Mitochondrial P450

• US 5,668,000 A
• Filed: 08/26/1996
• Issued: 09/16/1997
• Est. Priority Date: 09/26/1990
• Status: Expired due to Term

• Current Assignee: Sumitomo Chemical Company Limited
• Original Assignee: Sumitomo Chemical Company Limited
• Inventors: Sakaki, Toshiyuki, Akiyoshi, Megumi, Ohkawa, Hideo, Yabusaki, Yoshiyasu
• Primary Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US08/702,795
• Time in Patent Office: 386 Days
• Field of Search: 435/69.1, 435/71.1, 435/172.3, 435/189, 435/254.21, 435/320.1, 536/23.2
• US Class Current: 435/189
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/07 containing a mitochondrial localisation signal
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/0077 with a reduced iron-sulfur protein as one donor (1.14.15)
• CPC Class Codes: C12N 9/0085 Steroid 17 alpha-monooxygenase (1.14.99.9)
• CPC Class Codes: C12P 33/00 Preparation of steroids
• CPC Class Codes: C12P 7/02 containing a hydroxy group
• CPC Class Codes: C12Y 114/99009 Steroid 17-alpha-monooxygenase (1.14.99.9), i.e. cytochrome-P450-steroid-17-alpha-hydroxylase

Abstract

The expression of mitochondrial P450 in yeast is disclosed. The mitochondrial P450 includes a chimeric P450 in which a signal sequence present at the N-terminus of a mammalian mitochondrial P450 has been substituted by a signal of a yeast mitochondrial protein and a chimeric P450 in which the latter signal has been further substituted by a targeting signal sequence to microsome.

An expression plasmid for producing the enzyme in a large scale, a recombinant yeast strain carrying the expression plasmid, a process for producing the enzyme by the use of the recombinant yeast, and a process for producing 5β-cholestane-3α, 7α, 12α, 27-tetrol, 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ are also disclosed.

Claims

1. A yeast expression plasmid comprising DNA encoding a mitochondrial P450 which is a chimeric cytochrome P450₁₇α

-c25 which consists of P450_c25 having its targeting signal substituted by DNA encoding the N-terminal 15 amino acids of bovine adrenal cytochrome P450₁₇α

, operably linked to a promoter and terminator that is operative in yeast.

2. The yeast expression plasmid according to claim 1, wherein said promoter and terminator are the yeast alcohol dehydrogenase promoter and terminator.

3. The yeast expression plasmid according to claim 1, which further comprises DNA encoding the mature form of bovine adrenal adrenodoxin and DNA encoding the mature form of bovine adrenal NADPH-adrenodoxin reductase.

4. The yeast expression plasmid according to claim 3, wherein said promoter and terminator are the yeast alcohol dehydrogenase promoter and terminator, and said DNA encoding the mature form of bovine adrenal adrenodoxin is operatively linked between a phosphoglycerate kinase promoter and terminator and said DNA encoding the mature form of bovine adrenal NADPH-adrenodoxin reductase is operatively linked between a glyceraldehyde-[e]3-phosphate dehydrogenase promoter and terminator.

7. Yeast containing the yeast expression plasmid of claim 1.

8. A yeast containing the yeast expression plasmid of claim 2.

9. A yeast containing the yeast expression plasmid of claim 4.

12. A process for producing a chimeric cytochrome P450 17α

-25 which comprises cultivating a yeast strain containing the expression plasmid of claim 1.

13. A process for producing 5 β

-cholestane-3α

,7α

,12α

,27-tetrol, 25-hydroxyvitamin D₃ or 1α

,25-dihydrovitamin D₃, which process comprises reacting 5-β

-cholestane-3α

,7α

,12α

-triol, vitamin D₃ or 25-hydroxyvitamin D₃ respectively with a culture of a yeast containing the expression plasmid of claim 1.

14. A process for making 5-β

-cholestane-3α

, 7α

, 12α

-27-tetrol, 25-hydroxyvitamin D₃ or 1α

,25-dihydroxy-vitamin D₃, which comprises contacting 5-β

-cholestane-3α

,7α

,12α

-triol, vitamin D₃ or 25-hydroxy vitamin D₃, respectively, with a microsomal fraction obtained from a yeast of claim 1.

5. The yeast expression plasmid pAMS25.

10. A yeast containing the yeast expression plasmid of claim 5.

6. The yeast expression plasmid pRXMS25.

11. A yeast containing the yeast expression plasmid of claim 6.