A basic study toward metabolic engineering of sesquiterpenes
Makoto KIMURA

Graduate School of Bioagricultural Sciences,
Nagoya University

Research aims
There are many useful sesquiterpenes, but their organic synthesis is often laborious
and costly. Through a series of enzymatic reactions, microorganisms can produce
various kinds of sesquiterpenes from farnesyl pyrophosphate (FPP). The initial step ofthese reactions is catalyzed by a pathway-specific cyclase. Microorganisms with anability to produce a large amount of natural sesquiterpenes may be useful as a basis forforeign sesquiterpene cyclase gene expression, with the aim to produce large amounts ofrelated sesquiterpene skeletons. Currently, these are difficult to provide at low costusing organic synthesis. In this study, we aimed to understand the regulatory
mechanisms of sesquiterpene trichothecene (TCN) biosynthesis in Fusarium
graminearum, focusing on a TCN biosynthesis transcription factor, FgTri6. The
ultimate goal of this research is to develop the organism as a host for the mass
production of heterologous sesquiterpenes.
Methods
We analyzed the mechanisms of Tri6 regulation using the following approaches:(1) Phylogenetically close Fusarium sporotrichioides does not require sucrose1) forTCN production
The promoter and coding region of the F. sporotrichioides Tri6 (FsTri6) gene wereintroduced into the corresponding region of F. graminearum. In addition, Tri6 wasconstitutively overexpressed in F. graminearum. The effects on TCN production in theabsence of sucrose were examined using these strains.
(2) The promoter region of Tri6 contains TRI6p and AreAp consensus bindingsites2)

The two TRI6p binding sites on the Tri6 promoter, 5ʹ-AGGCCT-3ʹ, were mutated to5ʹ-AGGgCT-3. Additionally, Tri4 was constitutively overexpressed outside of thecluster under the control of a TEF promoter. Each of the 15 AreAp consensus-bindingsites located on in Tri6 promoter was mutated.
When the core region of TCN gene cluster is extended by introducing a vector
fragment3), or by moving the promoter region outside of the gene cluster4), the Tri genepromoters do not function properly. Therefore, to engineer the Tri6 locus, we used apositive and negative selection system that creates transgenic strains possessing only thepreferred gene replacements, with mutations at the original locus5).
Results
Each of the transgenic F. graminearum strains that carried either the promoter orcoding region of FsTri6 failed to produce TCN in the absence of sucrose (Fig. AB).
Additionally, the Tri6 overexpressor could not produce TCN in the absence of sucrose(Fig. 1C). Wild-type F. graminearum did not produce TCN in a medium supplemented
with 100 µM sucrose (Fig. 1C left panel), although TCN did accumulate in the mediumof the Tri6 overexpressor (Fig. 1C right panel).
When the TRI6p-binding sites in the Tri6 promoter were mutated, weak expression ofTri6, but not of other pathway Tri genes, was observed. Consistent with this observation,
no TCN accumulation was detected in the mutant culture. After constitutively
expressing Tri4 outside of the gene cluster, a small amount of TCN became detectable.
This indicates that, if sucrose is present in the medium, only a small amount of TRI6p is
sufficient to activate other pathway Tri gene promoters and eventually produce TCN.
The mutant strain, in which all the AreAp-binding sites on the Tri6 promoter weremodified, produced similar but slightly reduced amounts of TCN.
Conclusions
We found that neither the sequences of the FgTri6 promoter nor the FgTRI6p protein
were responsible for the sucrose-dependent induction of TCN production in F.
graminearum. Sucrose has been suggested to be important for post-translational
activation of TRI6p protein in F. graminearum, which is dependent on unknown signals
activated by sucrose. Controlling the timing of sesquiterpene production so that it
coordinates with the most appropriate stage of growth may mean that the fungus could
be exploited as a host for the metabolic engineering of sesquiterpenes.

References
1) Nakajima, Y. et al. (2016) Oligosaccharides containing an α-(1 → 2)
(glucosyl/xylosyl)-fructosyl linkage as inducer molecules of trichothecenebiosynthesis for Fusarium graminearum. Int. J. Food Microbiol. 238:215-221.
2) Merhej, J., Richard-Forget, F., Barreau, C. (2011) Regulation of trichothecenebiosynthesis in Fusarium: recent advances and new insights. Appl. Microbiol.
Biotechnol. 91: 519-528.
3) Chen, L., McCormick, S.P., Hohn, T.M. (2000) Altered regulation of15-acetyldeoxynivalenol production in Fusarium graminearum. Appl. Environ.
Microbiol. 66:2062-2065.
4) Tokai, T. et al. (2013) A promoter of Fusarium graminearum Tri4 does not function
when placed at the end of the trichothecene gene cluster. Mycotoxins 63:17-25.
5) Nakajima, Y., Ichikawa, H., Maeda, K., Nishiuchi, T., Kobayashi, T., Kimura, M.
(2013) Genome engineering of Fusarium species by using positive and negative
selection approaches for studying regulation of mycotoxin production. (Review inJapanese) Mycotoxins 63:85-92.

Fig.

1.

Sucrose-dependent

expression of Tri genes and TCN
production in F. graminearum.
YS_60 and YG_60 media containsucrose and glucose as a carbon
source, respectively. The results ofnorthern blotting and TLC analyses
are shown. (A) A transgenic strain
in which the FgTri6 promoter wasreplaced by the FsTri6 promoter.
(B) A transgenic strain in which theFgTri6 coding region was replacedby the FsTri6 coding region. (C) A
transgenic strain in which Tri6 wasconstitutively overexpressed. The effects of 100 µM sucrose (in YG_60 medium) on Tri gene
expression and TCN production were analyzed.

Fig. 2. Generation of transgenic strains carrying mutations on the Tri6 promoter region. (A) PTri6_M1,a transgenic stain in which the two TRI6p-binding sites on the Tri6 promoter were mutated;
PTri6_M1/ PTEF::Tri4, a transgenic strain in which the two TRI6p-binding sites on the Tri6 promoter
were mutated and Tri4 was overexpressed from outside of the gene cluster. (B) Tri gene expression
and TCN production by transgenic strains. The results of northern blotting and TLC analyses areshown.