FIELD OF INVENTION
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)
BACKGROUND OF THE INVENTION
Cytochrome P450 (P450s) 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 + it + 02 + RH ==> NADP+ + H2O + R-OH
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for generating large amount of cytochrome P450 Within the last 15 years, in vivo experiments using animal models have been replaced by in vitro studies using human enzymes from different sources Recombinant expression methods for the production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
A wide variety of 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
There are a lot of factors which affect the overall yield of any recombinant protein in any organism One of the most important parts for the system to be successful is the expression plasmid / vector used for the expression of a 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 cob based systems
One of the common concerns for effective high protein production and purification in any host is the proteolytic degradation of the recombinant gene products by host-specific proteases (Ahmjan Idins, Kewei Bi, Hideki Tohda, Hiromichi Kumagai, Yuko Giga-Hama, 2006 Jan 30,23(2) 83-99) De novo proteins synthesis in any system is a continuous cycle of synthesis and degradation over time (in situ protease dependent degradation/cleavage), based on the requirement of that particular cellular system 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 Gleeson, Christopher E White, David P Meininger and Elizabeth A Komives in Methods in Molecular Biology, Pichia Protocols, 10 1385/0-89603-421-6 81) These findings demonstrate that construction of a protease-deficient host system is useful in effective protein production and purification of any recombinant protein
Heterologous expression of cytochrome P450s has been established in several organisms Saecharomyces 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 Other literature reviews suggests that expression of mammalian cytochorme P450s is usually performed without the addition of tagged sequences at either end It has been observed that the addition of c-myc tag at the C terminus leads to abolition of protein expression (R Soni, unpublished results) It has been reported that the expression efficiency of P450s in S cerevisiae can be improved by appropriate alteration of the yeast strain or the expression plasmid (Urban P, Cullin C, Pompon D , Biochimie 1990 Jun-Jul, 72(6-7) 463-72 and Gonzalez FJ, Korzekwa KR, Ann Rev Pharmacol Toxicol 1995, 35 369-90 Review)
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
In addition to the characteristics described above for the higher expression of heterologous proteins, 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 (Y Batard et al, Arch Biochem Bwphys 379,161(2000)) However, 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 It occurs on mRNAs and is recognized by the nbosomes as the translational start site In vivo, this site is often not matched exactly on different mRNAs and the amount of protein synthesized from a given mRNA is strongly dependent on the strength of the Kozak sequence, defined by the nature and physiological relevance of the protein to be expressed
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
SUMMARY OF THE INVENTION
The present invention relates to development of a piotease-deficient haploid yeast stiain by respective disruption ot major protease encoding genes of S cerevwae 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 SEQIDNO 41
Another aspect of the present invention relates to 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
Another aspect of the present invention relates to 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
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 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, SEQIDNO 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, SEQIDNO 11, SEQIDNO 12, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQIDNO 18, SEQIDNO 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 having nucleotide sequence selected from a group consisting of SEQ ID NO 38, SEQ ID NO 39 and SEQ ID NO 40, contacting the substrate with the yeast strain, and analyzing the metabolites of the substrate
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, SEQIDNO 10, SEQIDNO 11, SEQ ID NO 12, SEQIDNO 14, SEQIDNO 15 SEQIDNO 16, SEQIDNO 18, SEQIDNO 19, SEQIDNO 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
Further aspect of the present invention relates to 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
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 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 yeast strain, and analyzing the metabolites of the substrate
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 11, SEQ ID NO 12. SEQ ID NO 14, SEQ ID NO 15 SFQIDNO 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
Further aspect of the present invention relates to use of the 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 BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS Figure 1 shows construction of PYPD strain with stable integration of modified human cytochrome P450 reductase
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
BRIEF DESCRIPTION OF NUCLEOTIDE AND PROTEIN SEQUENCES
SEQ ID NO 1 shows nucleotide sequence of human CYP1A2 (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 CYP1A2 as set forth in SEQ ID NO 1
SEQ ID NO 4 shows partially modified and partially optimized nucleotide sequence
of human CYP1A2 as set forth in SEQ ID NO 1
SEQ ID NO 5 shows nucleotide sequence of human CYP2B6 (Wild type)
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 torth in SEQ ID NO 9
SEQ ID NO 11 shows partially modified and partially optimized nucleotide
sequence of human CYP2C8 as set forth in SEQ ID NO 9
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)
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 CYP2C9 as set forth in SEQ ID NO 13
SEQ ID NO 16 shows partially modified and partially optimized nucleotide
sequence of human CYP2C9 as set forth in SEQ ID NO 13
SEQ ID NO 17 shows nucleotide sequence of human CYP2C19 (Wild type)
SEQ ID NO 18 shows modified and optimized nucleotide sequence of human
C YP2C19 as set forth in SEQ ID NO 17
SEQ ID NO 19 shows partially modified and partially optimized nucleotide
sequence of human C YP2C19 as set forth in SEQ ID NO 17
SEQ ID NO 20 shows partially modified and partially optimized nucleotide
sequence of human CYP2C19 as set forth in SEQ ID NO 17
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 CYP2D6 as set forth in SEQ ID NO 21
SEQ ID NO 24 shows partially modified and partially optimized nucleotide
sequence of human CYP2D6 as set forth in SEQ ID NO 21
SEQ ID NO 25 shows nucleotide sequence of human CYP2E1 (Wild type)
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 CYP2E1 as set forth in SEQ ID NO 25
SEQ ID NO 28 shows partially modified and partially optimized nucleotide
sequence of human CYP2E1 as set forth m 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 CYP3A4 as set forth in SEQ ID NO 29
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 (Wild type)
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 GAL 1 promoter along with CYC1 terminator
SEQ ID NO 42 shows amino acid sequence of human CYP1A2 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
SEQ ID NO 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
DETAILED DESCRIPTION OF THE INVENTION
The term "DNA", "polynucleotide" and "nucleotide" used herein can be used interchangeably
The term "protease deficient yeast strain" used herein refers to the genetically engineered yeast strain with deletion of protease A and protease B coding genes
The term "modified" used herein refers to modification (s) carried out in the DNA sequences coding for human cytochrome P450 reductase, human cytochrome P450 oi human cytochrome b5, wherein the sequence was modified using codon usage of the yeast for higher expression of the protein
The term "modified" and "yeast sequence biased" used herein can be used interchangeably
The term "optimized" used herein 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
In the present invention 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 Surprisingly, the specific activity of human cytochrome P450 in the presence of human cytochrome P450 reductase encoded by their respective modified DNA sequences was significantly higher than the activities of human cytochrome P450 (s) described above (Table 1)
Furthermore, it was observed that the presence of endogenous yeast cytochrome P450 reductase in the yeast strain comprising the integrated heterologous yeast sequence biased human cytochrome reductase did not show any inhibitory or negative effect on expression of the human cytochrome P450
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
In one embodiment, 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 torth in SEQ ID NO 1, coding for human cytochrome P450 having amino acid sequence as set forth in SEQ ID NO 42
In another embodiment 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
In another embodiment 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
In another embodiment 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
In another embodiment 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
In another embodiment 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 ammo acid sequence as set forth in SEQ ID NO 49
In another embodiment 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
In another embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided a plasmid comprising a modified polynucleotide sequence coding for cytochrome P450
In another embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided a plasmid comprising a modified polynucleotide sequence coding for cytochrome P450 and a modified polynucleotide sequence coding for cytochrome b5
In another embodiment of the present invention there is provided a plasmid comptising 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
In another embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided 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
In one embodiment of the present invention there is provided a Protease A and Protease B deficient yeast strain
In accordance with the present invention, in one embodiment there is provided 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
In another embodiment there is provided 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, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment of the present invention there is provided 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
In another embodiment there is provided 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
In another embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided 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, SEQ ID NO 31 and SEQ ID NO 32 and a polynucleotide sequence coding for cytochrome b5, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment of the present invention there is provided 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, SEQ ID NO 31 and SEQ ID NO 32 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, wherein the yeast strain is protease A and protease B deficient strain
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 32 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 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
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 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
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 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
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
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, 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 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, 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, SEQIDNO 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 nucleotide sequence selected from a group consisting of SEQ ID NO 38, SEQIDNO 39 and SEQ ID NO 40 coding for cytochrome b5
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 30, SEQ ID NO 31 and SEQ ID NO 32 coding for cytochrome P450, contacting the substrate with the yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 coding for cytochrome b5, contacting the substrate with the yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 30, SEQ ID NO 31 and SEQ ID NO 32 coding for cytochrome P450 and a polynucleotide sequence coding for cytochrome b5, contacting the substrate with the yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In another embodiment, 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 30, SEQ ID NO 31 and SEQ ID NO 32 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 yeast strain and analyzing the metabolites of the substrate, wherein the yeast strain is protease A and protease B deficient strain
In further embodiment, the present invention provides use of the yeast strain(s) as disclosed in the present invention for analysis of a substrate
We have performed experiments to compare the specific activities of human cytochrome P450 reductase and human cytochrome P450 with wild type and modified DNA sequences and the results are as follows
In case of human cytochrome P450 reductase, wild type sequence exhibited little significant activity over yeast endogenous reductase levels However, optimized sequence led to about 50 fold increase in specific activity over the endogenous yeast reductase
In case of human cytochrome P450, wild type sequence exhibited no significant activity However, modified DNA sequence led to a significant level of specific activity for a particular CYP450
In addition, we have also observed that the specific activity of human cytochrome P450 is directly correlated with the amount of human cytochrome P450 reductase
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 1) the yeast strain comprising wild type human cytochrome P450 along with the endogenous cytochrome P450 reductase and n) the yeast strain comprising wild type human cytochrome P450 along with the wild type human cytochrome P450 reductase
In one of the embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided 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 GAL1
In yet another embodiment of the present invention there is provided 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
In further embodiment of the present invention there is provided 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 isADH2orGALl
The recombinant DNA molecule as disclosed in the present invention further comprises a Kozak sequence
In one of the embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided 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
In yet another embodiment of the present invention, there is provided 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
Another 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 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
In yet another embodiment of the present invention there is provided 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
Another 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 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
Another 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 and 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 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
Another 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 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 accession
number 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 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 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 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 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 a group consisting of the nucleotide sequence as set forth in SEQ ID NO 38, SEQ ID NO 39 and SEQ ID NO 40, contacting the substrate with the yeast strain, and analyzing metabolites of the substrate,
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 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 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 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 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 having nucleotide sequence selected from a group consisting of SEQ ID NO 38, SEQ ID NO 39 and SEQ ID NO 40, wherein the process comprises growing said yeast strain by using the conventional method to obtain human cytochrome P450,
In an additional embodiment of the present invention there is provided use of the 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
Further embodiment of the present invention provides 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
In another embodiment of the present invention there is provided 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
In another embodiment 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 m 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
In >et another embodiment of the present invention there is provided 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 GAL1
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 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 is integrated in the genome of said yeast strain
In another embodiment of the present invention there is provided 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
In another embodiment of the present invention there is provided 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 recombinant DNA molecule is integrated in the genome of said yeast strain
In another embodiment of the present invention there is provided 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 recombinant DNA further comprises a polynucleotide sequence coding for cytochrome b5
In another embodiment of the present invention there is provided 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 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 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 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 yeast strain, and analyzing the metabolites of the substrate, wherein the modified DNA sequence coding for cytochrome P450 is integrated into the genome of said yeast strain
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, SEQ ID NO 39 and SEQ ID NO 40, contacting the substrate with the yeast strain, and analyzing the metabolites of the substrate
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,
Another embodiment of the present invention provides use of the 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 Sacchai omyces 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 The selection of recombinant clones in the absence of the particular amino acid in the media confirms the disruption of particular protease gene The process involving the disruption of both the protease coding genes was sequential Protease B coding 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
Similarly 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
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 Integration of modified human cytochrome P450 reductase gene in Protease deficient strain (PYPD)
Construction of protease deficient strain with integrated human cytochrome P450 reductase gene was carried out by the integration of plasmid pYRI113KRN 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 PYPD113KRN The integrated cytochrome P450 reductase gene is under the control of GAL 1 promoter along with CYC1 terminator
Development of 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 GAL1 promoter along with CYC1 terminator
Construction of yeast episomal vector comprising cytochrome P450 3A4
The yeast episomal vector, pYREl 13K34 (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 BgRl and Xbal restriction enzyme The
fragment was purified and hgated into BamHl- Xbal digested pYRElOO vector to
obtain the recombinant plasmid pYREl 13K34
Construction of yeast episomal vector comprising cytochrome P450 3A4 and
cytochrome b5
Recombinant plasmid pYREl 13K34 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 BspEl and BssRll restriction
enzymes Fragment of human cytochrome b5 of 1302 bp was purified and ligated into BspEl-BssHII digested pYRE113K34 vector to obtain pYRE113K34SIIb5 (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
Similarly 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 GAL1 or ADH2 promoter along with CYC1 terminator were constructed as described above
Further 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 GAL 1 or ADH2 promoter along with CYC 1 terminator, and cytochrome b5 having nucleotide sequence as set forth in SEQ ID NO 39 or SEQ ID NO 40 under the control of GAL 1 or ADH2 promoter along with CYC1 terminator were constructed as described above
Recombinant vector construct comprising cytochrome P450 1A2 (CYP1A2) Yeast episomal vector comprising cytochrome P450 1A2 (CYP1A2) 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 CYP1A2 is similar to CYP3A4 cloning as described above, but using restriction
enzymes BamHl, 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 pYREl 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 pYRE113K29 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 2C19 (CYP2C19) with or without cytochrome b5
Yeast episomal vector comprising cytochrome P450 2C19 (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 Hmdlll, Xbal for CYP2C19 resulting in the constructs pYREl 13K219 of 6843 bp and using restriction enzymes BspEl, 5ssHII for CYP2C19 with cytochrome b5 to generate construct pYREl 13K219SIIb5 of 8136 bp
Recombinant vector construct comprising cytochrome P450 2E1 (CYP2E1) with or without cytochrome b5
Yeast episomal vector comprising cytochrome P450 2E1 (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 pYRE113K21 of 6852 bp and using restriction enzymes BspEl, BssHll 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 11 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 Hindlll, Xhol for CYP2C8 resulting in the constructs pYRE113K28 of 6831 bp and using restriction enzymes BspEl, BssHll 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 SIIb5 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 GAL1 promoter along with CYC1 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 GAL1 promoter along with CYC1 terminator in the yeast strain
Development of 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 CYC1 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 hgated to pYRE200 digested with BamWl, 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
ZJssHII 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)
Methodology for the cloning and expression of CYP1A2 is similar to CYP3A4 cloning as described above, but using following restriction enzymes BamHl, Xbal, resulting in the construct pYRE213K12 of 7044 bp
The lesulting yeast episomal vectoi comprises yeast sequence biased human CYP1A2 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~)
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 m 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 2C19 (CYP2CT9) 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 Hmdlll, Xbal for CYP2C19 resulting in the construct pYRE213K219 of 6984 bp and restriction enzyme BssWW 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 38, SEQ ID NO 39 or SEQ ID NO 40
Recombinant vector construct comprising cytochrome P450 2E1 (CYP2E1) with or without cytochrome b5
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 Hmdlll, Xbal for CYP2E1 resulting in the construct pYRE213K21 of 6993 bp and restriction enzyme BssHll for CYP2E1 with cytochrome b5 resulting in the construct pYRE213K21SIIb5 of 8436 bp
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 Sllb5
cassette is similar to CYP3A4 and CYP3A4SIlb5 cloning as described above, but
using restriction enzymes HindIII, A7?oI for CYP2C8 resulting in the construct
pYRE213K28 of 6972 bp and restriction enzymes BssHIII 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 38, SEQ ID NO 39 or SEQ ID NO
40
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 BssHIII for CYP2B6 with cytochrome b5 resulting in the construct pYRE213K62SIIb5 of 8418 bp
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 1 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 CYC 1 terminator in the yeast strain
Preparation of the 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 80°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 mm at 37°C) NADPH regeneration reagents containing glucose 6 phosphate, glucose 6 phosphate dehydrogenase, NADP and MgCl2 in 100mM tribasic Total mixture was incubated for another 20 min at 37°C The activity was measured using flounmeter at particular excitation and emission wavelength
EXAMPLES
The following examples are 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 Construction of Protease deficient yeast strain PYPD
The 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 (22ug) of 4113 bp carrying protease B coding disruption gene was digested with Kpnl and Sacl 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 2ug 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
Similarly lOug of plasmid (5707 bp) DNA carrying Protease A disruption gene was digested with Pstl enzyme to obtain linearized plasmid and was analysed (250ng) on 1 2% analytical agarose gel The digested mixture was purified using Qiagen purification column and purified DNA was quantified for further processing
Protease B disrupted recombinant yeast strain with genotype UKAHL obtained as described above was transformed with 2ug 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
Development of Protease deficient strain comprising integrated modified human cytochrome P450 reductase gene
Plasmid DNA, pYRI113KRN (7723 bp) (Figure 1) (5jxg) carrying cytochrome P450 reductase gene under the control of GALl promoter along with CYCl terminator was digested with BstEll enzyme to obtain hneranzed plasmid and was analysed (250ng) for digestion on 1 2% analytical agarose gel The digested mixture was purified using Qiagen purification column and purified DNA was quantified for further processing using methods well known in the art
Protease disrupted yeast strain, genotype UKAL was transformed with 2(j.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°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 PYPD113KRN 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
Construction of yeast episomal vector containing cytochrome P450 3A4
The yeast episomal vector, pYRE113K34 (6870 bp) comprising of human CYP3A4 (SEQ ID NO 30) was prepared as follows
Episomal expression vector pYRElOO (5µ) and the plasmid containing human CYP3A4 (SEQ ID NO 30) (10µg) were digested with BamHl Xbal and BgIII, Xbal 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°C O/N The hgated mixture was transformed in DH5-alpha cells to obtain recombinant transformant comprises the
recombinant vector designated as pYRE113K34, wherein pYRE113K34 comprises CYP3A4 cDNA under the control of GAL 1 promoter along with CYC1 terminator.
Transformation of pYRE113K34 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 ug of plasmid pYRE113K34 carrying yeast sequence biased respective cytochrome P450 under the control of GAL 1 promoter along with CYC1 terminator was transformed into the yeast cells to obtain the yeast strain comprising integrated cytochiome 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°C for 2-3 days The transformants were confirmed by streaking them on same selective media agar plates Example 4
Development of 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 GAL1 promoter along with CYC1 terminator
Construction of yeast episomal vector containing cytochrome P450 3A4 and cytochrome b5
Five ug of pYRE113K34 comprising of human CYP3A4 (SEQ ID NO 30) and 10µg of plasmid containing synthetic cassette for expiession of human cytochrome b5 (SEQ ID NO 41) were digested with BspEl and BssHll restriction enzymes as pei standard procedures well known in the art 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°C O/N The
ligated mixture was transformed in DH5-alpha cells to obtain recombinant transformants comprising the recombinant vector pYRE113K34SIIb5, wherein pYREl 13K34SIIb5 comprises CYP3A4 cDNA and cytochrome b5 cDNA (SEQ ID NO 41) under the control of GAL 1 promoter along with CYC1 terminator
Transformation of pYRE113K34SIIb5 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 µg of plasmid pYRE113K34SIIb5 carrying yeast sequence biased respective cytochiome P450 under the control of GAL1 promoter along with CYC1 terminator and cytochrome b5 under the control of GAL1 promoter along with CYC 1 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 GAL 1 promoter along with CYC1 terminator (Figure 3) Transformants were selected on KA SD agar selection plates The plates were incubated at 30°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 CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 with and without cytochrome b5 under the GAL1 promoter along with CYC1 terminator were generated in a similar manner as described for CYP3A4 above
Similarly the recombinant constructs and yeast strains comprising the constructs for expression of CYP1A2, CYP2B6 CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 with and without cytochrome b5 under the ADH2 promoter
along with CYCl terminator were generated in a similar manner as described for
CYP3A4 above Details are depicted in Figure 4 and 5
Example 5
Cytochrome P450 content
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 As an example, specific content of CYP2D6 was estimated
to be 85 pmol/mg of microsomal protein content
Example 6
Cytochrome P450 enzyme assay using yeast strain
One ml of yeast cell (10 OD600 nm) culture was incubated with 50µl of 10mM of substrate bufuralol (100 µM final concentration) for lhr at 37°C and 250 rpm After 1hr 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
The assays for determining the activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9,
CYP2C19 CYP3A4 and CYP2E1 enzymes were performed in a similar manner as
described for CYP2D6 above using the respective strains and their respective
substrates
Example 7
Assay for determining drug metabolism using microsomes isolated from the
yeast strain
Enzyme assay for CYP2D6
Microsomal p450 activities were determined in an incubation mixture of final volume 0 2 ml A cocktail of 100 mM potassium phosphate buffer pH 7 4, lµM of substrate EOMCC, 0 3 pmol of P450 was prepared Contents were mixed and mixture was pre-incubated at 37°C for 10 min Added another cocktail called NADPH regeneration system containing NADP (20mg/ml), Glucose 6 phosphate (20mg/ml), MgCl2 hexahydrate (13 3mg/ml), Tribasic (5mM), Glucose-6-phosphate dehydrogenase (40U/ml) Contents were mixed and mixture was incubated at 37°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 CYP1A2. 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)
Table 1. Results of specific activity and content of CYP2D6 in protease deficient strain
(Table Removed)

We Claim:
1. 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.
2. A recombinant DNA molecule comprising the modified DNA sequence as claimed in claim 1, wherein the modified sequence is operably linked to a promoter sequence.
3. The recombinant DNA molecule as claimed in claim 2, wherein the promoter is ADH2 or GALl.
4. 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 sequence codes for cytochrome b5.
5. A recombinant DNA molecule comprising the modified DNA sequence as claimed in claim 4, wherein the modified sequence is operably linked to a promoter sequence.
6. A recombinant DNA molecule comprising the polynucleotide sequence having nucleotide sequence as set forth in SEQ ID NO: 41.
7. A recombinant DNA molecule as claimed in claim 5, wherein the promoter is ADH2orGALl.
8. The recombinant DNA molecule as claimed in claim 2 or 5 further comprises a Kozak sequence.
9. A yeast strain comprising the modified DNA sequence as claimed in claim 1, wherein the yeast strain is a protease A and protease B deficient strain.
10. A yeast strain comprising the modified DNA sequence as claimed in claim 1 and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, wherein the yeast strain is protease A and protease B deficient strain.
11. The yeast strain as claimed in claim 9 or 10, wherein the modified DNA sequence is integrated in the genome of said yeast strain.
12. A yeast strain comprising the recombinant DNA molecule as claimed in claim 2, wherein the yeast strain is a protease A and protease B deficient strain.
13. A yeast strain comprising the recombinant DNA molecule as claimed in claim 2 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.
14. The yeast strain as claimed in claim 6, wherein the recombinant DNA molecule is integrated in the genome of said yeast strain.
15. The yeast strain as claimed in claim 13, wherein the plasmid further comprises a polynucleotide sequence coding for cytochrome b5.
16. The yeast strain as claimed in claim 13, wherein the polynucleotide sequence of human cytochrome P450 is 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.
17. The yeast strain as claimed in claim 15, 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.
18. A process of determining metabolite of a substrate, said 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 said substrate with said yeast strain; and
analyzing metabolites of said substrate.
19. The process as claimed in claim 18, wherein the polynucleotide sequence coding for cytochrome P450 is 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.
20. The process as claimed in claim 18, wherein the polynucleotide sequence coding for cytochrome b5 is 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.
21. A process of determining metabolite of a substrate, said 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, 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 having nucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40;
contacting the substrate with the yeast strain; and
analyzing the metabolites of the substrate.
22. A process of producing cytochrome P450 using protease A and protease B deficient strain yeast 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 sequence codes for human cytochrome P450 reductase; and a plasmid comprising a polynucleotide sequence coding for cytochrome P450, wherein the process comprises growing said yeast strain by using the conventional method to obtain cytochrome P450.
23. A process as claimed in claim 22, wherein the nucleotide sequence of cytochrome P450 is 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.
24. The process as claimed in claim 22, wherein said plasmid further comprises a polynucleotide sequence selected from a group consisting of SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, wherein the polynucleotide sequence codes for cytochrome b5.
25. Use of the yeast strain as claimed in claim 10 for substrate analysis.
26. Use of the yeast strain as claimed in claim 13 for substrate analysis.