Presentation at Pacifichem 2015.

1. 12/25/2015
1
Amy L. Lane, Ph.D.
Assistant Professor
University of North Florida
Department of Chemistry
Amy.Lane@unf.edu
Probing the biosynthetic capabilities
of a marine Nocardiopsis sp.
regulationortransportoxidoreductase
nsnR1 R2 I H T1T2 orf7
cytochromeP450
F L
hypotheticalorother
E
nonribosomalpeptide
synthetase(NRPS)
A B C D G
epoxidehydrolase
lysinecyclodeaminase
R3 R4
orf17-18
polyketide
synthase(PKS)
Pacifichem 2015
Raju et al. Org. Lett. 2011;
Tetrahedron 2013; Eur. J. Chem. 2010
Nocardiopsis sp. CMB-M0232: Talented natural product chemist

2. 12/25/2015
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Nocardiopsins:
Newest rapamycin class natural products
• FKBP modulators.
• Nocardiopsins
structurally distinct
from other class
members.
FK506
rapamycin
nocardiopsin A
Vezina et al. (1975) J. Antibiot.;
Kino et al. (1987) J. Antibiot.;
Raju et al. (2010) Eur. J. Chem.
Nocardiopsins:
Newest rapamycin class natural products
FK506
rapamycin
nocardiopsin A
Vezina et al. (1975) J. Antibiot.;
Kino et al. (1987) J. Antibiot.;
Raju et al. (2010) Eur. J. Chem.
Macrolide ring closure via
3o C-OH with non-PKS
derived hydroxy group
THF
group

3. 12/25/2015
3
Nocardiopsin Polyketide Core Assembly
Bis et al. ChemBioChem (2015)
regulation or transportoxidoreductase
nsnR1 R2 I H T1 T2 orf7
cytochrome P450
F L
hypothetical or other
E
nonribosomal peptide
synthetase (NRPS)
A B C D G
epoxide hydrolase
lysine cyclodeaminase
R3 R4
orf17-18
polyketide
synthase (PKS)
N O
O
O
O
O
OH
OH5
6
N O
O
O
O
O
OH
OH
10
Oxidoreductase
and/or
P450
(NsnH,
NsnI)
nocardiopsin B
6
8
Nonribosomal peptide incorporation and macrolide formation
Bis et al. ChemBioChem (2015)
regulation or transportoxidoreductase
nsnR1 R2 I H T1 T2 orf7
cytochrome P450
F L
hypothetical or other
E
nonribosomal peptide
synthetase (NRPS)
A B C D G
epoxide hydrolase
lysine cyclodeaminase
R3 R4
orf17-18
polyketide
synthase (PKS)
NH2
COOHH2N
lysine
cyclodeaminase
(NsnL)
N
H
COOH
L-lysine L-pipecolic acid
NRPS,
L-pipecolic
acid
NRPS
(NsnE)
Suggests novel biosynthetic route to
prepare molecule for macrolide closure.
nsnG
nsnF

4. 12/25/2015
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Nocardioazines: Anticancer natural products
from a marine Nocardiopsis sp.
• Uniquely prenylated
indole alkaloid
diketopiperazine (DKP).
• New class of bridged DKP.
• Reverse drug resistance in
cancer cells by inhibiting
P-glycoprotein.
Raju et al. (2011) Org. Lett.
Probing the enzymology of the nocardioazine
biosynthetic pathway
Biosynthesis of cyclo(Trp-Trp) core:
Hypothesis 1: Core assembled via nonribosomal peptide synthetase.
Hypothesis 2: Core assembled via cyclodipeptide synthase (CDPS). Two
putative CDPSs encoded by genome.
James et al. ACS Synthetic Biology. Articles ASAP.

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Cyclodipeptide synthases (CDPSs):
Candidates for Biosynthesis of Nocardioazine Core
• First CDPS was characterized by Gondry and coworkers in 2009.
Reviewed by Belin et al. (2012) Nat. Prod. Rep.
CDPS-encoding gene clusters from Nocardiopsis sp.
• The noz cluster encodes CDPS as well as
additional enzymes with putative roles in
nocardioazine assembly.
• The ncd cluster encodes only a CDPS
homolog and no other enzymes
homologous to biosynthetic enzymes.
James et al. ACS Synthetic Biology. Articles ASAP.

6. 12/25/2015
6
time (min)
• Expression of
either NozA or
NcdA in E. coli
resulted in
biosynthesis of
cyclo(Trp-Trp).
• No other DKP
products were
observed.
• In vitro assays with
purified enzymes
also supported
tryptophanyl-tRNA
as sole substrate of
both CDPSs.
Evaluating the
biosynthetic
function of
NozA and NcdA
A
James et al.
ACS Synthetic Biology.
Articles ASAP
Both nozA and ncdA are transcribed
by Nocardiopsis sp.
• RT-PCR suggests both NozA and NcdA catalyze production of
cyclo(L-Trp-L-Trp) by Nocardiopsis sp.
• NozA and NcdA provide uncommon example of multiple
phylogenetically distinct enzymes from one organism yielding the
same secondary metabolite. James et al. ACS Synthetic Biology. Articles ASAP.
RT-PCR Accumulation of cWW in
Nocardiopsis cultures

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Conclusions
• The unique ensemble of natural products of Nocardiopsis sp. CMB-
M0232 is assembled by an equally unique suite of biosynthetic
enzymes.
• NozA and NcdA catalyze formation of cyclo(L-Trp-L-Trp) and add to the
group of substrate specific CDPSs.
• Both NozA and NcdA are candidates for biosynthesis of cyclo(L-Trp-L-
Trp) as a nocardioazine precursor.
• Nocardiopsin assembly includes an unprecedented biosynthetic
strategy to prepare the linear PKS-NRPS precursor for macrolide ring
closure.
Acknowledgements
Funding:
• Research Corporation Cottrell College Science Award
• UNF Transformative Learning Opportunities Grants
• UNF Faculty Fellowship Award
Lane lab students
Collaborators
• Dr. Robert Capon (U Queensland)
• Dr. Rajesh Viswanathan (Case
Western Reserve U)
• Dr. Jonathan Karty (Indiana U)
• Dr. Bryan Knuckley (UNF)
Elle James
Dana Bis
Yang Ban