Investigating Biosynthetic Steps of an Angucycline Antifungal

74 Pages·2017·2.29 MB·English

by  S. Gabrielle Gladstone

UUttaahh SSttaattee UUnniivveerrssiittyy
DDiiggiittaallCCoommmmoonnss@@UUSSUU
All Graduate Theses and Dissertations Graduate Studies
5-2016
IInnvveessttiiggaattiinngg BBiioossyynntthheettiicc SStteeppss ooff aann AAnngguuccyycclliinnee AAnnttiiffuunnggaall
S. Gabrielle Gladstone
Utah State University
Follow this and additional works at: https://digitalcommons.usu.edu/etd
Part of the Biological Engineering Commons
RReeccoommmmeennddeedd CCiittaattiioonn
Gladstone, S. Gabrielle, "Investigating Biosynthetic Steps of an Angucycline Antifungal" (2016). All
Graduate Theses and Dissertations. 4952.
https://digitalcommons.usu.edu/etd/4952
This Thesis is brought to you for free and open access by
the Graduate Studies at DigitalCommons@USU. It has
been accepted for inclusion in All Graduate Theses and
Dissertations by an authorized administrator of
DigitalCommons@USU. For more information, please
contact [email protected].
INVESTIGATING BIOSYNTHETIC STEPS OF AN ANGUCYCLINE ANTIFUNGAL
by
S. Gabrielle Gladstone
A thesis submitted in partial fulfillment
of the requirements for the degree
of
MASTER OF SCIENCE
in
Biological Engineering
Approved:
Jixun Zhan Ron Sims
Major Professor Committee Member
Anhong Zhou Mark R. McLellan
Committee Member Vice President for Research and
Dean of the School of Graduate Studies
UTAH STATE UNIVERSITY
Logan, Utah
2016
ii
Copyright © S. Gabrielle Gladstone 2016
All Rights Reserved
iii
ABSTRACT
Investigating Biosynthetic Steps of an Angucycline Antifungal
by
S. Gabrielle Gladstone, Master of Science
Utah State University, 2016
Major Professor: Jixun Zhan
Department: Biological Engineering
From the bacterium Streptomyces sp. SCC-2136 (ATCC 55186), two angucycline nat-
ural products are produced, designated Sch 47554 and Sch 47555. These compounds are
produced through a type II polyketide biosynthetic pathway. The early biosynthetic steps
to these molecules were confirmed. These include the minimal polyketide synthase (PKS),
the C-9 ketoreductase, the first-ring aromatase, the subsequent ring cyclase, and two oxy-
genases. Also confirmed were the biosynthetic genes responsible for production of the first
amicetose moiety, as well as the glycosyltransferase that creates a C-glycosidic bond be-
tween the angucyclic scaffold and the amicetose moiety. In confirming these pathways, two
new natural products were produced: GG31, an amitosylated rabelomycin, and GG53, ra-
belomycin hydroxylated at C-12b. Future work will be to understand the late biosynthetic
steps and generate new angucyclines through combinatorial biosynthesis.
(64 pages)
iv
PUBLIC ABSTRACT
Investigating Biosynthetic Steps of an Angucycline Antifungal
S. Gabrielle Gladstone
The species of bacterium Streptomyces sp. SCC-2136 which has the American Type
Culture Collection index 55186 naturally produces two chemical compounds, labeled Sch
47554 and Sch 47555. These compounds were previously reported to posess antifungal
activity. This thesis sets out to confirm the mechanism by which the bacterium produces
these compounds; specifically which genes are responsible for producing the enzymes that
make and shape the molecules.
The genes and enzymes that were characterized are the minimal polyketide synthase,
the ketoreductase that specifically acts on the ninth carbon, the first-ring aromatase, the
subsequent ring cyclase, and two oxygenases. Also elucidated were the genes responsible
for production of the first sugar (called amicetose) in the chain of two sugars attached to
carbon 9 as well as the glycosyltransferase enzyme that attaches this sugar to the rest of
the molecule.
In confirming these pathways, two new compounds were produced: GG31, which has
a base structure of the antibiotic rabelomycin but is attached at carbon 9 to a molecule of
the deoxysugar amicetose; and GG53 which has the base structure of rabelomycin but with
a hydroxyl group at carbon 12b. Future work will be to understand the later biosynthetic
steps and generate new related molecules through combinatorial biosynthesis.
v
Maria Montessori once said, ”The preparations for life are indirect”. This is certainly true.
This thesis is dedicated to my parents, Melissa and Russell Gladstone; to Andrew Tori
Hamblin; to Kandy Napan-Hsieh; and to Aerin Richardson.
Wasn’t this a wild ride.
vi
ACKNOWLEDGMENTS
I must thank Dr. Jixun Zhan for his guidance and patience while I learned about
molecular biology and combinatorial biosynthesis. I also wish to thank the members of my
Masterscommittee, Drs. AnhongZhouandRonSimsfortheirattentionandhelptheselast
two semesters. Paul Veridian, the graduate coordinator in BioEngineering, was invaluable
in the management of all the paperwork, deadlines, and requirements. I could not have
finished this without you.
Additionally, I am eternally grateful for all of the people who were friendly and en-
couraging over the last four years. These include my labmates: Dr. Zeng Jia, Dr. Kandy
Napan, Dr. Siyuan Wang, Dr. Jiachen Zi, Dr. Lei Shao, Dr. Tong Zhou, Dr. Bingji Ma,
Dr. Shuwei Zhang, Dr. Dayu Yu, Dr. Jianfeng Mei, Dr. Anfeng Xiao, Dr. Riming Yan,
Dr. Huiwen Yan, Lei Sun, and Fuchao Xu.
For their help outside the lab, I wish to thank the Painter family, Kat Combs, Jessica
Bills, Steven Collins, Stetson Barnhouse, Kassie and Rick Cressal, Jesse Walker, the Smurf
family, the USU library staff, and anyone who gave me a reassuring smile.
And most importantly, I thank my parents, my grandparents, and my partner Andrew
Hamblin: we made it.
S. G. Gladstone
Logan, UT
2016
vii
CONTENTS
Page
ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
PUBLIC ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2 Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1 Type I and Type III PKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 Type II PKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 Angucycline Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.1 Angular Hydroxyls . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3.2 Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Experimental Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.1 Extraction of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.2 Polymerase Chain Reaction and Amplification of Genes . . . . . . . . . . . 16
3.3 Creating Plasmids for Heterologous Expression . . . . . . . . . . . . . . . . 17
3.4 Extraction of Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1 Rabelomycin Pathway Elucidation . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.1 Minimal PKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.1.2 Ketoreductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.1.3 Aromatases and Cyclases . . . . . . . . . . . . . . . . . . . . . . . . 22
4.2 Glycosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.3 Angular Hydroxyls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
5 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
6.1 Glycodiversification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
6.2 Co-expression of Two Plasmids to Assist with Combinatorial Biosynthesis . 35
APPENDICES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
A Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
B Nuclear Magnetic Resonance Spectra for Structure Confirmation of GG31 . 47
viii
LIST OF TABLES
Table Page
3.1 Sequences of primers used for plasmid construction . . . . . . . . . . . . . . 17
3.2 General PCR mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.1 Nuclear magnetic resonace spectroscopy (NMR) data for GG31 . . . . . . . 25
4.2 Plasmid construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
5.1 Gene functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
A.1 Numbered compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
ix
LIST OF FIGURES
Figure Page
1.1 Structures of 1, 2, 3, 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1 The proposed organization of the herboxidiene biosynthetic gene cluster . . 5
2.2 Polyketide chains of certain lengths spontaneously form predictable structures 7
2.3 Biosynthesis of oxytetracycline and reported shunt products . . . . . . . . . 8
2.4 Oxidoreductase UrdM catalyzes a 2-step reaction . . . . . . . . . . . . . . . 13
2.5 Catalysis of 12 and 12b oxidation . . . . . . . . . . . . . . . . . . . . . . . . 14
2.6 Examples of glycosylated natural products . . . . . . . . . . . . . . . . . . . 14
2.7 Deoxysugars that can be synthesized from common intermediates . . . . . . 15
2.8 The four urdamycin glycosyltransferases . . . . . . . . . . . . . . . . . . . . 15
4.1 Putative gene cluster of ATCC 55186 . . . . . . . . . . . . . . . . . . . . . . 20
4.2 The vector used to build all of the expression plasmids . . . . . . . . . . . . 20
4.3 HPLC traces of expressed biosynthetic products. . . . . . . . . . . . . . . . 27
4.4 Structures of urdamycin A and simocyclinone D8 . . . . . . . . . . . . . . . 28
4.5 Structure elucidation of GG31 . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.6 Comparison of retention times of 18 to 16 . . . . . . . . . . . . . . . . . . . 29
4.7 Proposed pathway to 1 and 2 . . . . . . . . . . . . . . . . . . . . . . . . . . 30
6.1 Putative route from glucose-6-phosphate to the three sugars . . . . . . . . . 34