Reprogramming alkaloid biosynthesis in Catharanthus roseus : synthetic biology in plants

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.The medicinal plant Madagascar periwinkle (Catharanthus roseus) produces over 130 monoterpene indole alkaloid (MIA) natural products. Many of these compounds have pharmaceutical value, such as the anticancer agents vinblastine and vincristine. Unnatural modifications can impart novel bioactivity to the parent natural product. Advances in synthetic biology and microbial engineering have allowed overproduction of natural products and their analogs in non-native organisms such as yeast and E. coli. However, re-engineering of plant pathways to yield "novel" products has been limited, particularly when compared to the successes achieved in prokaryotic systems. This thesis describes several strategies to re-engineer MIA biosynthesis in periwinkle to produce novel alkaloids. The first strategy involves the introduction of a biosynthetic enzyme with redesigned substrate specificity into periwinkle. The resulting transgenic plant culture produces a variety of unnatural alkaloid compounds when co-cultured with precursors that the re-engineered enzyme has been designed to accept. The second strategy improves upon this work by enabling periwinkle to autonomously synthesize precursor analogs in situ. Specifically, the prokaryotic halogenation machinery was introduced into the genome of periwinkle, which lacks the biosynthetic ability to produce halogenated compounds. These halogenases function within the context of the plant cell to generate halogenated precursor, which is then shuttled into MIA metabolism to yield halogenated alkaloids. Altogether, a new functional group-an organohalide-was introduced into plant secondary metabolism in a regioselective and predictable manner. The third strategy involves RNAi-mediated suppression of MIA biosynthesis in periwinkle. Alkaloid production was obliterated in the resulting transgenic plant culture. The silenced plant culture produces a variety of fluorinated alkaloids when co-cultured with fluorinated starting substrate. The yields of some unnatural alkaloids were improved since the natural precursor was absent. Finally, the fourth strategy describes chemical functionalization of halogenated MIAs. Postbiosynthetic chemical derivatizations of halogenated MIAs using palladium-catalyzed Suzuki-Miyaura cross-coupling reactions robustly afforded aryl and heteroaryl analogs of MIAs. Altogether, the work described in this thesis demonstrates the versatility of medicinal plants in the generation of unnatural alkaloids. Thus, despite their genetic complexity, plants are a viable platform for synthetic biology efforts.by Weerawat Runguphan.Ph.D

Diversification of Monoterpene Indole Alkaloid Analogs through Cross-Coupling

<i>Catharanthus roseus</i> monoterpene indole alkaloid analogs have been produced via a combination of biosynthetic and chemical strategies. Specifically, introduction of a chemical handlea chlorine or a bromineinto the target molecule by mutasynthesis, followed by postbiosynthetic chemical derivatization using Pd-catalyzed Suzuki-Miyaura cross-coupling reactions robustly afforded aryl and heteroaryl analogs of these alkaloids

Integrating carbon–halogen bond formation into medicinal plant metabolism

Halogenation, once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways1. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants2. Given the impact that halogenation can have on the biological activity of natural products1, we rationalized that introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group– a halide– is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore appear to be a viable platform for synthetic biology efforts

Redesign of a Dioxygenase in Morphine Biosynthesis

Opium poppy (Papaver somniferum) produces medicinally important benzylisoquinoline alkaloids, including the analgesics codeine and morphine, in the morphinan pathway. We aligned three dioxygenases that were recently discovered in P. somniferum and subsequently identified the nonconserved regions. Two of these enzymes, codeine O-demethylase (PsCODM) and thebaine O-demethylase (PsT6ODM), are known to facilitate regioselective O-demethylation in morphinan biosynthesis. We systematically swapped the residues that were nonconserved between the PsCODM and PsT6ODM sequences to generate 16 mutant PsCODM proteins that could be overexpressed in Escherichia coli. While wild-type PsCODM can demethylate both codeine and thebaine, one engineered PsCODM mutant selectively demethylates codeine. Use of this reengineered enzyme in the reconstitution of morphine biosynthesis could selectively disable a redundant pathway branch and therefore impact the yields of the downstream products codeine and morphine in subsequent metabolic engineering efforts.National Science Foundation (U.S.) (Predoctoral Fellowship)John Innes CentreUniversity of East Angli

Opportunities in metabolic engineering to facilitate scalable alkaloid production

Numerous drugs and drug precursors in the current pharmacopoeia originate from plant sources. The limited yield of some bioactive compounds in plant tissues, however, presents a significant challenge for large-scale drug development. Metabolic engineering has facilitated the development of plant cell and tissue systems as alternative production platforms that can be scaled up in a controlled environment. Nevertheless, effective metabolic engineering approaches and the predictability of genetic transformations are often obscured due to the myriad cellular complexities. Progress in systems biology has aided the understanding of genome-wide interconnectivities in plant-based systems. In parallel, the bottom-up assembly of plant biosynthetic pathways in microorganisms demonstrated the possibilities of a new means of production. In this Perspective, we discuss the opportunities and challenges of implementing metabolic engineering in various platforms for the synthesis of natural and unnatural plant alkaloids

Metabolic Engineering of Saccharomyces cerevisiae for Production of Fragrant Terpenoids from Agarwood and Sandalwood

Sandalwood and agarwood essential oils are rare natural oils comprising fragrant terpenoids that have been used in perfumes and incense for millennia. Increasing demand for these terpenoids, coupled with difficulties in isolating them from natural sources, have led to an interest in finding alternative production platforms. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce fragrant terpenoids from sandalwood and agarwood. Specifically, we constructed strain FPPY005_39850, which overexpresses all eight genes in the mevalonate pathway. Using this engineered strain as the background strain, we screened seven distinct terpene synthases from agarwood, sandalwood, and related plant species for their activities in the context of yeast. Five terpene synthases led to the production of fragrant terpenoids, including α-santalene, α-humulene, δ-guaiene, α-guaiene, and β-eudesmol. To our knowledge, this is the first demonstration of β-eudesmol production in yeast. We further improved the production titers by downregulating ERG9, a key enzyme from a competing pathway, as well as employing enzyme fusions. Our final engineered strains produced fragrant terpenoids at up to 101.7 ± 6.9 mg/L. We envision our work will pave the way for a scalable route to these fragrant terpenoids and further establish S. cerevisiae as a versatile production platform for high-value chemicals

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris

Concerns over climate change have led to increased interest in renewable fuels in recent years. Microbial production of advanced fuels from renewable and readily available carbon sources has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the yeast Pichia pastoris, an industrial powerhouse in heterologous enzyme production, to produce the advanced biofuel isobutanol from sugarcane trash hydrolysates. Our strategy involved overexpressing a heterologous xylose isomerase and the endogenous xylulokinase to enable the yeast to consume both C5 and C6 sugars in biomass. To enable the yeast to produce isobutanol, we then overexpressed the endogenous amino acid biosynthetic pathway and the 2-keto acid degradation pathway. The engineered strains produced isobutanol at a titer of up to 48.2 ± 1.7 mg/L directly from a minimal medium containing sugarcane trash hydrolysates as the sole carbon source. To our knowledge, this is the first demonstration of advanced biofuel production using agricultural waste-derived hydrolysates in the yeast P. pastoris. We envision that our work will pave the way for a scalable route to this advanced biofuel and further establish P. pastoris as a versatile production platform for fuels and high-value chemicals

MOESM1 of Metabolic engineering of Pichia pastoris for production of isobutanol and isobutyl acetate

Additional file 1. Supplementary information for metabolic engineering of the methylotrophic yeast pichia pastoris for production of isobutanol and isobutyl acetate