Introduction of Research Interests in Plant Metabolic Engineering Lab.

Topic 1. Carotenoid Metabolic Engineering in Plants

The first generation of Korean Golden rice (PAC rice) producing the carotenoids, terpene pigment metabolites with a variety of
functionality and color, has been developed by applying two types of multicistronic expression technology, F2A and IRES, capable of
simultaneously expressing two carotenogenic genes under a single promoter (Ha et al., 2010). Using this PAC (Psy:F2A:R1Tp-CrtI)
recombinant gene, , we have also developed a high content of beta-carotene-producing soybean (Kim et al. 2012).
In the follow-up study, the second-generation of stPAC rice was developed by optimizing PAC gene into stPAC (stPsy:F2A:R1Tp-
stCrtI) recombinant gene by elucidating the effect of a codon-optimized gene according to rice codon usage on the enhancement
of carotenoid contents (Jeong et al. 2017). We have further advanced it into the complex multicistronic expression technology and
applied to the development of various colorful rice varieties having diverse functional carotenoids of zeaxanthin, astaxanthin and
capsanthin by simultaneous use of the conventional interbreeding. In addition, we have developed several novel technologies
requiring for plant metabolic engineering: the novel chloroplast-targeting peptides of PTp and its synthetic peptide of stPTp
(You et al. 2016) and three multicistronic expression sequences of T2A, I2A1 and I2A2 being verified via the multi-expression of
2, 3 and 4 genes of carotenoid biosynthetic genes under a single promoter. Taken all technologies tigether, the recombinant genes of
stPTAC (stPsy:T2A:PTp-stCrtI) and stPTARC (stPsy:F2A:R3Tp-stCrtI) were constructed and introduced into rice to develop the
beta-carotene rice events for practical use and these rice plants have been on the beginning stage for the safety evaluation.
And, the large-scale approaches to improve beta-carotene rice event and to develop zeaxanthin, astaxanthin, and capsanthin
rice events are also being conducted for practical uses.

Topic 2. Modulating the biosynthetic mechanisms of functional terpene metabolites for the construction of the precursors-enhanced platform plants

Terpene metabolites are composed of two isoprene building blocks (C5), isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate
(DMAPP), that are biosynthesized via a plastidial methylerythritol 4-phosphate (MEP) pathway and a cytosolic mevalonate (MVA)
pathway in plants. The isoprene building blocks (C5) are sequentially condensed by rice prenyltransferase (PTases) of GPS
(GPP synthase), FPS (FPP synthase) and GGPS (GGPP synthase) to generate the initiative terpene precursors of geranyl diphosphates
(GPP, C10), farnesyl diphosphates (FPP, C15) and geranylgeranyl dihphosphates (GGPP, C20), respectively.
And then those precursors are modified into diverse terpene metabolites based on the monoterpene (C10), sesquiterpene(C15),
diterpene (C20), triterpene (C30), and tetraterpene (C40) structures by various actions of terpene synthase (TPS) mediating
condensation, cyclization, reduction, glycosylation and so on.

To elucidate the rice specified mechanism supplying the terpene precursors such as isoprenes (IPP/DMAPP) and
prenyl-pyrophosphates (GPP/FPP/GGPP), we have analyzed the multi-omics data of the isoprene-enriched rice transgenic plants
and the GGPP-enriched rice cultivar plants for the biosynthetic networking of terpene metabolites. Also, we have investigated the
biosynthetic mechanism of rice terpenoids, through the functional analyses of rice PTases on the biosynthesis of GPP, FPP and GGPP,
protein-protein interaction between PTase and TPS, and subcellular localization in rice.

Topic 3. Mastering of Regulatory Mechanism for the Plant Functional Metabolism

For increasing plant metabolism toward favorable directions for either humans or plant itself, direct increment of structural gene
expressions or applying transcriptional regulators which enhance the structural gene expressions have been reported as the main
strategies in plant metabolic engineering fields. However in case of terpene metabolism, small numbers of transcriptional factors
(TFs) have been studied as mainly negative regulators including only few positive regulators. Therefore, it is necessary to acquire the
TF candidates related to the regulation of terpene metabolism. Through the study of their functions, elucidation of the regulatory
mechanism might be very useful to enhance the plant value by increasing the functional metabolite production.

So far, several phenomenological reports that plant secondary metabolites have effects on the resistance of abiotic environmental
stresses such as drought (water deficit), excessive watering (water-logging/flooding), extreme temperatures (cold, frost and heat)
and salinity have been reported. But there is little to the report to explain the resistant mechanisms and we are doing strategic
approaches to elucidate them. Firstly, we obtained the candidate TF genes to be up- or down-regulated under blue-light condition
that simultaneously increased phenolic compounds and terpene metabolites (Lakshmanan et al. 2015). Among them, a TF is mainly
being studied to clarify the regulatory mechanism that affects the expression of genes being involved in terpene metabolism when
it was over-expressed in plant body. On the basis of its cold-resistant phenotype, the action mechanism of terpene metabolism under
the abiotic stress condition is trying to be interpreted via the establishment of systems biological network by integration between
transcriptomic and metabolomic data. Through this, we would like to develop the environmental stress tolerant crops by secondary
metabolite control eventually. It will be the one of novel mechanisms related to abiotic stress resistance of plant systems. Finally,
we aim to identify transcriptional regulatory networks between〈transcription factor - specific secondary metabolism - abiotic stress
resistances〉.