US2003022322A1PendingUtilityA1
SAM operon
Priority: Nov 13, 1996Filed: Aug 31, 2001Published: Jan 30, 2003
Est. expiryNov 13, 2016(expired)· nominal 20-yr term from priority
C12N 9/1085C07K 14/36C12P 19/40
49
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
The invention provides isolated nucleic acid compounds encoding a novel SAM synthetase of Streptomyces fradiae. Also provided are vectors and transformed heterologous host cells for expressing the SAM synthetase and a method for preparing S-adenosylmethionine from recombinant host cells transformed with the SAM synthetase gene.
Claims
exact text as granted — not AI-modifiedWe claim;
1 . A substantially pure SAM symthetase from Streptomyces fradiae having the amino acid sequence;
Met Ser Arg Arg Leu Phe Thr Ser Glu Ser Val Thr Glu Gly His Pro
1 5 10 15
Asp Lys Ile Ala Asp Arg Ile Ser Asp Thr Val Leu Asp Ala Leu Leu
20 25 30
Ala Arg Asp Pro Arg Ala Arg Val Ala Val Glu Thr Leu Ile Thr Thr
35 40 45
Gly Gln Val His Ile Ala Gly Glu Val Thr Thr Thr Ala Tyr Ala Pro
50 55 60
Ile Ala Gln Leu Val Arg Asp Thr Val Leu Ser Ile Gly Tyr Asp Ser
65 70 75 80
Ser Ala Lys Gly Phe Asp Gly Ala Ser Cys Gly Val Ser Val Ser Ile
85 90 95
Gly Ala Gln Ser Pro Asp Ile Ala Arg Gly Val Asp Thr Ala Tyr Glu
100 105 110
Arg Arg Gly Gly Gly Thr Ala Pro Gly Gly Pro Gly Asp Glu Leu Asp
115 120 125
Arg Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Cys Asp
130 135 140
Glu Thr Pro Glu Leu Met Pro Leu Pro Ile Asn Leu Ala His Arg Leu
145 150 155 160
Ser Arg Arg Leu Ser Glu Val Arg Lys Asn Gly Thr Ile Pro Tyr Leu
165 170 175
Arg Pro Asp Gly Lys Thr Gln Val Thr Ile Glu Tyr Asp Gly Asp Lys
180 185 190
Ala Val Arg Leu Asp Thr Val Val Val Ser Ser Gln His Ala Ser Gly
195 200 205
Ile Asp Leu Asp Ser Leu Leu Ala Pro Asp Ile Arg Arg His Val Val
210 215 220
Glu Pro Val Leu Ala Gly Leu Ala Glu Asp Gly Ile Lys Leu Asp Thr
225 230 235 240
Ala Gly Tyr Arg Leu Leu Val Asn Pro Thr Gly Arg Phe Glu Ile Gly
245 250 255
Gly Pro Met Gly Asp Ala Gly Leu Thr Gly Arg Lys Ile Ile Ile Asp
260 265 270
Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys
275 280 285
Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Met Arg Trp Val
290 295 300
Ala Lys Asn Val Val Ala Ala Gly Leu Ala Ser Arg Cys Glu Val Gln
305 310 315 320
Val Ala Tyr Ala Ile Gly Lys Ala Glu Pro Val Gly Leu Phe Val Glu
325 330 335
Thr Phe Gly Thr Ala Thr Val Asp Val Glu Arg Ile Glu Gln Ala Ile
340 345 350
Gly Glu Val Phe Asp Leu Arg Pro Ala Ala Ile Ile Arg Asp Leu Asp
355 360 365
Leu Leu Arg Pro Ile Tyr Ala Lys Thr Ala Ala Tyr Gly His Phe Gly
370 375 380
Arg Glu Leu Pro Glu Phe Thr Trp Glu Arg Thr Asp Arg Thr Glu Gln
385 390 395 400
Leu Ile Ala Ala Ala Gly Leu *
405
which is SEQ ID NO 2.
2 . A substantially pure methyltransferase from Streptomyces fradiae having the amino acid sequence:
Met Arg Ile Ala Val Thr Gly Ser Ile Ala Thr Asp His Leu Met Ala
1 5 10 15
Phe Pro Gly Arg Phe Gly Asp Gln Leu Ile Pro Asp Gln Leu Ala Arg
20 25 30
Val Ser Leu Ser Phe Leu Val Asp Gly Leu Glu Val Arg Arg Gly Gly
35 40 45
Val Ala Val Gly Ile Ala Phe Gly Leu Gly Arg Pro Gly Pro Thr Pro
50 55 60
Leu Leu Val Gly Ala Val Gly Asn Asp Phe Ala Asp Tyr Gly Thr Trp
65 70 75 80
Pro Lys Glu His Gly Val Asp Thr Gly Gly Val Leu Val Pro Thr Glu
85 90 95
His Gln Thr Ala Arg Phe Leu Cys Ile Thr Asp Arg Asp Ala Asn Gln
100 105 110
Ile Ala Ala Ser Tyr Thr Gly Ala Met Arg Glu Ala Arg Asp Ile Gly
115 120 125
Leu Arg Arg Thr Gly Ala Leu Pro Ala Pro Arg His Gly Leu Val Leu
130 135 140
Ile Cys Pro Asp Asp Pro Ala Ala Met Val Arg His Thr Ala Gln Cys
145 150 155 160
Arg Glu Pro Gly Leu Pro Phe Val Ala Asp Pro Ser Gln Gln Leu Ala
165 170 175
Arg Leu Glu Thr Asp Glu Val Arg Ala Leu Val His Gly Ala His Trp
180 185 190
Val Phe Thr Asn Glu Tyr Glu Ala Ala Leu Leu Leu Glu His Ser Gly
195 200 205
Trp Lys His Ser Glu Thr Leu Glu Arg Val Gly Ala Trp Val Thr Thr
210 215 220
Leu Gly Gly Ala Gly Val Arg Ile Glu Arg Ala Gly Glu Pro Pro Leu
225 230 235 240
Thr Val Pro Ala Val Pro Asp Val Pro Val Val Asp Pro Thr Gly Ile
245 250 255
Gly Ala Ala Phe Arg Ala Gly Phe Leu Ala Gly Ala Gly Arg Gly Leu
260 265 270
Ser Ile Val Ser Ala Ala Arg Leu Gly Cys Val Leu Ala Ala Arg Ala
275 280 285
Leu Gly Thr Val Gly Pro Ala Asp Leu Pro Asp Arg Ser Gly Gly Ser
290 295 300
Ala Arg His Gly Glu Gly Arg Val Arg Arg Gly Arg Gly Gly Ala Ala
305 310 315 320
Arg Pro Arg Ala Gly Arg Pro His Met Thr Arg Pro Cys Pro Gly Ser
325 330 335
Arg Arg Glu Pro Pro Ala Gly Arg Pro Ala Arg Ala Ala Ala Val Ile
340 345 350
Arg Arg Pro Gly Ala Gly Gly Pro Thr Ala Gly Gly Cys Arg *
355 360 365
which is SEQ ID NO 3.
3 . A substantially pure MTHR from Streptomyces fradiae having the amino acid sequence:
Met Arg Thr Thr Leu Arg Glu Ile Leu Gly Ser Gly Arg Leu Ser Phe
1 5 10 15
Ser His Glu Phe Phe Pro Pro Arg Thr Glu Ala Gly Thr Arg Thr Leu
20 25 30
Trp Asn Ala Ile Arg Arg Ile Glu Pro Leu Ala Pro Thr Phe Val Ser
35 40 45
Val Thr Tyr Gly Ala Gly Gly Ser Ser Arg Asp Arg Thr Val Glu Val
50 55 60
Thr Lys Arg Ile Ala Thr Asp Thr Thr Leu Arg Pro Val Ala His Leu
65 70 75 80
Thr Ala Val Gly His Ser Val Ala Glu Leu Arg Arg Ile Ile Gly Gln
85 90 95
Tyr Ala Asp Ala Gly Val Arg Asp Val Leu Ala Leu Arg Gly Asp Pro
100 105 110
Pro Gly Asp Pro Asn Ala Pro Trp Val Pro His Pro Glu Gly Leu Thr
115 120 125
His Ala His Glu Leu Val Ser Leu Val Arg Gly Ser Gly Gly Phe Gly
130 135 140
Val Gly Val Ala Ala Phe Pro Glu Arg His Pro Arg Ser Pro Asp Trp
145 150 155 160
Asp Ser Glu Ile Arg His Phe Val Arg Lys Cys Arg Ala Gly Ala Asp
165 170 175
Tyr Ala Ile Thr Gln Met Phe Phe Arg Val Glu Asp Tyr Leu Arg Leu
180 185 190
Arg Asp Arg Val Ala Ala Ala Gly Cys Cys Thr Pro Val Ile Pro Gly
195 200 205
Ile Met Pro Ala Thr Asp Val Arg Gln Ile Ala Arg Phe Ala Glu Leu
210 215 220
Ser His Ala Thr Phe Pro Glu Gly Leu Ala Arg Arg Leu Glu Ala Ala
225 230 235 240
Arg Gly Asn Pro Ala Glu Gly His Arg Ile Gly Val Glu Tyr Ala Thr
245 250 255
Ala Met Ala Gly Arg Leu Leu Ala Glu Gly Ala Pro Gly Leu His Tyr
260 265 270
Ile Thr Leu Asn Arg Ser Thr Ala Thr Leu Glu Ile His Arg Asn Ile
275 280 285
Leu Gly Thr Pro Ala Pro Gly Ser Ala Arg Gln Val Leu Ala Ala Pro
290 295 300
Leu *
305
which is SEQ ID NO 5.
4 . An isolated nucleic acid compound encoding the protein of claim 1 .
5 . An isolated nucleic acid compound comprising a sequence encoding the protein of claim 1 or fragment thereof wherein said compound has a sequence selected from the group consisting of:
(a) residues 986 through 2209 of SEQ ID NO:1;
(b) residues 986 through 2209 of SEQ ID NO:6
(c) a nucleic acid compound complementary to (a) or (b); and
(d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO. 2.
6 . An isolated nucleic acid compound of claim 5 wherein the sequence is (a) or a sequence complementary to (a).
7 . An isolated nucleic acid compound of claim 5 wherein the sequence of said compound is (b) or a sequence complementary to (b).
8 . An isolated nucleic acid compound encoding the protein of claim 2 .
9 . An isolated nucleic acid compound comprising a sequence encoding the protein of claim 2 or fragment thereof wherein said compound has a sequence selected from the group consisting of:
(a) residues 2241 through 3341 of SEQ ID NO:1;
(b) residues 2241 through 3341 of SEQ ID NO:6
(c) a nucleic acid compound complementary to (a) or (b); and
(d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO. 3.
10 . An isolated nucleic acid compound of claim 9 wherein the sequence is (a) or a sequence complementary to (a).
11 . An isolated nucleic acid compound of claim 9 wherein the sequence of said compound is (b) or a sequence complementary to (b).
12 . An isolated nucleic acid compound encoding the protein of claim 3 .
13 . An isolated nucleic acid compound comprising a sequence encoding the protein of claim 3 or fragment thereof wherein said compound has a sequence selected from the group consisting of:
(a) residues 3338 through 4255 of SEQ ID NO:4;
(b) residues 3338 through 4255 of SEQ ID NO:6
(c) a nucleic acid compound complementary to (a) or (b); and
(d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO. 5.
14 . An isolated nucleic acid compound of claim 13 wherein the sequence is (a) or a sequence complementary to (a).
15 . An isolated nucleic acid compound of claim 13 wherein the sequence of said compound is (b) or a sequence complementary to (b).
16 . An isolated nucleic acid compound encoding the SAM operon from Streptomyces fradiae , wherein said compound is SEQ ID NO.1 or SEQ ID NO.6.
17 . A vector comprising an isolated nucleic acid compound of claim 16 wherein said compound is SEQ ID NO.1.
18 . A vector comprising an isolated nucleic acid compound of claim 5 in operable linkage to a promoter sequence.
19 . A vector comprising an isolated nucleic acid compound of claim 9 in operable linkage to a promoter sequence.
20 . A vector comprising an isolated nucleic acid compound of claim 13 in operable linkage to a promoter sequence.
21 . A host cell containing a vector of claim 17 .
22 . A host cell containing a vector of claim 18 .
23 . A host cell containing a vector of claim 19 .
24 . A host cell containing a vector of claim 20 .
25 . A method for constructing a recombinant host cell having the potential to express SEQ ID NO:2, SEQ ID NO.3, and SEQ ID NO.5 said method comprising introducing into a host cell by any suitable means a vector of claim 16 .
26 . A method as in claim 25 wherein said host cell is an Actinomycete.
27 . A method for expressing SEQ ID NO:2, SEQ ID NO.3 and SEQ ID NO.5 in the recombinant host cell of claim 26 , said method comprising culturing said recombinant host cell under conditions suitable for gene expression.
28 . A method for synthesizing S-adenosylmethionine, comprising the steps of:
a) admixing a suitable amount of the following reagents:
i) a substantially pure SAM synthetase as claimed in claim 1 ,
ii) ATP;
iii) methionine; and
b) purifying said S-adenosylmethionine by any suitable means.
29 . A method for producing S-adenosylmethionine comprising the steps of:
a) preparing a crude extract from the recombinant cells of claim 22; b) adding to said extract suitable amount of ATP and methionine; and c) recovering said S-adenosylmethionine by any suitable means.
30 . A method for producing S-adenosylmethionine comprising the steps of:
a) culturing the host cell of claim 21 under conditions suitable for synthesis of S-adenosylmethionine, and b) recovering S-adenosylmethionine from said host cell by any suitable means.Join the waitlist — get patent alerts
Track US2003022322A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.