学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 西山 真, 東京大学教授 松永 茂樹, 東京大学特任教授 尾仲 宏康, 東京大学准教授 作田 庄平, 東京大学准教授 葛山 智久University of Tokyo(東京大学

THONG WEI LI

トン ウェイ リー

UTokyo Repository

論文の内容の要旨 
 
応用生命工学 専攻 
平成２５年度博士課程  進学 
氏     名 トン ウェイ リー 
指導教員名 西山 真 
 
論文題目 
Studies on novel polyketides discovered via activation of cryptic gene clusters in Streptomyces 
（放線菌の潜在的遺伝子クラスターの活性化により発見した         
新奇ポリケタイドに関する研究） 
 
Actinomycetes, a group of Gram-positive bacteria with high G+C content, is well known as a 
prolific source of a wide range of bioactive secondary metabolites. The intense pursuit of 
bioactive metabolites from actinomycetes started back in the 1940s and early 1950s, when 
important antibiotics, such as the aminoglycosides, tetracyclines and macrolides, were 
discovered. To date, actinomycetes remain an important source for the production of 
naturally derived antibiotics that are used clinically. Despite the success of the 
microorganism as a source of natural products, conventional bioactive compounds screening 
have been deemed stagnant, and efforts are focused on the generation of synthetic compounds 
of their structural analogs instead. This is possibly due to the high rates of rediscovery of 
known compounds. In contrast, recent genome sequencing revealed that there are over 20 
biosynthetic gene clusters (BGCs) in an actinomycete strain, predicted to encode different 
structural classes of secondary metabolites. Intriguingly, most of the predicted BGCs are 
classified as cryptic or orphan, because the metabolites produced by these cryptic BGCs are 
yet to be known. Hence, a growing number of efforts have been expended to develop 
methods that aim to unlock the cryptic pathways in actinomycetes. 
While genome sequences have opened the doors for many successful cryptic gene 
activation approaches, I opted for a broader, sequence-independent strategy in this study. 
Here, the screening of mutants showing rifampicin resistance was applied to explore the 
potential novel compounds in Streptomyces. Spontaneous rifampicin-resistant (rif) mutants 
were reported to have mutation(s) in their rpoB gene (encoding RNA polymerase β-subunit) 
and the rpoB mutations are able to activate the production of bioactive compounds as 
demonstrated in the discovery of a cryptic new type of antibiotic called piperidamycin. 
Chapter 1 Screening of rifampicin-resistant mutants and comparative metabolic 
analysis 
The determination of minimum inhibitory concentrations (MIC) of rifampicin on 20 
actinomycete strains used in this study showed that rifampicin was potent against most of the 
strains used at 3.125 μg mL−1 and below. Streptomyces sp. RM72 and Streptomyces sp. ND90, 
however, were found to have high MIC values of 25 μg mL−1 and 100 μg mL−1, respectively, 
suggesting the possibility of natural resistance. The MICs obtained were then used as 
reference to choose the concentration of rifampicin needed for the generation of rif mutants in 
each strain. Spontaneous rif mutant generation in 20 actinomycete strains produced 278 
mutants.  Then, comparative metabolic analyses were performed on 200 rif mutants selected, 
along with their wild-type strains. Several improved production of metabolites by the rif 
mutants were detected in the screening. Among them, 4 overproduced metabolites 
(metabolites 1, 3, 6 and 7) detected in the rif mutants (WK2057-10-39, TW-R50-13, S45-50-
3 and S55-50-5), were selected for isolation and structural elucidation. The selection was 
based on the preliminary structure searches of these metabolites on SciFinder
®
 and the 
Dictionary of Natural Products on DVD ver. 21:1, which suggested that they are potentially 
novel compounds. 
Chapter 2 Identification of the overproduced secondary metabolites 
Following the comparative metabolic screening in the chapter 1, isolations and structural 
elucidations were performed on the 4 metabolites 1, 3, 6 and 7 selected. Isolation of 
metabolite 1 from WK2057-10-39 resulted in 4 known phenoxazinones. On the other hand, 
purification from 6 L culture of TW-R50-13 yielded 2 novel compounds (compounds 3 and 
3a) with 2 distinct substituents, the 2-amino-3-hydroxycyclopent-2-enone (C5N) and 
methylbenzene moieties (Figure 1). Besides that, metabolite 6 was elucidated as cinerubin A, 
while metabolite 7 was isolated as an unknown compound with a novel skeleton featuring an 
isoindolinone structure (Figure 1). 
 
 
Figure 1. Novel compounds isolated in this study. 
The novel compounds isolated were then evaluated for their biological activities. 
Compound 7 was found to show potent cytotoxicity against all cell lines tested (human 
ovarian adenocarcinoma SKOV-3, malignant pleural mesothelioma Meso-1, and T 
lymphoma Jurkat cells) and antimicrobial activity against Staphylococcus aureus. Compound 
7 was then named as isoindolinomycin. On the contrary, no biological activity was detected 
1-13C sodium acetate
1,2-13C2 sodium acetate
2-13C sodium acetate
1 2 3
1 kbp
Contig12
Contig19
1 kbpKetosynthase
C5N
Methylbenzene
3 5 6 7 15 16 21 22
for compounds 3 and 3a in all assays tested. Both rif mutants producing the novel compounds, 
TW-R50-13 and S55-50-5 have a point mutation in their rpoB gene at the position 437, which 
is a mutation point known to be effective in activating the production of secondary 
metabolites. 
Chapter 3 Biosynthesis of the new compounds 3 and 3a isolated from the rif mutant 
TW-R50-13 
Compounds 3 and 3a are novel compounds featuring a C5N unit and a methylbenzene moiety. 
Of these partial structures, the biosynthesis of the latter has not been reported. BLAST 
searches against the draft genome sequence of Streptomyces sp. SANK 60404 using a 5-
aminolevulinate (ALA) synthase homologue, identified a BGC containing genes that may be 
related to the methylbenzene moiety (type I polyketide synthases) and C5N unit (amide 
synthetase, ALA synthase, and acyl CoA ligase). The deletion of amide synthetase abolished 
the production of 3 and 3a, confirming the relevance of the gene cluster. However, further 
analysis on the BGC showed that the deletion of the polyketide synthase (PKS) gene did not 
abolish the compounds, indicating that the genes are not responsible for the biosynthesis of 3 
and 3a. Because there is no other C5N gene cluster found in the draft genome sequence of 
SANK 60404, I concluded that the BGC is only responsible for the C5N moiety and that the 
genes for the methylbenzene moiety are located at a different locus. Then, a feeding 
experiment using 
13
C-labeled precursors demonstrated that the methylbenzene moiety is most 
likely synthesized by the action of PKS. In silico screening on the draft genome sequence 
indicated that there are at least 7 BGCs encoding different types of PKS. Gene inactivation in 
5 of these PKS gene clusters revealed that a BGC in Contig19 is involved in producing the 
methylbenzene moiety (Figure 2).  
 
 
 
 
Figure 2. 
13
C-labeling pattern and BGCs identified to synthesize compounds 3 and 3a. 
Unlike any other known PKSs, the gene cluster in Contig19 is made up of 8 discrete 
ketosynthases (KSs). A construction of phylogenetic tree showed that these KSs are 
categorized into 2 new clades distant from the other existing KSs. Further analysis on the 
gene cluster suggested that is it potentially a new type of PKS with unprecedented 
mechanism. The deletion of 4 genes in the BGC coupled with in silico analysis provided 
insight into the biosynthetic mechanism that produces the compounds 3 and 3a. 
4 17 18 21 24 26 30 39
Halogenase
Methyltransferase
Type II PKS
Adenylation
1 kbp
Glycosyl
transferase
Methyl
transferase
Methyltransferase
Chapter 4 Biosynthesis of isoindolinomycin isolated from the rif mutant S55-50-5 
To identify the BGC for isoindolinomycin, BLAST searches on the draft genome sequence of 
Streptomyces sp. SoC090715LN-16 was performed using a halogenase homologue as a query. 
The search returned two BGCs, each bearing a gene with close homology to tryptophan 
halogenase. Further analysis on the flanking regions showed that one of the BCG contains 
type II PKS, glycosyltransferase and methyltransferases, which are highly rational for the 
production of isoindolinomycin (Figure 3). The relevance of this BGC was then confirmed by 
gene disruption experiments. The biosynthetic studies of isoindolinomycin were mainly 
focused on 2 aspects: 1) the attachment of the methyl moiety to the core structure and 2) the 
formation of the 5-membered ring in the tetracyclic structure. For the first aspect, 3 
methyltransferases (idmB4, idmB24 and idmB39) found in the gene cluster were deleted to 
identify the gene responsible for the methyl moiety. The deletion of the methytransferases 
indicated that idmB4 is the gene most likely responsible for the attachment of the methyl 
moiety to the tetracyclic structure, while idmB39 may be involved in the O-methylation at the 
sugar moiety. Deletion of idmB24, however, did not cease the production of 
isoindolinomycin. In addition, no accumulation of intermediates was detected in the culture 
of idmB4 deletion mutant, suggesting a possible unprecedented methylation by idmB4 that 
occurs before the tetracyclic is formed. On the other hand, the formation of the 5-membered 
ring may involves the incorporation of an amino acid, presumably, glycine as the starter unit 
for the type II PKS system. To prove this assumption, a malachite green phosphate assay was 
performed to determine the substrate for the stand-alone adenylation domain (idmB21), which 
is known for its specific amino acid recognition activation as adenylyl amino acids. As 
expected, IdmB21 was found to display strict substrate specificity toward glycine only among 
the 20 amino acids tested. Taken together all the data obtained from in silico analysis, gene 
deletions and in vitro assay, the biosynthetic pathway for the unprecedented skeleton of 
isoindolinomycin was proposed. 
 
 
Figure 3. Organization of the BGC identified for isoindolinomycin. 
 
Reference 
Thong W. L, Shin-ya K, Nishiyama M, Kuzuyama T. Methylbenzene-containing polyketides 
from a Streptomyces that spontaneously acquired rifampicin resistance: Structural elucidation 
and biosynthesis. J. Nat. Prod. In revision. 


放線菌の潜在的遺伝子クラスターの活性化により発見した新奇ポリケタイドに関する研究

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放線菌の潜在的遺伝子クラスターの活性化により発見した新奇ポリケタイドに関する研究

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