Applications of Fungal Prenyltransferases in the Chemoenzymatic Synthesis Anwendungen von

190 Pages·2017·54.54 MB·English

by  Kang Zhou

Applications of Fungal Prenyltransferases in the
Chemoenzymatic Synthesis

Anwendungen von pilzlichen Prenyltransferasen
in der Chemoenzymatischen Synthese

Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)

dem

Fachbereich Pharmazie
der Philipps-Universität Marburg
vorgelegt von
Kang Zhou
aus Hunan, China

Marburg/Lahn Jahr 2017
Erstgutachter: Prof. Dr. Shu-Ming Li
Zweitgutachter: Prof. Dr. Peter Kolb
Eingereicht am 21. März 2017
Tag der mündlichen Prüfung am 05. Mai 2017

Hochschulkennziffer: 1180
Dedicated to my family
TABLE OF CONTENTS
Table of contents
List of publications ................................................................................................................ III
Share of author contributions .............................................................................................. IV
Abbreviations .......................................................................................................................... V
Summary ................................................................................................................................... 1
Zusammenfassung.................................................................................................................... 3
1. Introduction .......................................................................................................................... 5
1.1.Prenylated aromatic secondary metabolites ..................................................................... 5
1.1.1. Prenylated acylphloroglucinols ................................................................................ 6
1.1.2. Prenylated flavonoids............................................................................................... 7
1.1.3. Prenylated indole alkaloids ...................................................................................... 8
1.2. Biosynthetic pathways of prenylated acylphloroglucinols, prenylated flavonids, and
fumitremorgins ..................................................................................................................... 10
1.2.1. Biosynthetic pathway of prenylated acylphloroglucinols ...................................... 10
1.2.2. Biosynthetic pathway of prenylated flavonoids ..................................................... 12
1.2.3. Biosynthetic pathway of fumitremorgins............................................................... 13
1.3. Prenyltransferases ......................................................................................................... 14
1.3.1. Membrance-bound prenyltransferases for aromatic substrates.............................. 15
1.3.2. Soluble prenyltransferases for aromatic substrates ................................................ 16
1.4. Chemoenzymatic synthesis of prenylated aromatic compounds .................................. 21
1.4.1. Chemoenzymatic synthesis of prenylated indole alkaloids and other prenylated
derivatives ........................................................................................................................ 21
1.4.2. Chemoenzymatic synthesis of aromatic substrates by altering the prenyl donors. 22
1.4.3. Chemoenzymatic synthesis of aromatic substrates with enzymes from saturation
mutagenesis experiments ................................................................................................. 23
2. Aims of this thesis............................................................................................................... 25
2.1. Biochemical investigations on the acceptance of acylphloroglucinols in the presence
of DMAPP, GPP, and FPP catalyzed by fungal prenyltransferases .................................... 25
2.2. Chemoenzymatic synthesis of prenylated flavonoids by fungal prenyltransferases .... 25
2.3. Creation of FtmPT1 mutants with strongly increased activity for production of C3-
prenylated cyclo-Trp-Pro stereoisomers by saturation mutagenesis .................................... 26
3. Results and discussion ....................................................................................................... 27
3.1. Biochemical investigations on the acceptance of acylphloroglucinols in the presence of
DMAPP, GPP, and FPP catalyzed by fungal prenyltransferases......................................... 27
3.2. Chemoenzymatic synthesis of prenylated flavonoids by fungal prenyltransferases .... 32
I
TABLE OF CONTENTS
3.3. Creation of FtmPT1 mutants with strongly increased activity for production of C3-
prenylated cyclo-Trp-Pro stereoisomers by saturation mutagenesis .................................... 35
4. Publications ........................................................................................................................ 39
4.1. Friedel-Crafts alkylation of acylphloroglucinols catalyzed by a fungal indole
prenyltransferase .................................................................................................................. 39
4.2. Gem-diprenylation of acylphloroglucinols by a fungal prenyltransferase of the
dimethylallyltryptophan synthase superfamily .................................................................... 64
4.3. Complementary flavonoid prenylations by fungal indole prenyltransferases ............ 107
4.4. Saturation mutagenesis on Tyr205 of the cyclic dipeptide C2-prenyltransferase
FtmPT1 results in mutants with strongly increased C3-prenylating activity ..................... 132
5. Conclusions and future prospects................................................................................... 162
6. References ......................................................................................................................... 164
Acknowledgements .............................................................................................................. 178
Erklärung.............................................................................................................................. 179
Curriculum vitae .................................................................................................................. 180

II
LIST OF PUBLICATIONS
List of publications
Kang Zhou, Carsten Wunsch, Jungui Dai, and Shu-Ming Li (2017). Gem-diprenylation of
acylphloroglucinols by a fungal prenyltransferase of the dimethylallyltryptophan
synthase superfamily. Org. Lett., 19 (2): 388–391
Wei Zhao, Aili Fan, Sylwia Tarcz, Kang Zhou, Wen-BingYin, Xiao-Qing Liu, and Shu-Ming
Li (2017). Mutation on Gly115 and Tyr205 of the cyclic dipeptide C2-prenyltransferase
FtmPT1 increases its catalytic activity toward hydroxynaphthalenes. Appl Microbiol
Biotechnol, 101: 1989–1998
Kang Zhou, Wei Zhao, Xiao-Qing Liu, and Shu-Ming Li (2016). Saturation mutagenesis on
Tyr205 of the cyclic dipeptide C2-prenyltransferase FtmPT1 results in mutants with
strongly increased C3-prenylating activity. Appl Microbiol Biotechnol, 100 (23):9943-
9953
Kang Zhou, Xia Yu, Xiulan Xie, and Shu-Ming Li (2015). Complementary flavonoid
prenylations by fungal indole prenyltransferases. J. Nat. Prod., 78 (9): 2229–2235
Kang Zhou, Lena Ludwig, and Shu-Ming Li (2015). Friedel-Crafts alkylation of
acylphloroglucinols catalyzed by a fungal indole prenyltransferase. J. Nat. Prod., 78 (4):
929–933

III
SHARE OF AUTHOR CONTRIBUTIONS
Share of author contributions
Estimated
Publication (status) Authors equity ratio
[%]
Gem-diprenylation of acylphloroglucinols by a fungal
Zhou, K.; Wunsch, C.; Dai, J.;
prenyltransferase of the dimethylallyltryptophan 50
Li, S.-M.
synthase superfamily. Org. Lett., (published)
Mutation on Gly115 and Tyr205 of the cyclic dipeptide
Zhao, W.; Fan, A.; Tarcz, S.;
C2-prenyltransferase FtmPT1 increases its catalytic
Zhou, K.; Yin, W.-B.; Liu, X.-Q; 15
activity toward hydroxynaphthalenes. Appl Microbiol
Li, S.-M.
Biotechnol. (published)
Saturation mutagenesis on Tyr205 of the cyclic dipeptide
C2-prenyltransferase FtmPT1 results in mutants with Zhou, K.; Zhao, W.; Liu, X.-Q;
45
strongly increased C3-prenylating activity. Appl Li, S.-M.
Microbiol Biotechnol. (published)
Complementary flavonoid prenylations by fungal indole Zhou, K.; Yu, X.; Xie, X.; Li, S.-
60
prenyltransferases. J. Nat. Prod. (published) M.
Friedel-Crafts alkylation of acylphloroglucinols
catalyzed by a fungal indole prenyltransferase. J. Nat. Zhou, K.; Ludwig, L.; Li, S.-M. 65
Prod. (published)

……………………………………… ………………………………………
Signature candidate Signature supervisor
IV
ABBREVIATIONS
Abbreviations
For units of measurements, the international system of units and units derived thereof have
been used. Acronyms and abbreviations are explained in detail in the corresponding passage
in the text.
2D NMR two dimensional nuclear magnetic resonance
4HB 4-hydroxybenzoate
5-DMATS 5-dimethylallyl tryptophan synthase
5-DMATS 5-dimethylallyltryptophan synthase from Streptomyces coelicolor
Sc
6-DMATS 6-dimethylallyl tryptophan synthase
6-DMATS 6-dimethylallyltryptophan synthase from Streptomyces ambofaciens
Sa
6-DMATS 6-dimethylallyltryptophan synthase from Streptomyces violaceusniger
Sv
7-DMATS 7-dimethylallyl tryptophan synthase
APs acylphloroglucinols
AnaPT indole diterpene prenyltransferase anaPT
AtaPT Aspergillus terreus aromatic prenyltransferase
A. fumigatus Aspergillus fumigatus
A. terreus Aspergillus terreus
br broad (NMR signal)
CD OD deuterated methanol
3
CDCl deuterated chloroform
3
CoA coenzyme A
CCL carboxyl CoA ligase
CHS chalcone synthase
Comp. compound
Conc. concentration
COSY correlation spectroscopy
Cyclo-L-Trp- L-Leu cyclo-L-tryptophanyl-L-leucinyl
Cyclo-L-Trp-L-Pro cyclo-L-tryptophanyl-L-prolinyl
D O deuterium oxide
2
d doublet
DCM dichloromethane
DMA dimethylallyl
V
ABBREVIATIONS
DMAPP dimethylallyl diphosphate
DMATS dimethylallyltryptophan synthase
DMSO-d deuterated dimethyl sulfoxid
6
DNA deoxyribonucleic acid
EI-MS electron impact mass spectrometry
ESI-MS electrospray ionization spectrometry
E. coli Escherichia coli
FPP farnesyl diphosphate
GPP geranyl diphosphate
GGPP geranylgeranyl diphosphate
His pentahistidine
5
His hexahistidine
6
His octahistidine
8
HMBC heteronuclear multiple bond correlation
HPLC high performance liquid chromatography
HR high resolution
HSQC heteronuclear single quantum coherence
Hz hertz
HIPT-1 aromatic prenyltransferase from Humulus lupulus
HIPT1L aromatic prenyltransferase HIPT
HIPT2 aromatic prenyltransferase HIPT
H. lupulus Humulus lupulus
H. monopetalus Hexalobus monopetalus
IPP isopentenyl diphosphate
IPTG isopropyl β-thiogalactopyranoside
J coupling constant
k turnover number
cat
kDa kilo Dalton
K Michaelis-Menten constant
M
LB lysogeny broth
m multiplet
MAPP monomethylallyl diphosphate (also methylallyl diphosphate)
MHz mega hertz
MVA mevalonate
VI
ABBREVIATIONS
MS mass spectrometry
multi multiplicity
n × C5 number of C5 units, minimum 1
Ni-NTA nickel-nitrilotriacetic acid
NMR nuclear magnetic resonance
NOESY nuclear overhauser effect spectroscopy
NRPS non-ribosomal peptide synthase
N. fischeri Neosatorya fischeri
A absorption at 600 nm
600
PT prenyltransferase
PKS polyketide synthase
PIBP phlorisobutyrophenone
PIVP phlorisovalerophenone
PBZP phlorbenzophenone
PPi inorganic diphosphate
ppm parts per million
q quartet
rel. conv. relative conversion
RP reverse phase
rpm revolutions per minute
s singlet
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
sp. species (sing.)
spp. species (pl.)
syn. synonymous
t triplet
TB Terrific-Broth
TBA tetrabutylammonium
Tris 2-amino-2-(hydroxymethyl)-propan-1,3-diol
v/v volume per volume
VPS valerophenone synthase
× g gravitational acceleration
VII