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Noda Institute for Scientiﬁc Research GRANT
2011 Research Grant

Establishing a new methodology for genome mining andbiosynthesis of natural products and their analogs through ayeast molecular genomics
Kenji WATANABE
Department of Pharmaceutical Sciences, University of Shizuoka

Research aims
Fungal genome sequencing revealed the presence ofmany genes coding for natural product biosynthetic enzymes, including polyketide synthases and nonribosomal
peptide synthetases. However, it remains a challenge tocharacterize these enzymes and identify the compounds
they synthesize, either in their original hosts or more
tractable heterologous hosts, such as yeast. Here, we havedeveloped a streamlined method for isolating biosynthetic
genes from fungal sources and producing bioactive molecules using an engineered Saccharomyces cerevisiae strain
as a host. We used overlap extension PCR and yeast homologous recombination technology to clone a desired fungal
polyketide synthase or nonribosomal peptide synthetasegene (5–20 kb) into a yeast expression vector quickly andefﬁciently. This approach was used successfully to clone
ﬁve polyketide synthases and one nonribosomal peptidesynthetase from various fungal species. Subsequent detailed
chemical characterizations of the resulting natural products
identiﬁed 6 polyketide and 2 nonribosomal peptide products, some of which were new compounds. Our systemshould facilitate the efforts in mining the uncharacterized
fungal biosynthetic potential for identifying novel naturalproducts and rationally engineering in the biosynthetic
pathways for production of analogs possessing desiredbioactivity.
Methods
Assembling the PKS and NRPS biosynthetic genes onplasmids
To construct the expression vector in yeast, GAL1 promoter–ADH1 terminator cassette was taken from thegenome of S. cerevisiae and cloned into pTOWug2-836
vector to yield pKW1250. The HIS3 gene responsible for Lhistidine biosynthesis was cloned out from pRS423 and integrated into pTOWug2-836 replacement with URA3,
uracil biosynthetic gene, yielding pKW1113 by the homologous recombination activity. The TEF1 promoter–CYC1
terminator cassette was inserted into pKW1113(HIS3) vector to yield pKW18101,2). In pKW1810, the expressed ORF
will have at least one Flag-tag at the N terminus. A C-terminal hexahistidine tag-coding fragment and multiple

cloning sites originating from pET32a plasmid werealso present in pKW1810. Each ORF, CHGG_00542,
CHGG_10027,
CHGG_10128,
CC1G_05377
andAfu6g12080, was predicted based on the sequence information available from the Broad Institute database, and theirpredicted functions were determined by comparison toknown proteins using the BLAST peptide sequence database search program and the FFAS03 protein sequence proﬁle-proﬁle alignment and fold recognition program.
A full-length cDNA of the putative PKS or NRPS gene
was synthesized by reverse transcriptase with a oligo(dT)20
Primer using the corresponding mRNA in the isolated total
RNA as a template. To construct the yeast expression vector carrying one of the target biosynthetic gene, the gene
was ampliﬁed from the full-length cDNA by PCR in overlapping smaller fragments no more than 3 kb in length
using multiple pairs of primers. Subsequently, these DNA
fragments were reconstituted into a full-length double
strand DNA coding for the desired ORF by the overlap extension PCR method. This ampliﬁed ORF was combinedwith pKW1810 linearized by restriction enzyme digestion.
Both DNA fragments were then simultaneously introduced
into S. cerevisiae BY4741. The two fragments were joinedin situ by the endogenous homologous recombination
activity of S. cerevisiae through the 25-bp homologous
sequences present in both DNA fragments. The desiredtransformants were selected by identifying the plasmid vector marker, HIS3, on an L-histidine-deﬁcient plate. Theplasmid now carrying the desired ORF was recovered fromthe yeast transformant and transferred to E. coli. The plasmid was ampliﬁed for examining its structure by restriction
enzyme digestion, and later sequenced to conﬁrm identityof the plasmid.
Results
Characterization of polyketide products biosynthesized byChaetomium globosum PKS CHGG_00542 in yeast 3)
We cultivated the SCKW5 strains on an YPD liquidmedium at a large scale and puriﬁed the compounds using
column chromatography and HPLC when necessary.
Fractions obtained from the HPLC eluent of thepKW14050(CHGG_00542)/SCKW5 culture extract weredried in vacuo to furnish pure solid 13). Subsequently, 1 was49

Figure 1. Chemical structures of the compounds biosynthesized by theengineered yeast. A) Compounds 1–5 were biosynthesized by the wild
type CHGG_00542 PKS. B) Compounds 6 and 7 were biosynthesized by
the S2010A mutant CHGG_00542 PKS. C) Compound 9 was biosynthesized by CHGG_10027 and CHGG_10128 PKSs. D) Compound 10 wasbiosynthesized by CC1G_05377 PKS. E) Compounds 11 and 12 werebiosynthesized by Afu6g12080 NRPS.

reacted with diazomethane to give a methyl ester 2 at
1.3 mg/L (Figure 1A). Furthermore, the engineered yeastallowed us to biosynthesize 3, which was converted into amethyl ester 4 by dimethyl sulfate at 32.5 mg/L of culture
for further chemical structure characterizations. Additionally, 3 was presumably oxidized spontaneously to yield 5 at
25 mg/L as another biosynthetic product produced by
CHGG_00542. Based on in vitro studies, Ebizuka et al.
have proposed a biosynthetic pathway for 1 involving a PKS called PKS1 from A. oryzae4). Therefore,
CHGG_00542 could play a functionally equivalent role as
PKS1. Interestingly, the authors were able to rationally reengineer the biosynthetic pathway to produce 6 (Figure 1B)
by inactivating the TE/CLC domain of PKS1 via a sitedirected mutagenesis of the catalytic Ser2009 to an Ala.
Amino acid sequence comparison between PKS1 andCHGG_00542 indicates 61% identity over the entire
sequence. On the other hand, 74% identity was observed between their TE/CLC domains. To determine if
CHGG_00542 is functionally similar to PKS1, we prepareda S2010A mutant of CHGG_00542, where the residue
S2010 corresponds to S2009 in PKS1. We designated thismutant as CHGG_00542* and named the yeast expression
vector carrying this mutant gene pKW14051. As predicted,
we were able to isolate 6 from the culture ofpKW14051(CHGG_00542*)/SCKW5 that was prepared
under essentially the same condition used to produce 1.
However, we also unexpectedly detected the presence of another compound 7 in this culture.

Characterization of polyketide products biosynthesized by
Chaetomium globosum PKSs CHGG_10027 and CH
GG_10128 in yeast 3)
We were also able to isolate nonpolar compounds fromthe cultures of pKW14051(CHGG_10027)/SCKW5 andpKW14051(CHGG_10128)/SCKW5 at less than 0.5 andapproximately 1.7 mg/L of culture, respectively. Thesecompounds were characterized by HR-ESI-MS and NMR
following similar procedures as described earlier. Surprisingly, spectroscopic analysis of these products indicatedthat CHGG_10027 and CHGG_10128 biosynthesized anidentical compound, 6-methylorsellinic acid 9 (Figure 1C).
CHGG_10027 is a type I iterative PKS with seven modules
as ascertained by in silico analysis: SAT (starter unit: ACPtransacylase), KS (ketosynthase), MAT (malonyl-CoA acyltransferase), PT (product template), ACP (acyl carrier protein), MT (methyltransferase) and RED (reductase). On theother hand, CHGG_10128 is predicted to have eight modules: SAT, KS, MAT, PT, double ACP, MT and EST (esterase/lipase). The product structure suggests that the SAT
domain in both CHGG_10027 and CHGG_10128 accepts
an acetyl-CoA as the only type of starting unit for the synthesis of the polyketide core structure. Also, both of thesePKSs would be categorized as a non-reducing PKS due thelack of domains capable of catalyzing a reduction and/ordehydration reaction, but neither has a conventional C-terminal thioesterase domain. CHGG_10027 has a reductase
domain, and CHGG_10128 carries an esterase/lipase-likedomain. The only example of a type I iterative PKS with aC-terminal esterase/lipase-like domain reported was MpaC
from Penicillium brevicompactum involved in the biosynthesis of mycophenolic acid. In this recent report, MpaC
was proposed to synthesize 9, the same compound produced by CHGG_10128, as an intermediate during the mycophenolic acid biosynthesis. Comparison of the domainarchitecture of CHGG_10128 and MpaC shows that bothPKSs are highly homologous to each other except for theabsence of the second ACP domain in MpaC. Since theamino acid sequence between the corresponding domainsof CHGG_10128 and MpaC are on average approximately
42% identical and 60% similar, it is plausible thatCHGG_10128 produces the same polyketide product 9 that
MpaC synthesizes. However, it was unexpected that both
CHGG_10027 and CHGG_10128 would produce the same
polyketide compound despite that these PKSs had clearly
different domain architectures. In particular, CHGG_10027
carries a C-terminal RED domain, suggesting that
CHGG_10027 is expected to generate an aldehyde-containing product. It is possible that the aldehyde is accidentally
oxidized to a carboxylic acid by some yeast oxidoreductase.
Conclusion
In this study, we established an innovative approach forbiosynthesizing bioactive compounds of fungal origin byfocusing on previously uncharacterized biosynthetic geneclusters and using an engineered S. cerevisiae strain as asurrogate host. We exploited the overlap extension PCR
method to quickly synthesize full-length PKS and NRPS
genes, which are usually 5- to 20-kb or longer, using a poolof cDNA reverse-transcribed from a total RNA isolated

50

from the source fungus. The ampliﬁed gene was subsequently cloned into a yeast expression vector using the recombination capability of yeast. This series of procedures
allowed fast and efﬁcient establishment of a yeast systemfor expression of biosynthetic genes of unknown function
and production of corresponding natural products. Our results clearly demonstrated successful expression of fourtype I iterative PKS genes and one NRPS gene from threedifferent fungal species in S. cerevisiae, all of which led tothe production of a total of six compounds whose identities
were characterized spectroscopically and veriﬁed the speculated functions of these biosynthetic mega-enzymes. Our
plasmid-based system provides an advantage over fungal
systems in terms of ease and speed of cloning the target
genes, and it also tolerates handling of substantially large
genes. Also, the use of a plasmid-borne system simpliﬁes
the effort of engineering biosynthetic pathways for production of various analogs using traditional molecular biological techniques. By streamlining the process of translating

uncharacterized fungal biosynthetic genes into structurally
characterized compounds, our methodology should facilitate the efforts in isolating novel natural products and rationally engineering in the biosynthetic pathways for production of analogs possessing comparable or even more potent bioactivity.
References
1.

Tsunematsu, Y. et al. (2012) Overexpressing transcriptional regulator
in Chaetomium globosum activates a silent biosynthetic pathway:
evaluation of shanorellin biosynthesis. J. Antibiot. 65: 377–380.
2. Nakazawa, T. et al. (2012) Overexpressing transcriptional regulator
in Aspergillus oryzae activates a silent biosynthetic pathway to produce novel polyketide. ChemBioChem 13: 855–861.
3. Ishiuchi, K. et al. (2012) Establishing a new methodology forgenome mining and biosynthesis of polyketides and peptides throughyeast molecular genetics. ChemBioChem 13: 846–854.
4. Ebizuka, Y. et al. (2004) Unprecedented mechanism of chain length
determination in fungal aromatic polyketide synthases. Chem. Biol.
11: 1101–1106.