Associate Professor
Plant Natural Products (Terpenoid) Biosynthesis
Enzyme Mechanisms and Engineering
Biochemical Pathway Identification (Functional Genomics)
Metabolic Engineering
Natural products provided the basis for foundation of the chemical
and pharmaceutical industries, and still provide a rich source of clinically
useful compounds. Among the known natural products, terpenoids or isoprenoids,
composed of variable numbers of isoprene (5 carbon) units, stands out
as the largest class of natural products, with nearly 50,000 distinct
compounds. Within the terpenoids, a common bicyclic core structure can
be found in a substantial fraction (~7,000), which we have grouped together
as the labdanes and related diterpenoids. Notably, a number of these
exhibit medically relevant effects, including anti-biotic, anti-inflammatory,
and anti-cancer activity. Unfortunately, the compounds of interest most
often can be isolated in very small quantities and their generally complex
structures are refractory to synthesis and systemic modification, hindering
pharmacological investigation and use. Thus, despite a number of known
applications, labdane-related diterpenoids are significantly underutilized.
Increased understanding of the enzymatic mechanisms underlying biosynthesis
of these intriguing natural products is expected to enable enzymatic
and metabolic engineering for the production of targeted compounds.
My laboratory is taking an interdisciplinary approach towards three
interconnected themes within this area. First, understanding the diverse
biochemical mechanisms contributing to the production of these natural
products. Second, identifying the biosynthetic enzymes involved in producing
terpenoid natural products of interest. Third, metabolic engineering,
in microbial systems, to produce plant derived terpenoids. The long-term
goal of this research program is enzymatic and metabolic engineering
to enable the production of targeted libraries and specific terpenoid
'natural' products.
Despite the daunting array of chemical structures observed among the
terpenoid natural products, certain common reactions can be observed
in the biosynthesis of all members of this family. Formation of a generally
cyclic hydrocarbon backbone, catalyzed by terpene synthases (cyclases),
defines the basic structural type, while subsequent oxygenation reactions,
generally catalyzed by P450s, sets the pattern for derivation of the
final bioactive compound. Thus, terpene synthases and cytochromes P450
play key roles in terpene biosynthesis. Through detailed biochemical
study of specific examples of these classes of biocatalysts we expect
to specifically enable the enzymatic engineering of terpenoid biosynthesis
that is an integral part of our long-term goal. As a convenient entry
into such biochemical studies we are utilizing the defined metabolic
pathway for production of the gibberellin plant growth hormones as a
model system. The initial steps in this pathway are mediated by two
terpene synthases (cyclases) and two cytochrome P450 oxidases. The two
cyclases catalyze cyclization using very different mechanisms, yet are
related members of the conserved terpene synthase enzyme family. Thus,
these two cyclases are being studied to dissect the distinct catalytic
requirements for their respective cyclization reactions. The two cytochrome
P450 oxidases catalyze multiple sequential reactions in each producing
a carboxylate moiety. The underlying determinants for the intriguing
specificity and reactivity of these multifunctional P450s are under
investigation.
Because rice produces a number of labdane-related diterpenoid natural
products in addition to the ubiquitous gibberellins and its genomic
sequence is available, we have used this plant species as a model system
for investigating functionally novel enzymes and metabolic pathways.
In particular, the well-conserved nature of the diterpene synthases
has led to significant advances in the last year, with five novel cyclases
having been identified through this functional genomics approach by
our group, as well as others [1-7]. Further, because plant secondary
metabolites are often produced in dedicated secretory cells and these
cells express the entire biosynthetic pathway, they provide a highly
enriched source for the enzymes and corresponding genes of the relevant
pathway. Isolation of these cells provides a useful entry into biochemical
pathway identification. However, this has only been attempted with readily
isolated secretory structures, such as the glandular trichomes found
on leaf surfaces. We are developing laser-capture microdissection as
a more general method that should applicable to any targeted secretory
cell. Currently, we are engaged in further elucidation of rice allelochemical
biosynthesis that presumably occurs in root epidermal cells. It is anticipated
that the results of this project will provide additional novel biosynthetic
gene products for enzymatic and metabolic studies and engineering efforts.
Finally, the complex structure exhibited by many terpenoid natural products
defies practical chemical synthesis. This is frequently coupled to low
yields from native sources, resulting in severe bottlenecks in the use
of otherwise promising compounds. Recombinant engineering of microbial
systems offers the possibility of bypassing this restriction. Thus,
we are currently modifying bacteria (E. coli) to produce plant derived
terpenoid natural products. For example, all the labdane-related diterpenes
found in rice. While others are focused on increasing the flux to isoprenoid
biosynthesis in such systems, we are more specifically concerned with
pathway reconstruction demonstrating novel production of valuable diterpenoid
metabolites. Expression of these pathways in strains engineered to produce
large quantities of isoprenoids is expected to alleviate some of the
issues associated with natural product availability.
All of these projects involve recombinant cloning of biosynthetic genes
from plants, their functional expression in microbial systems (bacterial
and/or yeast). Mutants will also be generated and functionally expressed
in the same systems. The enzymatic studies further require protein purification
and biochemical and biophysical characterization in vitro.
References
1. Cho, E.-M., et al., Molecular cloning and characterization of a cDNA
encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin
biosynthetic enzyme, from suspension-cultured rice cells treated with
a chitin elicitor. Plant J., 2004. 37(1): p. 1-8.
2. Otomo, K., et al., Diterpene Cyclases Responsible for the Biosynthesis
of Phytoalexins, Momilactones A, B, and Oryzalexins A-F in Rice. Biosci.
Biotechnol. Biochem., 2004. 68(9): p. 2001-2006.
3. Otomo, K., et al., Biological functions of ent- and syn-copalyl diphosphate
synthases in rice: key enzymes for the branch point of gibberellin and
phytoalexin biosynthesis. Plant J., 2004. 39(6): p. 886-893.
4. Prisic, S., et al., Rice contains disparate ent-copalyl diphosphate
synthases with distinct metabolic functions. Plant Physiol., 2004. 136(4):
p. 4228-4236.
5. Wilderman, P.R., et al., Identification of syn-pimara-7,15-diene
synthase reveals functional clustering of terpene synthases involved
in rice phytoalexin/allelochemical biosynthesis. Plant Physiol., 2004.
135(4): p. 2098-2105.
6. Xu, M., et al., Functional identification of rice syn-copalyl diphosphate
synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic
natural products. Plant J., 2004. 39(3): p. 309-318.
7. Nemoto, T., et al., Stemar-13-ene synthase, a diterpene cyclase involved
in the biosynthesis of the phytoalexin oryzalexin S in rice. FEBS Lett,
2004. 571: p. 182-186.
