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Biomolecular Engineering------Exploitation of Enzymes toward Synthesis of
Pharmaceuticals and Biofuels
College of Chemistry Advisory Board Meeting Presentation, 10/24/2011
Wenjun ZhangDepartment of Chemical and Biomolecular Engineering
University of California, Berkeley
Natural Products
doxorubicinerythromycin
vancomycin penicillin artemisinin
O
O
O
HO O
H
lovastatin
O
ONHO OO
O
O
O
HOHO
OH
OH
paclitaxel
Pharmaceuticals
Biofuels
OHOH OHOH
OH
HN O
O N
N
H
physostigmine
Enzyme and PathwayEngineering
Genome Mining
Predict New Candidate
Discovery
Combinatorial Biosynthesis
Prerequisite
Biotransformation
Research Themes
Natural Products
Pharmaceuticals and Biofuels
Enzyme and PathwayCharacterization
genome sequencing
……….atgagtctgcagaacgcaaaggcgccggaacgcgtcgcggtcctgacccggatctggcgagaggttctcggagatcccgccctcgacgaggatgcggatctcttcgagaagaacggatcgtcgttgcaggtcctgcagatcaccggtcctcgccgcgcaggtacgcctcgcctgagtgcgccgga……….
mutagenesis
X
cofactorscoenzymes
pure enzyme(s)buffer
in vitro analysis
purified proteins
in vivo analysis
producing organismgenome analysis
gene cluster
heterologous expression
substrates
engineered biosynthesis
structure
pharmaceuticals
biofuels
genome analysis
Research Approach
Example 1: Engineered Biosynthesis of Bacterial Aromatic Polyketides
OH O OH O O
NH2
OH
N
OH
HOH
HHO
OxytetracyclineD-Glucose
O
HO
OH
HO
OHOH
?
Streptomyces rimosus
oxyA oxyC oxyE oxyG oxyI oxyK oxyM oxyO oxyQ oxyS
otrB oxyB oxyD oxyF oxyH oxyJ oxyL oxyN oxyP otrAoxyR oxyT
Resistance Minimal PKS Initiation Module Cyclase Tailoring Unknown
0 2 4 6 8 10 12 14 16 18 20 22 24 kB
KR
Oxytetracycline Biosynthetic Pathway
OH OH OH OH
NH2
O
OH
OH OH O O
NH2
O
OH
O
OHOH OH O O
NH2
O
OH
NH2
OHOH OH O O
NH2
O
OH
N
OH
4-Keto-ATC 4-amino-ATC anhydrotetracycline (ATC)
OxyF OxyL OxyQ OxyT
MT OXY MTAMT
OH O O O
NH2
O
OH
N
OH
OH
OH O O O
NH2
O
OH
N
OH
OHOH
OH O OH O
NH2
O
OH
N
OH
OHOH
5a-11a-dehydrotetracycline (DHTC) 5a-11a-dehydrooxytetracyclineoxytetracycline
OxyS
OXY OXY RED
O O O
O O O O O
NH2
S
O
O E
O O O
O O O O O
NH2
S
O
HO EHO
O
S
O
CoA
9 X
OxyD
OxyAOxyBOxyC OxyJ
KS CLFACP
KRAMT
O O
OH O O O O
NH2
S
O
E
O
OH OH O O O
NH2
S
O
E
OH OH OH O O
NH2
S
O
E
OxyK OxyN OxyHOH OH OH OH
NH2
O
OH
pretetramid
ARO/CYC CYC
Zhang et al, AEM 2006; Zhang et al, JNP 2006; Zhang et al, JBC 2007; Zhang et al, JACS 2008
Combinatorial Biosynthesis: modification of natural product biosynthetic pathways toward the production of “unnatural” natural products.
1
2
3
COMBINED PATHWAY
Engineered Biosynthesis 1
ABCD
J
KN
FLQT
ABCD
J
KN
FLQT
OH OH O O
NH2
O
OH
N
OH
6-desmethyl-ATC
OH OH O O
NH2
O
OH
N
OH
ATC
(yield: ~10 mg/L)
Zhang et al, JACS 2008
Engineered Biosynthesis 2
S
OO
CoA
9 X
O O O
O O
NH2
O
S
O
OxyC
O
O
O
N
O
OHO
OHOHOH
HO
WJ85
OxyABC
OxyDHO
O
OHO
OOHNH
OOH
HO
WJ35
O
O
OHO
OH
OH
O
WJ78
OH OH OH OH
NH2
O
OH
pretetramid
Engineered Biosynthesis 2
HO
R
H2NO
O
HO
OH1614
9 7
pannorin
O
O
HO
OH
OH
A C9-C14 and C7-C16 cyclase from the tetracenomycin pathwayTcmN
S
OO
CoA
9 X
O O O
O O
NH2
O
S
O
OxyC
O
O
O
OxyABC
OxyDHO C-7/C-16cyclizations
HO
O
OHO
H2NO
O
HO
OHO
O
OHO
O
HO
OH
OH
WJ150
C-9/C-14 O O
OH O
NH2
O
S
O
9
OH
HO
14
7
16 1614
9 7
TcmN
OxyC
pannorin analog
(yield: ~50 mg/L)
O
O
OHO
O
HO
OH
OH
Zhang et al, JACS 2007
E. coli as a Host for Bacterial Aromatic Polyketide Production
OH O OH O O
NH2
OH
N
OH
HOH
HHO
Oxytetracycline
OOH
OH
O
OO O
O
OHNH2
OH
Daunorubicin
Bacterial Aromatic Polyketides
E. coliActinomycetes
Macrolides Cyclic Peptides Terpenoids Alkaloids
6-deoxyerythronolide B
O
OH
OHO
O
OH
ON
N
HN
NH
O
NH
NN
OHN
O
N
N
O
O
O
O
O
O O
O
N
N
S S
Echinomycin
H
HHO
O
Artemisinic acid
N
O
HO
OH
O
Reticuline
OOH
HO
O
OH
R
R = Me, Et, iPr, Pr
R1128
O
OH O
O COOH
HO
H
Frenolicin
OOH
HO
O
OH
R
R = Me, Et, iPr, Pr
R1128
O
OH O
O COOH
HO
H
Frenolicin
Fungal type I PKS
Malonyl-CoA
(SAT) KS MAT PT ACP TE/CYC
ACPS
OOOO
O O O
O
O
HO
O
OH
OHOH
O
(SAT) KS MAT PT ACP TE/CYC
Backbone Assembly
Tailoring No crosstalk
Bacterial type II PKS
Malonyl-CoA
KS CLF
ACP
MAT
S
OOOO
O O O
O
O
ACP
CYC
Bacterial and Fungal Aromatic Polyketides Biosynthesis
OOHOHOH
COOHHO
Bacterial Aromatic Polyketides Fungal Aromatic Polyketides
Bacterial type II PKS Fungal type I PKSMalonyl-CoA Malonyl-CoA
KS CLF
ACP
MAT
S
OOOO
O O O
O
O
ACP
HO
O
OH
OHOH
O
Bacterial Aromatic Polyketides Fungal Aromatic Polyketides
Biosynthesis of Bacterial Aromatic Polyketides in Escherichia coli
KS MAT ACP
ACPS
OOOO
O O O
O
O
CYC
KR
OOHOHOH
COOH
Zhang et al, PNAS 2008
pacidamycinsmureidomycins
napsamycinssansanmycins
NH
O
ON
OOPOO-
O
OH OH
PO
O-O
O
HNOHO
OH
HN
O
NHO
O
-O
HNO
NHO
NH3+
HNO
-OOC
O
UDP-MurNAc-pentapeptide
substrate
inhibitors
HN
O
NH
HN
-OOC
N
O
NH
NH
O
ON
O
OH
O
O
OH
AA4
AA5
AA1
MraY
liposidomycinscaprazamycins
capuramycin
tunicamycinsstreptoviridinscorynetoxins
muraymycins
O N
HO OH
HN O
O
OOH2N
HO O
HN COOH
HN
O
NH
OROH
N
HN
NH
HN
O
HO
OH
H
HN
H
O N
HO OH
HN O
O
OO
RO OR
H2N
N
NO
HOOC
O
O
R
O N
O OH
HN O
OCONH2
OO
OH
O
HNHN
O OH
O N
HO OH
HN O
OOH
O
OHOH
HN
O
O
O NH O
OHHO
HO
Example 2: Biosynthesis of Uridyl Peptide Antibiotics
Pacidamycin Biosynthesis
?
Streptomyces coeruleorubidus
O
N
HN
AA1 AA2
HN
ONH
O
NH
NH
OAA5
O
OH
N
HN O
O
pacidamycins
Pacidamycin Scaffold Assembly Summary
B C D E F G I J K L M N O P Q R S T U VA H
2 kbStreptomyces coeruleorubidus NRRL 18370
W X
S
T
PacH
ONHN
H
HN
HN
O
O
COOHHN
ONH
ONHN
H
HN
HN
O
O
COOHHN
O
O
HO
NHN
O
O
C
PacI
aminouridine
OH
OHOH
O
H2N
OHOH
O
H2N
APacW
NH2
O
O
SH
T
PacP
TE*
NH2
NH2
O
OH
SH
T
PacH
mTyrA
DABA
SH SH
A C AT TC*
PacJ
PacO PacN PacL
Ala ArAA
NH2
O
OHNH2
O
OH
C
PacD
OH
NH2
O
OH
OH
tRNA
AAT
PacB
Zhang et al, PNAS 2010; Zhang et al, Biochemistry 2010; Zhang et al, JACS 2011; Zhang et al, Biochemistry 2011; Zhang et al, PNAS 2011
NRPS Assembly Line
S
H2N
O
S
H2N
O
S
HN
O
HNO
-O
O
OH
S
H2N
O NH2
S
HN
O
A
C
A
A
C
TT
T
T
T
C*
PacJPacOPacN
PacN
PacL
PacP
TE*
PacD
NHO
NH
O
COO-
HN
O
NH2
O
NH
HN
ONH
O
NH
NH
O
O
OH
N
HN O
O
H2NCOO-
S
H2N
O NH2
T
PacH
A
PacW
NH2
PacI
PacH
C
C
O
OAMP
OH
NH2
-O
O
OH
OH
OH
HO
OH
HO
NH2
O
OH
N
HN O
O
HO
OH
OH
-OOC
APacW
SH
T
PacP
TE*
SH
T
PacH
mTyrA
DABA
SH SH
A C AT TC*
PacJ
PacO PacN PacL
Ala ArAAC
PacD
C
PacI
O
NH
RHN
AA1 AA2
HN
ONH
O
NH
NH
OAA5
O
OH
N
HN O
O
HO
O
NH
RHN
AA1 AA2
HN
OO
O
NH
NH
OAA5
O
OH
N
HN O
O
HO
O
NH
RHN
AA1 AA2
HN
OO
O
NH
NH
OAA5
O
OH
N
HN O
O
AA1 = alanyl or HAA2 = m-Tyr or AlaAA5 = Phe or Trp or m-TyrR = CH3 or H
In vitro Total Synthesis of >50 Pacidamycin Analogs
Ongoing Projects
O
ONHO OO
O
O
O
HOHO
OH
OH
paclitaxel
HN O
O N
N
H
physostigmineundecene
uridyl peptide antibiotics
S S
O
NH
O
NH
NH
O
O
HO
O
OHN
O
OHHN
O
O
O
R1O
R2
O
N
N
sparsomycin
antimycin4-methylquinazoline
O
N
HN
AA1 AA2
HN
ONH
O
NH
NH
OAA5
O
OH
N
HN O
O
O
OHN
HO
HN
O
O
OH
O
asukamycin
Acknowledgements
HMSWalsh Lab
Christopher T. WalshHeidi ImkerSteven MalcolmsonBrian Ames
Yi TangJixun ZhanSuzanne M. MaYanran LiUCLA
Tang Lab
BerkeleyZhang Lab
Zhe RuiMoriah M. Sandy
Funding:Berkeley Startup, EBI
