  • the plasmid containing human CYP3A4 (SEQ ID NO: 30) was digested with BgHl and Xba ⁇ restriction enzyme. The fragment was purified and ligated into BamWl- Xba ⁇ digested pYRElOO vector to obtain the recombinant plasmid pYRE1 13K34.
  • yeast episomal vector comprising cytochrome P450 3A4 and cytochrome b5
  • Recombinant plasmid pYRE113K34 comprising of human CYP3A4 (SEQ ID NO: 30) and a plasmid containing synthetic cassette for expression of human cytochrome b5 (SEQ ID NO: 41, SIIb5) were digested with Bsp ⁇ and itesHII restriction enzymes. Fragment of human cytochrome b5 of 1302 bp was purified and ligated into Bsp ⁇ -BssHW digested pYRE113K34 vector to obtain pYRE1 13K34SIIb5 (8163 bp) comprising catalytic domain CYP3A4 and cytochrome b5.
  • Recombinant plasmids namely pYRE113K34 and pYRE113K34SIIb5 were transformed in protease deficient yeast strain having integrated human cytochrome P450 reductase for the expression of catalytically active cytochrome 3A4 and/or cytochrome b5. Transformation was carried out using standard lithium chloride method well known in the art. The transformation of episomal plasmid containing catalytic domain CYP3A4 and/or cytochrome b5 enables the yeast cells to grow on a medium devoid of Uracil, resulting in the changed genotype from UKA to KA. Thus the recombinant yeast strain is selected on KA, SD media agar selection plates.
  • yeast episomal vector comprising cytochrome P450 3A4 having nucleotide sequence as set forth in SEQ ID NO: 31 or SEQ ID NO: 32 under the control of GALl or ADH2 promoter along with CYCl terminator were constructed as described above.
  • yeast episomal vector comprising cytochrome P450 3A4 having nucleotide sequence as set forth in SEQ ID NO: 31 or SEQ ID NO: 32 under the control of GALl or ADH2 promoter along with CYCl terminator; and cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 39 or SEQ ID NO: 40 under the control of GALl or ADH2 promoter along with CYCl terminator were constructed as described above.
  • Recombinant vector construct comprising cytochrome P450 1A2 (CYP 1A2)
  • Yeast episomal vector comprising cytochrome P450 1A2 (CYP 1A2) having nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 were constructed as described above.
  • Methodology for the cloning and expression of CYPl A2 is similar to CYP3A4 cloning as described above, but using restriction enzymes BamUl, Xbal for both the vector and the insert plasmid resulting in the construct pYREl 13K12 of 6903 bp.
  • Recombinant vector construct comprising cytochrome P450 2D6 (CYP2D6)
  • Yeast episomal vector comprising cytochrome P450 2D6 (CYP2D6) having nucleotide sequence as set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 were constructed as described above.
  • Methodology for the cloning and expression of CYP2D6 is similar to CYP3A4 cloning as described above, but using restriction enzymes BamHl, Xbal resulting in the construct pYRE1 13K26 of 6846 bp.
  • Recombinant vector construct comprising cytochrome P450 2C9 (CYP2C9) with or without cytochrome b5
  • Yeast episomal vector comprising cytochrome P450 2C9 (CYP2C9) having nucleotide sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16 were constructed as described above. Methodology for the cloning and expression of CYP2C9 and CYP2C9 with
  • SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes Kpnl, Xbal for CYP2C9 resulting in the constructs pYRE 113K29 of 6858 bp and using restriction enzymes BspEl, BssHll for CYP2C9 with cytochrome b5 to generate construct pYREl 13K29SIIb5 of 8151 bp.
  • Recombinant vector construct comprising cytochrome P450 2Cl 9 (CYP2C19) with or without cytochrome b5 Yeast episomal vector comprising cytochrome P450 2Cl 9 (CYP2C19) having nucleotide sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 were constructed as described above. Methodology for the cloning and expression of CYP2C19 and CYP2C19 with
  • SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes Hin ⁇ lll, Xbal for CYP2C19 resulting in the constructs pYRE113K219 of 6843 bp and using restriction enzymes BspEl, BssHll for CYP2C19 with cytochrome b5 to generate construct pYREl 13K219SIIb5 of 8136 bp.
  • Recombinant vector construct comprising cytochrome P45Q 2El (CYP2E1) with or without cytochrome b5
  • Yeast episomal vector comprising cytochrome P450 2El (CYP2E1) having nucleotide sequence as set forth in SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28 were constructed as described above.
  • Methodology for the cloning and expression of CYP2E1 and CYP2E1 with SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes Hindlll, Xbal for CYP2E1 resulting in the constructs pYREl 13K21 of 6852 bp and using restriction enzymes BspEl, BssHU for CYP2E1 with cytochrome b5 to generate construct pYRE113K21SIIb5 of 8145 bp.
  • Recombinant vector construct comprising cytochrome P450 2C8 (CYP2C8) with or without cytochrome b5
  • Yeast episomal vector comprising cytochrome P450 2C8 (CYP2C8) having nucleotide sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 1 1 or SEQ ID NO: 12 were constructed as described above.
  • Methodology for the cloning and expression of CYP2C8 and CYP2C8 with SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes HmdIII, Xhol for CYP2C8 resulting in the constructs pYREl 13K28 of 6831 bp and using restriction enzymes BspEl, BssHU for CYP2C8 with cytochrome b5 to generate construct pYREl 13K28SIIb5 of 8124 bp.
  • Recombinant vector construct comprising cytochrome P450 2B6 (CYP2B6) with or without cytochrome b5 Yeast episomal vector comprising cytochrome P450 2B6 (CYP2B6) having nucleotide sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 were constructed as described above.
  • Methodology for the cloning and expression of CYP2B6 and CYP2B6 with SNb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes BamHl, Xbal for CYP2B6 resulting in the constructs pYREl 13K62 of 6834 bp and using restriction enzymes BspEl, BssHll for CYP2B6 with cytochrome b5 to generate construct pYREl 13K62SIIb5 of 8127 bp.
  • Figure 2 illustrates development of modified protease A and protease B deficient yeast strain comprising modified and optimized human cytochrome P450 reductase polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 34 integrated at LEU2 locus of the yeast genome and a plasmid comprising yeast biased human cytochrome P450 polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 30.
  • Both the genes i.e. cytochrome P450 reductase and cytochrome P450 are operably linked to GALl promoter along with CYCl terminator.
  • Figure 3 illustrates development of modified protease A and protease B deficient yeast strain comprising modified and optimized human cytochrome P450 reductase polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 34 integrated at LEU2 locus of the yeast genome a plasmid comprising yeast sequence biased human cytochrome P450 polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 30 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 38. All these three genes are expressed under the control of GALl promoter along with CYCl terminator in the yeast strain.
  • protease deficient yeast strain comprising integrated modified human cytochrome P450 reductase gene and an episomal cytochrome P450(s) and/or cytochrome b5 under the control of alcohol dehydrogenase promoter along with CYCl terminator Construction of yeast episomal vector containing cytochrome P450 3A4
  • the yeast episomal vector containing yeast sequence biased human CYP3A4, pYRE213K34 of size 7011 bp was prepared as follows.
  • the plasmid containing synthesized yeast sequence biased human CYP3A4 was digested with BgRl, Xbal restriction enzyme as per standard procedures well known in the art and the desired fragment was purified and ligated to pYRE200 digested with BamHl, Xbal restriction enzymes.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP3A4 having nucleotide sequence as set forth in SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32. Construction of yeast episomal vector containing cytochrome P450 3A4 and cytochrome b5
  • Recombinant plasmid pYRE213K34 and plasmid containing synthesized cassette for expression of yeast sequence biased human cytochrome b5 were digested with BssHll restriction enzymes as per standard procedures well known in the art. Fragments were purified and ligation was performed to obtain CYP3A4 with cytochrome b5.
  • the recombinant plasmid is named pYRE213K34SIIb5 of size 8454 bp.
  • Transformation of pYRE213K34 and pYRE213K34 with b5 recombinant constructs using standard Lithium chloride method was performed in protease deficient yeast strain having integrated human cytochrome P450 reductase for the expression of catalytically active cytochrome 3A4 with and without cytochrome b5.
  • This transformation of episomal plasmid containing catalytic unit with or without cytochrome b5 will cause the ability of the cells to grow without Uracil. This will change the final genotype from UKA to KA and hence the recombinant strain selection on KA, SD media agar selection plates.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP3A4 having nucleotide sequence as set forth in SEQ ID NO: 30, SEQ ID NO: 31 or SEQ ID NO: 32 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • Recombinant vector construct comprising cytochrome P450 1A2 (CYP1A2)
  • CYPl A2 Methodology for the cloning and expression of CYPl A2 is similar to CYP3A4 cloning as described above, but using following restriction enzymes BamHl, Xbal, resulting in the construct pYRE213K12 of 7044 bp.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP 1A2 having nucleotide sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
  • Recombinant vector construct comprising cytochrome P450 2D6 (CYP2D6)
  • CYP2D6 Methodology for the cloning and expression of CYP2D6 is similar to CYP3A4 cloning as described above, but using restriction enzymes BamHl, Xbal, resulting in the construct pYRE213K26 of 6987 bp.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2D6 having nucleotide sequence as set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.
  • Recombinant vector construct comprising cytochrome P450 2C9 (CYP2C9) with or without cytochrome b5
  • Methodology for the cloning and expression of CYP2C9 and CYP2C9 with SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes Acc65I, Xbal for CYP2C9 resulting in the construct pYRE213K29 of 6999 bp and restriction enzymes BssHll for CYP2C9 with cytochrome b5 resulting in the construct pYRE213K29SIIb5 of 8442 bp.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2C9 having nucleotide sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2C9 having nucleotide sequence as set forth in SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • Recombinant vector construct comprising cytochrome P450 2Cl 9 (CYP2C19) with or without cytochrome b5
  • Methodology for the cloning and expression of CYP2C19 and CYP2C19 with SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above above, but using restriction enzymes Hwdlll, Xba ⁇ for CYP2C19 resulting in the construct pYRE213K219 of 6984 bp and restriction enzyme /frs ⁇ II for CYP2C19 with cytochrome b5 resulting in the construct pYRE213K219SIIb5 of 8427 bp.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2C19 having nucleotide sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2C19 having nucleotide sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ
  • Recombinant vector construct comprising cytochrome P450 2El (CYP2E1) with or without cytochrome b5
  • Methodology for the cloning and expression of CYP2E 1 and CYP2E 1 with SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes HmdIII, Xbal for CYP2E1 resulting in the construct pYRE213K21 of 6993 bp and restriction enzyme BssWW for CYP2E1 with cytochrome b5 resulting in the construct
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2E1 having nucleotide sequence as set forth in SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2E1 having nucleotide sequence as set forth in SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • Recombinant vector construct comprising cytochrome P450 2C8 (CYP2C8) with or without cytochrome b5
  • Methodology for the cloning and expression of CYP2C8 and CYP2C8 with SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes HmdIII, Xho ⁇ for CYP2C8 resulting in the construct pYRE213K28 of 6972 bp and restriction enzymes ifasHII for CYP2C8 with cytochrome b5 resulting in the construct pYRE213K28SIIb5 of 8415 bp.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2C8 having nucleotide sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2C8 having nucleotide sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ
  • Recombinant vector construct comprising cytochrome P450 2B6 (CYP2B6) with or without cytochrome b5
  • Methodology for the cloning and expression of CYP2B6 and CYP2B6 with SIIb5 cassette is similar to CYP3A4 and CYP3A4SIIb5 cloning as described above, but using restriction enzymes BamHl, Xbal for CYP2B6 resulting in the construct pYRE213K62 of 6975 bp and restriction enzymes BssHM for CYP2B6 with cytochrome b5 resulting in the construct
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2B6 having nucleotide sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the resulting yeast episomal vector comprises yeast sequence biased human CYP2B6 having nucleotide sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO: 40.
  • Figure 4 illustrates development of modified protease A and protease B deficient yeast strain comprising modified and optimized human cytochrome P450 reductase polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 34 integrated at LEU2 locus of the yeast genome and a plasmid comprising yeast sequence biased human cytochrome P450 polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 30.
  • Both the genes i.e. cytochrome P450 reductase and cytochrome P450 are operably linked to alcohol dehydrogenase promoter along with CYC 1 terminator.
  • Figure 5 illustrates development of modified protease A and protease B deficient yeast strain comprising modified and optimized human cytochrome P450 reductase polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 34 integrated at LEU2 locus of the yeast genome a plasmid comprising yeast biased human cytochrome P450 polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 30 and yeast sequence biased human cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO: 38. All these three genes are expressed under the control of alcohol dehydrogenase promoter along with CYCl terminator in the yeast strain.
  • microsomal fraction for cytochrome P450 enzyme assay Transformants containing episomal plasmid with and without cytochrome b5 in PYPD strain with stably integrated cytochrome P450 reductase were grown in KA selection SD media with glucose as carbon source. The functional enzyme was produced under galactose induction media. Microsomal fraction containing functional enzymes was prepared after cell fractionation. The prepared microsomes were stored at minus 8O 0 C and were checked for enzyme activity. Enzyme assay
  • the P450 content was checked using standard carbon monoxide difference spectra method of Omura and Sato, 1964 to estimate the P450 amount in microsomal fraction.
  • the total protein content was checked using Bradford reagent by methods well known in the art.
  • the enzyme activity was checked using respective standard fluorescent substrates.
  • the enzyme assay mixture to check the enzyme activity contained microsomal enzyme, respective standard fluorescent substrate, phosphate buffer, (pre-incubation 10 min at 37°C) NADPH regeneration reagents containing glucose 6 phosphate, glucose 6 phosphate dehydrogenase, NADP and MgCl 2 in 10OmM tribasic. Total mixture was incubated for another 20 min at 37°C. The activity was measured using flourimeter at particular excitation and emission wavelength.
  • Example 1 is put forth so as to provide those of ordinary skill in the art with a complete disclosure and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as there invention nor are they intended to represent that the experiments below are all and only experiments performed.
  • Example 1 is put forth so as to provide those of ordinary skill in the art with a complete disclosure and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as there invention nor are they intended to represent that the experiments below are all and only experiments performed.
  • Example 1 is put forth so as to provide those of ordinary skill in the art with a complete disclosure and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as there invention nor are they intended to represent that the experiments below are all and only experiments performed.
  • protease deficient strain was constructed by sequential disruption of protease A and protease B coding genes by the method of site specific recombination.
  • Protease B coding gene disruption Plasmid DNA (22 ⁇ g) of 4113 bp carrying protease B coding disruption gene was digested with Kpnl and Sad restriction enzymes. Fragments were separated on 1.2% preparatory agarose gel. Desired band size of 1260 bp was excised and DNA was extracted from gel using Qiagen gel extraction column purified DNA was quantitated for further processing.
  • Yeast cells (strain YPH500; genotype UKAWHL) were transformed with 2 ⁇ g of purified DNA fragment (1260 bp) using lithium chloride method. Other methods well known in the art can be used for yeast transformation. Transformants were selected on UKAHL SD agar selection plates, which were reconfirmed by re-streaking on same selection plate. Protease A coding gene disruption
  • Protease B disrupted recombinant yeast strain with genotype UKAHL obtained as described above was transformed with 2 ⁇ g of linearized purified DNA fragment (5707 bp) using lithium chloride method and other methods well known in the art. Transformants were selected on UKAL SD agar selection plates, which were reconfirmed by re-streaking on same selection plate. The genetically modified yeast strain thus obtained was designated as PYPD.
  • Example 2
  • Protease disrupted yeast strain genotype UKAL was transformed with 2 ⁇ g of linearized and purified DNA fragment of 7723 bp using lithium chloride method. Other methods well known in the art can be used for yeast transformation.
  • the transformed cells were plated on desired SD media selection plates (UKA) and incubated at 30 0 C for 2-3 days.
  • the recombinant clones obtained were confirmed by streaking them on selective media agar plates and the resulting clones were designated as PYPDl 13KRN. Details are depicted in Figure 1.
  • Example 3 Development of protease deficient yeast strain comprising integrated modified cytochrome P450 reductase gene and an episomal modified cytochrome P450 under the control of GALl promoter along with CYCl terminator
  • the yeast episomal vector, pYRE113K34 (6870 bp) comprising of human CYP3A4 (SEQ ID NO: 30) was prepared as follows.
  • Episomal expression vector pYRElOO (5 ⁇ g) and the plasmid containing human CYP3A4 (SEQ ID NO: 30) (lO ⁇ g) were digested with BamHl, Xba ⁇ and BgR ⁇ , Xba ⁇ enzyme respectively. Fragments were separated on 1.2% preparatory agarose gel. Desired band size was excised and DNA was extracted from gel using Qiagen gel extraction column. Ligation was performed at 16 0 C O/N.
  • telomere sequence was transformed in DH5-alpha cells to obtain recombinant transformant comprises the recombinant vector designated as pYRE113K34, wherein pYREl 13K34 comprises CYP3A4 cDNA under the control of GALl promoter along with CYCl terminator. Transformation of pYRE1 13K34 in protease deficient yeast strain comprising integrated modified Cytochrome P450 reductase gene
  • the genetically modified UKA genotype protease deficient yeast cells comprising integrated modified yeast sequence biased cytochrome P450 reductase gene were inoculated into 5ml of the SD selective media. The cells were harvested and were used for transformation. Two ⁇ g of plasmid pYREl 13K34 carrying yeast sequence biased respective cytochrome P450 under the control of GALl promoter along with CYCl terminator was transformed into the yeast cells to obtain the yeast strain comprising integrated cytochrome P450 reductase gene and an episomal cytochrome P450 (Figure 2). Transformants were selected on KA SD agar selection plates. The plates were incubated at 30 0 C for 2-3 days. The transformants were confirmed by streaking them on same selective media agar plates.
  • protease deficient yeast strain comprising integrated modified human cytochrome P450 reductase gene and an episomal modified cytochrome P450 and modified cytochrome b5 regulatory under the control of GALl promoter along with CYCl terminator Construction of yeast episomal vector containing cytochrome P450 3A4 and cytochrome b5
  • Five ⁇ g of pYREl 13K34 comprising of human CYP3A4 (SEQ ID NO: 30) and lO ⁇ g of plasmid containing synthetic cassette for expression of human cytochrome b5 (SEQ ID NO: 41) were digested with BspEl and i&sHII restriction enzymes as per standard procedures well known in the art.
  • the genetically modified UKA genotype protease deficient yeast cells comprising integrated modified yeast sequence biased cytochrome P450 reductase gene were inoculated into 5ml of the SD selective media. The cells were harvested and were used for transformation. Two ⁇ g of plasmid pYRE113K34SIIb5 carrying yeast sequence biased respective cytochrome P450 under the control of GALl promoter along with CYCl terminator and cytochrome b5 under the control of GALl promoter along with CYCl terminator was transformed into the yeast cells to obtain the yeast strain comprising integrates cytochrome P450 reductase gene and an episomal cytochrome P450 and cytochrome b5 under the control of GALl promoter along with CYCl terminator (Figure 3). Transformants were selected on KA SD agar selection plates. The plates were incubated at 30 0 C for 2-3 days. The transformants were confirmed by streaking them on same selective media agar plates.
  • the recombinant constructs and yeast strains comprising the constructs for expression of CYP 1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 with and without cytochrome b5 under the GALl promoter along with CYCl terminator were generated in a similar manner as described for CYP3A4 above.
  • Cytochrome P450 content was checked using standard carbon monoxide (CO) spectra method of Omura T and Sato R J. Biol. Chem. 239:2370-8, 1964 to estimate the amount of CYP450.
  • specific content of CYP2D6 was estimated to be 85 pmol/mg of microsomal protein content.
  • yeast cell (1.0 OD600 nm) culture was incubated with 50 ⁇ l of 1OmM of substrate bufuralol (100 ⁇ M final concentration) for lhr at 37 0 C and 250 rpm. After lhr of incubation, cells were pelleted and loopful of them were analyzed for fluorescent metabolite preparation by CYP2D6 in presence of cytochrome P450 reductase, in intact cells. The fluorescent metabolite conversion was determined using specific excitation and emission wavelengths and capturing the data using cooled CCD camera. This shows that the CYP2D6 is showing specific activity towards bufuralol.
  • NADPH regeneration system containing NADP (20mg/ml), Glucose 6 phosphate (20mg/ml), MgCl 2 hexahydrate (13.3mg/ml), Tribasic (5mM), Glucose-6- phosphate dehydrogenase (40U/ml). Contents were mixed and mixture was incubated at 37 0 C for 20 min. Readings were taken at specific excitation and emission wavelengths. Results were normalized using control membranes values and reaction efficiency was measured using standard metabolites reaction values.
  • the enzyme assays for measuring the activity of CYPl A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP2E1 enzymes were performed in a similar manner as described for CYP2D6 above using the respective enzymes and their respective substrates.
  • the specific activity of CYP2D6 was estimated to be 1.7 pmol/min/pmol of P450. This is in sharp contrast to the undetectable activity resulting from the expression of wild type sequence of CYP2D6.
  • the reductase activity was estimated to be 1560 nmol of cytochrome c reduced/min/mg of protein. This is again in contrast to undetectable limits with the wild type sequence (Table 1).

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Abstract

L'invention concerne des séquences polynucléotidiques modifiées codant pour le cytochrome P450 réductase humain, le cytochrome P450 humain ainsi que le cytochrome b5 humain. De plus, l'invention concerne une souche de levure génétiquement modifiée déficiente en protéases qui contient un gène du cytochrome P450 réductase humain modifié intégré. L'invention concerne également une souche de levure génétiquement modifiée déficiente en protéases qui contient un gène de cytochrome P450 réductase humain modifié intégré ainsi qu'un plasmide contenant une séquence polynucléotidique modifiée codant pour le cytochrome P450s et/ou une séquence polynucléotidique modifiée codant pour le cytochrome b5. Par ailleurs, l'invention a montré que la ou les souches de levure précitées expriment un cytochrome P450s catalytiquement actif qui présente une activité spécifique plus importante.
PCT/IN2010/000322 2009-05-19 2010-05-19 Souche de levure exprimant le cytochrome p450 réductase humain modifié et/ou un gène du cytochrome p450 Ceased WO2010134096A2 (fr)

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Citations (2)

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US5635369A (en) 1991-07-15 1997-06-03 Centre National De La Recherche Scientifique Yeast strains with stable integration of heterologous genes
US6579693B1 (en) 1995-09-15 2003-06-17 Aventis Pharma S.A. Genetically engineered yeast strains

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EP2158312A1 (fr) * 2007-06-12 2010-03-03 PomBio Tech GmbH Levure de fission exprimant la cytochrome p450 réductase

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US5635369A (en) 1991-07-15 1997-06-03 Centre National De La Recherche Scientifique Yeast strains with stable integration of heterologous genes
US6579693B1 (en) 1995-09-15 2003-06-17 Aventis Pharma S.A. Genetically engineered yeast strains

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Title
GONZALEZ FJ; KORZEKWA KR, ANNU REV PHARMACOL TOXICOL., vol. 35, 1995, pages 369 - 90
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