Kristin K. Wobbe

Recent Advances in Artemisinin Production Through Heterologous Expression

Artemisinin the sesquiterpene endoperoxide lactone extracted from the herb Artemisia annua, remains the basis for the current preferred treatment against the malaria parasite Plasmodium falciparum. In addition, artemisinin and its derivatives show additional anti-parasite, anti-cancer, and anti-viral properties. Widespread use of this valuable secondary metabolite has been hampered by low production in vivo and high cost of chemical synthesis in vitro. Novel production methods are required to accommodate the ever-growing need for this important drug. Past work has focused on increasing production through traditional breeding approaches, with limited success, and on engineering cultured plants for high production in bioreactors. New research is focusing on heterologous expression systems for this unique biochemical pathway. Recently discovered genes, including a cytochrome P450 and its associated reductase, have been shown to catalyze multiple steps in the biochemical pathway leading to artemisinin. This has the potential to make a semi-synthetic approach to production both possible and cost effective. Artemisinin precursor production in engineered Saccharomyces cerevisiae is about two orders of magnitude higher than from field-grown A. annua. Efforts to increase flux through engineered pathways are on-going in both E. coli and S. cerevisiae through combinations of engineering precursor pathways and downstream optimization of gene expression. This review will compare older approaches to overproduction of this important drug, and then focus on the results from the newer approaches using heterologous expression systems and how they might meet the demands for treating malaria and other diseases.

Reproductive Development Modulates Gene Expression and Metabolite Levels with Possible Feedback Inhibition of Artemisinin in Artemisia annua

The relationship between the transition to budding and flowering in Artemisia annua and the production of the antimalarial sesquiterpene, artemisinin (AN), the dynamics of artemisinic metabolite changes, AN-related transcriptional changes, and plant and trichome developmental changes were measured. Maximum production of AN occurs during full flower stage within floral tissues, but that changes in the leafy bracts and nonbolt leaves as the plant shifts from budding to full flower. Expression levels of early pathway genes known to be involved in isopentenyl diphosphate and farnesyl diphosphate biosynthesis leading to AN were not immediately positively correlated with either AN or its precursors. However, we found that the later AN pathway genes, amorpha-4,11-diene synthase (ADS) and the cytochrome P450, CYP71AV1 (CYP), were more highly correlated with AN’s immediate precursor, dihydroartemisinic acid, within all leaf tissues tested. In addition, leaf trichome formation throughout the developmental phases of the plant also appears to be more complex than originally thought. Trichome changes correlated closely with the levels of AN but not its precursors. Differences were observed in trichome densities that are dependent both on developmental stage (vegetative, budding, and flowering) and on position (upper and lower leaf tissue). AN levels declined significantly as plants matured, as did ADS and CYP transcripts. Spraying leaves with AN or artemisinic acid inhibited CYP transcription; artemisinic acid also inhibited ADS transcription. These data allow us to present a novel model for the differential control of AN biosynthesis as it relates to developmental stage and trichome maturation and collapse.

ARTEMISININ: THE BIOSYNTHETIC PATHWAY AND ITS REGULATION IN ARTEMISIA ANNUA, A TERPENOID-RICH SPECIES

SummaryArtemisinin is a sesquiterpene lactone isolated from the aerial parts of Artemisia annua L. plants. Besides being currently the best therapeutic against both drug-resistant and cerebral malaria-causing strains of Plasmodium falciparum, the drug has also been shown to be effective against other infections diseases including schistosomiasis and hepatitis. More recently, it has also been shown to be effective against numerous types of tumors. Although chemical synthesis of artemisinin is possible, it is not economically feasible. The relatively low yield (0.01–0.8%) of artemisinin in A. annua is a further serious limitation to the commercialization of the drug. Therffore, the enhanced production of artemisinin either in cell/tissue culture or in the whole plant of A. annua is highly desirable. A better understanding of the biochemical pathway leading to the synthesis of artemisinin and its regulation by both exogenous and endogenous factors is essential for facilitating increased yield. Two genes of the artemisinin biosynthetic pathway have now been identified. This critical review covers recent developments related to the biosynthesis of this important compound and related terpenoids, their regulation, and the production of these compounds both in vitro and in whole plants.

The biological response of hairy roots to O2 levels in bioreactors

SummaryThe efficient exchange of gases between roots and their environment is one of the biggest challenges in bioreactor design for transformed root cultures. Gas-phase reactors can alleviate this problem as well as provide a new tool for studying the biological response of roots and other differentiated tissues to changes in the gas phase composition. In our comparison of liquid- and gas-phase reactors, roots grown in liquid (shake flasks or bubble column reactors) are shown to be under hypoxic stress. Roots grown in a gas-phase reactor (nutrient mist), while not hypoxic, produced 50% less biomass. These results suggest that the response of the tissues to gas phase composition are complex and need further study.

The mevalonate-independent pathway is expressed in transformed roots of Artemisia annua and regulated by light and culture age

SummaryArtemisia annua produces a large number of unique terpenoids, making it of particular interest as a source of phytochemicals and a useful model plant for studying terpenoid metabolism. The ability to engineer fast-growing in vitro cultures to produce terpenoids in high yield would be a dramatic step towards commercial use. Two distinct pathways have been characterized in higher plants leading to the biosynthesis of isopentenyl diphosphate, the common precursor to all terpenes: the cytosolic mevalonate pathway and the plastid-localized mevalonate-independent pathway. While transformed roots of A. annua have been demonstrated to be superior to whole plants in terms of yield of the sesquiterpene artemisinin, they appear to lack functional chloroplasts, bringing into question the presence of a functional mevalonate-independent pathway. Using a cDNA library made from these roots, we isolated two clones encoding deoxy-d-xylulose-5-phosphate synthase (DXPS) and deoxy-d-xylulose-5-phosphate reductoisomerase (DXPR). The biochemical function of both enzymes was confirmed by complementing E. coli dxps- and dxpr-mutants. Northern blot analysis showed that the transformed root cultures expressed these genes at different levels during the culture cycle. In addition, cultures grown in continuous light showed substantial increases in DXPS transcript levels compared to dark-grown cultures. These results represent an important step towards demonstrating the presence of the plastid-localized terpenoid biosynthetic pathway in these easily engineered in vitro cultures.

Effect of Sugars on Artemisinin Production in Artemisia annua L.: Transcription and Metabolite Measurements

The biosynthesis of the valuable sesquiterpene anti-malarial, artemisinin, is known to respond to exogenous sugar concentrations. Here young Artemisia annua L. seedlings (strain YU) were used to measure the transcripts of six key genes in artemisinin biosynthesis in response to growth on sucrose, glucose, or fructose. The measured genes are: from the cytosolic arm of terpene biosynthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), farnesyl disphosphate (FPS); from the plastid arm of terpene biosynthesis, 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxyxylulouse 5-phosphate reductoisomerase (DXR); from the dedicated artemisinin pathway amorpha-4,11-diene synthase (ADS), and the P450, CYP71AV1 (CYP). Changes in intracellular concentrations of artemisinin (AN) and its precursors, dihydroartemisinic acid (DHAA), artemisinic acid (AA), and arteannuin B (AB) were also measured in response to these three sugars. FPS, DXS, DXR, ADS and CYP transcript levels increased after growth in glucose, but not fructose. However, the kinetics of these transcripts over 14 days was very different. AN levels were significantly increased in glucose-fed seedlings, while levels in fructose-fed seedlings were inhibited; in both conditions this response was only observed for 2 days after which AN was undetectable until day 14. In contrast to AN, on day 1 AB levels doubled in seedlings grown in fructose compared to those grown in glucose. Results showed that transcript level was often negatively correlated with the observed metabolite concentrations. When seedlings were gown in increasing levels of AN, some evidence of a feedback mechanism emerged, but mainly in the inhibition of AA production. Together these results show the complex interplay of exogenous sugars on the biosynthesis of artemisinin in young A. annua seedlings.

Artemisinin: Controlling Its Production in Artemisia annua

Artemisinin, a potent antimalarial sesquiterpene lactone, is produced in low quantities by the plant Artemisia annua L. We used inhibitors of both the mevalonate and nonmevalonate terpenoid pathways to study in both seedlings and hairy root cultures the source of isopentenyl diphosphate (IPP), the channeling of carbon from sterols to sesquiterpenes, and the role that sugars may play in controlling artemisinin biosynthesis. Together, our results indicated that artemisinin is likely biosynthesized from IPP pools originating in both the plastid and the cytosol and that channeling of carbon can be directed away from competing sterol pathways and toward sesquiterpenes. Although glucose stimulated artemisinin production, the response is very complex with ratios of glucose to fructose involved; artemisinin levels increased proportionate to increasing amounts of glucose. Disaccharides mainly inhibited artemisinin production, but the response was less definitive. Glucose also increased expression of some of the genes in the artemisinin biosynthetic pathway, thereby suggesting that this sugar is acting not only as a carbon source but also as a signal. As we develop a better understanding of the regulation of the artemisinin biosynthetic pathway, results suggest that many factors can possibly be harnessed to increase artemisinin production in A. annua.

Local Strategies: Creating and Nurturing Collaborative Communities of Practice

Kristin Wobbe is the associate dean of undergraduate studies at Worcester Polytechnic Institute and director of the Great Problems Seminars program which introduces first-year students to WPI’s intensely project-based curriculum. Thoughtful department chairs and deans are always seeking new ways to engage and prepare faculty to teach general education courses. The search for meaningful professional development opportunities for faculty can be particularly challenging at professionally-focused institutions. Liberal arts courses are sometimes viewed merely as service requirements that students eschew in favor of their major-specific courses. Creating and Nurturing Collaborative Communities of Practice

Mini workshop — Great problems lead to great projects: A first year seminar course

In 2007 WPI initiated the Great Problems Seminars to engage First Year students with current events, societal problems and human needs while developing skills that will facilitate subsequent project work. These seminars focus on large global issues — energy and its utilization, food and hunger, disease and healthcare delivery and the NAE Grand Challenges. Each is led by two faculty from disparate departments who expose the students to the complexity of the problem using a number of disciplinary perspectives. Students develop information literacy, effective writing and speaking skills, while working in teams on short assignments. For the final project student teams work with faculty supervision to analyze some aspect of the problem and/or develop a partial solution. The seminars culminate in a Project presentation day where all student groups present a poster on their work. We have assessment data that show that the courses achieve their desired outcomes. The special session will review the motivation to create these courses and provide information about course organization and logistics. Participants will review student projects, discuss the preparation required, and determine how elements of this program can be utilized on their home campus. Ideas generated by participants will be shared with all.