Introduction: Polyketide and Nonribosomal Peptide Natural Products Doxorubicin -Antitumor agent...
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Transcript of Introduction: Polyketide and Nonribosomal Peptide Natural Products Doxorubicin -Antitumor agent...
Introduction: Polyketide and Nonribosomal Peptide Natural Products
O
O
O
OH
OH
O
OH
O
O
OHNH2
OH
S
N
O
O
OH
O
O
H
O
O
O
OH
O
O
OHHO
HON(CH3)2
O
OCH3
OH
O
HNN
N
HN
N
NHHN
N
N
N
N
HO
O OO
O
OO
O
O
O
O
O
N
O
NO
N
O
OO
O
OO
O
N
S S
HN N
S
COO-OH
OH
Doxorubicin
-Antitumor agent
Cyclosporin
-Immunosuppressant
Yersiniabactin
-Siderophore
Erythromycin
-Antibiotic
Enniatin
-Antibiotic
Epothilone
-Antitumor agent
OH
Gene Expression and Active Enzyme Requirement
DEBS1+TE, DEBS2, or DEBS3
-T7 promoter
-lac operator
BL21(DE3) E. coli strain
SDS-PAGE
In vitro reaction Analytical TLC
DEBS1+TE DEBS2 DEBS3 Standard
-Enzyme Activity (Temperature Dependent)
sfp
KDa212158
97.2
66.4
DEBS1+TE DEBS2 DEBS3
-Gene Expression
In vivo Polyketide Production
BAP1AT ACP KS AT KR ACP KS AT KR ACP TE
O O
OH
14C-Propionate
SO
S
HO
O
HO
S
HO
O
Propionyl-CoA (2S)-Methylmalonyl-CoA
DEBS1+TE
PrpE PCC
PCC
DEBS1+TE
Triketide Lactone
O
OH
O
Standard Complete Negative Culture Control
-DEBS1+TE-Triketide Lactone Example
37°C
Induce/Feed
22°C
In vivo Polyketide Production
BAP1
Propionate
Propionyl-CoA (2S)-Methylmalonyl-CoA
Full DEBS
PrpE PCC
-DEBS-6dEB Example
SO
S
HO
O
HO
S
HO
O
HO
S
HO
O
O
HO
S
HO
O
O
HO
S
HO
HO
O
O
HO
HO
HO
HO
SO
O
O
O
O
OH
OH
OH
DEBS2 DEBS3
PCC DEBS1
DEBS2DEBS1DEBS3
PCC
DEBS2DEBS1DEBS3
PCCO
O
O
OH
OH
OH
O
O
O
OH
OH
OH
6dEB
Extending Production Capabilities-Using the yersiniabactin synthetase (YBTS) system
OH
O-
O
COO-
H3N+
CH2CH2
+S Adenosine
CH3
H 2N C O O H
S H
Salicylate
Cysteine
S-adenosylmethionine
S
OH O
S
OHO
S
N S
N S
OH
O
S
N S
N
OH
N
S S
HN N
S
COO-OH
OH
Yersiniabactin, Ybt
YbtE (60 kDa) HMWP2 (230 kDa) HMWP1 (350 kDa)
YbtU (41 kDa)NRPS NRPS NRPSPKS
O
SCoA
CO2H
Malonyl-CoA
HMWP2 YbtU
YbtE HMWP1 BAPint
Salicylate
S
OH O
S
OHO
S
N S
N
S
OH
O
S
N S
N
OH
S
OHO
S
N S
NN
S
N
S S
HN N
S
COO-OH
OH
S
OHO
S
N S
NN
S
OH
Extending Production Capabilities
HMWP1HMWP2
YbtE
YbtU
22C
-Gene Expression
-LC-MS Analysis
N
S S
HN N
S
COO-OH
OH
Fe3+
Summary
-Complex polyketide biosynthesis
+E. coli polyketide production
-Gene transfer
-Gene expression
-Post-translational modification and protein folding
-Substrate requirement
+First complex natural product generated through E. coli
-Process development/improvement
+Fed-batch bioreactor process
+Production improved ~200 fold
-New production capabilities
+Both polyketide and nonribosomal peptide products