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Kim Roberts Freedom Group

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Amorpha ((INSTALL))

FaunalAssociations:The flowers of Leadplant (Amorpha canescens) attract long-tongued bees, short-tongued bees,and wasps primarily. Among the bees are such visitors as bumblebees,Leaf-Cutting bees (Megachile spp.), Green Metallic bees, and Plastererbees (Colletes spp.); the Andrenid bee, Andrena quintilis,is an oligolege of Leadplant. The caterpillars of Coliascesonia (Dogface Sulphur) eat the foliage occasionally, butthis butterfly often fails to overwinter successfully in Illinois.Other insects that feed on the foliage, flowers, seeds, and other parts of Leadplant includegrasshoppers (Melanoplus spp. & others), broad-headed bugs (Alydus spp.), the plant bugs Lopidea instabilis and Plagiognathus amorphae, leaf beetles (Pachybrachis spp. & others), larvae of moths (Catocala spp. & others), and the leafhopper Scaphytopius cinereus (see Insect Table for a more complete list of these species). Many ofthese insects, especially grasshoppers, are an important source of food to insectivorous birdsand other animals. Mammalian herbivores, such as deer, rabbits, andlivestock, are very fond of this plant. It is high in protein and quitepalatable. This can make Leadplant difficult to establish in areaswhere these animals are abundant. Photographic Location:Photographs were taken at the webmaster's wildflower garden in Urbana,Illinois. Comments:This is a true prairie plant.


Bedding Dye Fodder Insecticide Oil Repellent Shelterbelt Soil stabilizationPlants have an extensive root system and are also fairly wind tolerant, they can be planted as a windbreak and also to prevent soil erosion[200]. Resinous pustules on the plant contain 'amorpha', a contact and stomachic insecticide that also acts as an insect repellent[57, 200]. The stems are used as bedding[61]. The plant contains some indigo pigment and can be used to make a blue dye[169]. Unfortunately, the pigment is only present in very small quantities, there is not enough to harvest commercially[169].

In addition to amorfrutins other compounds present in A. fruticosa have been evaluated for their potential as antidiabetic agents. In this context, the effects of amorphastilbol (APH), another constituent from A. fruticosa, was studied in vitro with 3T3-L1 adipocytes, as well as in vivo with db/db and high-fat-diet (HFD) mice (Lee et al., 2013, 2015). It was observed that the compound is able to stimulate the transcriptional activities of PPARγ and PPARα, resulting in beneficial effects on the metabolism of lipids and glucose without significant side effects that are notoriously associated with stimulation of the PPAR receptors, such as weight gain or hepatomegaly. APH was also able to improve insulin sensitivity via inhibition of protein tyrosine phosphatase 1B (Lee et al., 2015).

In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases). In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), amorpha-4,11diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1.0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.

Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step.

By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene.

Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.

Historically, heterologous production of small molecules has been hampered by the challenges in expressing complex functional pathways for the production of the non-native molecules. New tools in rapid gene synthesis and metabolite analysis have promoted the field of synthetic biology, which promises advances in pathway re-construction, and strives toward pathway and genome optimization. Recent success in reconstituting heterologous pathways in microorganisms for high-level production of small molecules has demonstrated the feasibility of achieving titers in the g/L range. Examples include production of polyketides such as 6-deoxyerythronolide B [6] and isoprenoids such as amorpha-4,11-diene [7] and lycopene [8] in Escherichia coli. Our work focuses on improving heterologous production of the artemisinin precursor amorpha-4,11-diene, which can be converted to artemisinin via chemical transformation.

Recently, the expression of a synthetic amorpha-4,11-diene synthase gene along with the mevalonate isoprenoid pathway native to S. cerevisiae was engineered in E. coli for the production of amorpha-4,11-diene (Figure 1) [9]. Production was increased with the use of a two-phase partitioning bioreactor which captures the product in the organic phase [7]. In this work, we present improvements in amorpha-4,11-diene production in E. coli by further strain engineering and fermentation process development. In the original heterologous mevalonate pathway, two key genes (HMG-CoA synthase (HMGS: ERG13, Genbank GeneID: 854913) and the catalytic domain of HMG-CoA reductase (tHMGR: HMG1, Genbank GeneID: 854900)) were derived from yeast [9]. It was subsequently demonstrated that the activity of tHMGR is insufficient to balance flux in the heterologous pathway leading to a pathway bottleneck [10]. In the current work HMGS and tHMGR were replaced with more active enzymes from Staphylococcus aureus, doubling amorpha-4,11-diene production. Amorpha-4,11-diene producing E. coli strains were grown in a defined, glucose restricted fed-batch process which achieved cell densities of 90 g/L dry cell weight. Titers were further improved in this process by simultaneously restricting ammonia and carbon. Nitrogen consumption rates depended on the components of the pathway that were expressed, thus the process was modified to accommodate the high production strain containing the modified mevalonate pathway. An amorpha-4,11-diene titer of greater than 25 g/L was achieved in a robust and reproducible fermentation process.

Genes in blue arrows are derived from E. coli, those in brown arrows from yeast, and ADS in red from A. annua. pMevT, pMBIS and pADS indicate the arrangement of genes on expression plasmids. Gene names and the enzymes they encode: atoB, acetoacetyl-CoA thiolase; ERG13, HMG-CoA synthase; tHMG1, truncated HMG-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; MVD1, mevalonate pyrophosphate decarboxylase; idi, IPP isomerase; ispA, farnesyl pyrophosphate synthase. Pathway intermediates: Ac-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; HMG-CoA, hydroxymethylglutaryl-CoA; Mev-P, mevalonate 5-phosphate; Mev-PP, mevalonate pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FDP, farnesyl pyrophosphate; ADS, amorphadiene synthase.

To test the effect of nitrogen restriction on cell growth and production of amorpha-4,11-diene, process A was modified whereby the ammonium sulfate was left out of the feed (process B). The ammonia in the medium was allowed to decrease to zero though nitrogen flow into the bioreactor was still positive due to base addition. The carbon consumption in process B was similar to that of process A (data not shown). Once the initial glucose was depleted, the glucose concentration was maintained at zero, thus preventing any accumulation of acetate in the culture (Figure 3b). Ammonium sulfate in the batch medium was utilized in the first 40 hours and the amount of ammonium hydroxide added to correct the pH thereafter did not result in ammonia accumulation until 95 hours (Figure 3b) while supporting maximum cell density of 235 OD600 (Figure 3a). Similar cell growth between processes A and B confirmed sufficient supply of nitrogen from the base to build cell mass in process B despite the fact that the measured ammonia in the culture was zero. The difference in amorpha-4,11-diene production between the two runs was evident after 50 hours where the production profiles diverged (Figure 3a). This correlated well with the ammonia concentrations measured in the culture between 50 hours and 100 hours. In process A, ammonia was in excess at 40 mM whereas in process B the ammonia concentration was negligible during most of the production period. Process B with strain B32 achieved a peak amorpha-4,11-diene titer of 16.7 g/L at 120 hours, a 2.5-fold improvement in titer compared to process A. 041b061a72


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