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Chapter 4. Conclusion
In this thesis, the potential of the secondary metabolite production of P. miuraensis SMH27-4 and the biosynthetic machinery for miuraenamide A were illustrated.
4.1. Genomic analysis of P. miuraensis SMH-27-4
Myxobacteria are common in terrestrial habitats and known for their potential to produce
novel natural products, whereas marine-derived (or halophilic) ones are quite rare and only
seven species (five genera) have been identified since the isolation of the first marine
myxobacteria H. ochraceum and P. pacifica in 1998. Although these marine myxobacteria
are regarded as a good factory of valuable secondary metabolites beyond the terrestrial
ones, their cultivation is generally difficult and takes a long period for enough growth. Their
genomic information is therefore important to elucidate their great potential to produce novel
leads with unique molecular scaffolds and bioactivities. P. miuraensis SMH-27-4 produces a
series of PKS/NRPS hybrid molecules named miuraenamides, but its metabolic profile
indicated a scarcity of metabolite diversity; no other distinct metabolites were detected in the
extracts. The genomic analysis of this strain was therefore performed in this study and
revealed the presence of 17 BGCs for producing metabolites, one of which was estimated to
encode the biosynthesis of miuraenamides. The complete genome sequence was not
available in this study due to the extremely difficult cultivation and DNA extraction from
aggregated mucous cells. Nevertheless, because of the high-quality sequence data, 93%
coverage of the complete genome (the rest could be repetition), and no overlooking of other
possible BGCs, the present draft genome information could contribute to improving the
inadequate expertise in the marine myxobacterial genomic functions, especially for hidden
biosynthetic machineries leading to brand-new natural products. Further studies will be
needed to reveal the mechanism of the miuraenamide biosynthesis as well as more precise
genomic analysis.
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4.2. Heterologous biosynthesis of miuraenamide A
Due to the difficulty in cultivation and the lack of genetic manipulation tools, the
biochemical studies on the native producer P. miuraensis SMH-27-4 are hard to process.
The heterologous biosynthesis became a promising methodology for the biosynthesis
mechanism elucidation and efficient production of miuraenamide A (1, Figure 4-1B). The
antiSMASH predicted the biosynthetic gene cluster of miuraenamide A (miu cluster, Figure
4-1A) was successfully cloned and heterologously expressed in the well-known terrestrial
myxobacterium Myxococcus xanthus with a productivity miuraenamide A of 6% of the
original strain. The proposed biosynthetic mechanism of the miu cluster was partially verified
by gene disruption experiments using the transformant. The type I PKSs (MiuA and MiuB),
one NRPS (MiuC), one O-methyltransferase (MiuE), and one tyrosine halogenase (MiuG)
were verified to be responsible for the biosynthesis. Besides miuraenamide A, four
congeners (2‒5, Figure 4-1B) were identified based on their mass spectra. The antifungal
activities of the miuraenamide E (3) or 4 were confirmed to be lower than the 1 due to the
lack of β-methoxyacrylate group or halogenation. Although the activities of 2 and 5 had not
been measures, they were estimated to be weaker than 1 due to the lack of the βmethoxyacrylate group. The BGC for 1 was narrowed to 20 orfs (orf11‒orf24 region)
extending over 62.1 kbp (corresponding to 72% of the original miu cluster). The removal of
the orf25‒29 and orf19‒23 regions resulted in a substantial increase of the yields of 1,
suggesting the presence of unknown gene(s) in the orf19‒23 region that are related to the
expression regulation or even affecting the substrate supply of the biosynthetic pathway. The
detailed mechanism is well worth exploring and may provide a new strategy for secondary
metabolite production boosting. Based on the results of in vivo experiments, it is likely that
the halogenase (MiuG) is tyrosine specific. It is also worthwhile to explore the potential
80
industrial applications and development of this enzyme.
miuE
miuA
miuB
miuC
miuG
orf1-10
orf19-23
Br
Br
HO
HO
HO
O O
O O
miuraenamide A (1) R = Br
debromomiuraenamide A (4) R = H
orf25-29
2R=H
4 R = OH
O O
miuraenamide E (3)
Figure 4-1. Organization of miu cluster (A) and strctures of miuraenamide A (1) and related
congeners (B).
81
Acknowledgements
The work for this thesis was carried out in the Bioactive Molecules Laboratory of the
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Japan. First, I would like to thank my supervisor, Professor Ojika, for
leading me into myxobacteria's wonderful and exciting world. Thank you for all your support
and patience. With your careful guidance, I made it to the end. My procrastination has
caused you a lot of trouble for the past four years. I always handed you the content that
needed to be revised just before the deadline was about to come. You never gave up on me,
which was the biggest motivation to support me in completing my coursework. Besides the
content presented in the thesis, many exploratory studies yielded no positive results.
Whenever I had ideas, Professor Ojika actively supported me in exploring and practicing.
This unwavering support has been a memorable experience for me. It will be a source of
motivation for my future life. If I had the opportunity to be a teacher for someone else, I
would give my students the diligence, trust, and patience I learned from Professor Ojika to
help them become better people.
I also want to thank Prof. Kita, Prof. Nishikawa, Prof. Tsunematsu, and Dr. Kondo for
reviewing my thesis and providing practical advice on my research. Also, thank all my lab
mates who had helped me with many things over the years.
I particularly thank Dr. Fudou and Dr. Iizuka from Ajinomoto Co. Inc. for their supply of
myxobacterium. Mr. Yamazaki, for his excellent previous work on genome library
construction and screening, made a solid foundation for my research.
I am deeply thankful to the Interdisciplinary Frontier Next-Generation Researcher Program
of the Tokai Higher Education and Research System, the Toyo Suisan Foundation
Scholarship, and the Hattori International Student Education Association for their financial
support and kind care. Without your support, I would not have been able to complete my
82
thesis, so I am genuinely grateful. I will remember the help and warmth I received in Japan,
work hard, and live well in the future, and try to be a bridge of friendship between China and
Japan.
Then again, I would like to thank my parents for their constant and unconditional trust and
support.
Finally, dearly cherish the memory of my grandmother Wang Xianhua, my grandfather Liu
Xuemin, and Shimajiri Shouta.
2023
LIU Ying
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