The regulation of Taxol production in Taxus suspension cell culture

Plant-derived specialized metabolites are used as pharmaceuticals, dyes and fragrances. Due to the complex nature of these compounds, the continued dependence on plants to synthesize these products has persisted. Many of these products are found at low abundance in plants, so increasing the production of these compounds through metabolic engineering is desirable. An example of a plant-derived pharmaceutical found at low abundance is taxol (Taxol® - Bristol-Myers Squib). Taxol is an anti-cancer agent that is in high demand. Plants in the genus Taxus produce Taxol. Taxus cell suspension cultures are currently being used to meet the demands, however, taxol accumulation is limited. The low yield found in Taxus cultures has prompted biologists and engineers to attempt increasing production of this valuable metabolite. In order to increase taxol accumulation, a better understanding of the metabolic pathway is required. There are unknown steps in the biosynthetic pathway, so cloning the genes involved with these steps is needed. Also, information regarding factors that regulate of the biosynthetic pathway must be collected. In this dissertation, novel genes are identified and the expression profiles of the known genes after methyl jasmonate (MJ) elicitation are revealed. The expression profiles of these genes are directly correlated with the taxol precursor accumulation. Transcription of these pathway genes occurs with MJ elicitation, demonstrating that the response to MJ elicitation is at the level of transcription; thus, taxol production is correlated with taxol pathway gene transcription. A transcription factor has been cloned from Taxus and characterized. This transcription factor is similar to regulators of the MJ-inducible responses in other plants. This Taxus transcription factor, TcJAMYC, activates transcription of taxol pathway gene and also binds to DNA elements found in the promoters of these pathway genes. This transcription factor has the potential to increase taxol accumulation in transgenic Taxus cultures

Jasmonate-responsive expression of paclitaxel biosynthesis genes in Taxus cuspidata cultured cells is negatively regulated by the bHLH transcription factors TcJAMYC1, TcJAMYC2 and TcJAMYC4

Taxus cell suspension culture is a sustainable technology for the industrial production of paclitaxel (Taxol®), a highly modified diterpene anti-cancer agent. The methyl jasmonate (MJ)-mediated paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Here, promoters of seven genes (TASY, T5αH, DBAT, DBBT, PAM, BAPT and DBTNBT), encoding enzymes of the paclitaxel biosynthetic pathway were isolated and used to drive MJ-inducible expression of a GUS reporter construct in transiently transformed Taxus cells, showing that elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. The paclitaxel biosynthetic pathway promoters contained a large number of E-box sites (CANNTG), similar to the binding sites for the key MJ-inducible transcription factor AtMYC2 from Arabidopsis thaliana. Three MJ-inducible MYC transcription factors similar to AtMYC2 (TcJAMYC1, TcJAMYC2 and TcJAMYC4) were identified in Taxus. Transcriptional regulation of paclitaxel biosynthetic pathway promoters by transient over expression of TcJAMYC transcription factors indicated a negative rather than positive regulatory role of TcJAMYCs on paclitaxel biosynthetic gene expression

Reengineering a Tryptophan Halogenase To Preferentially Chlorinate a Direct Alkaloid Precursor

Installing halogens onto natural products can generate compounds with novel or improved properties. Notably, enzymatic halogenation is now possible as a result of the discovery of several classes of halogenases; however, applications are limited because of the narrow substrate specificity of these enzymes. Here we demonstrate that the flavin-dependent halogenase RebH can be engineered to install chlorine preferentially onto tryptamine rather than the native substrate tryptophan. Tryptamine is a direct precursor to many alkaloid natural products, including approximately 3000 monoterpene indole alkaloids. To validate the function of this engineered enzyme in vivo, we transformed the tryptamine-specific RebH mutant (Y455W) into the alkaloid-producing plant Madagascar periwinkle (Catharanthus roseus) and observed the de novo production of the halogenated alkaloid 12-chloro-19, 20-dihydroakuammicine. While wild-type (WT) RebH has been integrated into periwinkle metabolism previously, the resulting tissue cultures accumulated substantial levels of 7-chlorotryptophan. Tryptophan decarboxylase, the enzyme that converts tryptophan to tryptamine, accepts 7-chlorotryptophan at only 3% of the efficiency of the native substrate tryptophan, thereby creating a bottleneck. The RebH Y45SW mutant circumvents this bottleneck by installing chlorine onto tryptamine, a downstream substrate. In comparison with cultures harboring RebH and WT RebF, tissue cultures containing mutant RebH Y455W and RebF also accumulate microgram per gram fresh-weight quantities of 12-chloro-19,20-dihydroakuarnmicine but, in contrast, do not accumulate 7-chlorotryptophan, demonstrating the selectivity and potential utility of this mutant in metabolic engineering applications

Monoterpene indole alkaloid pathway.

<p>The key intermediate strictosidine is formed by condensation of tryptamine, which contributes the indole ring, and secologanin, which is produced from the monoterpene geraniol. In various plants, strictosidine is further metabolized to generate over 2,500 monoterpene indole alkaloids. Solid lines indicate single enzymatic steps; dashed lines indicate multiple steps.</p

Expression patterns of known genes in monoterpene indole alkaloid biosynthesis across different tissues and treatments.

<p>Expression values in log₂ FPKM (fragments per Kilobase of transcript per million fragments mapped) were calculated, negative values were set to zero and then were clustered using R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-R1" target="_blank">[39]</a>. A) <i>Catharanthus roseus</i>: Expression values were sorted in three major groups: Developmental tissues, Yeast extract (YE) treatment of suspension cells (SC), and Methyl jasmonate (MJ) treatment of sterile seedlings (SS) and hairy roots (HR). B) <i>Rauvolfia serpentina</i>. Expression values shown represent the different developmental tissues.</p

Libraries and sequencing generated for <i>Rauvolfia serpentin</i>a.

<p>M: Million.</p

Summary of statistics of the transcriptome de novo assemblies of <i>Camptotheca acuminata</i>, <i>Catharanthus roseus</i>, and <i>Rauvolfia serpentina</i>.

<p>Summary of statistics of the transcriptome de novo assemblies of <i>Camptotheca acuminata</i>, <i>Catharanthus roseus</i>, and <i>Rauvolfia serpentina</i>.</p

Cluster of orthologous and paralogous genes families in <i>Camptotheca acuminata</i>, <i>Catharanthus roseus,</i> and <i>Rauvolfia serpentina</i> species as identified by OrthoMCL.

<p>Predicted peptides from the <i>Camptotheca acuminata</i>, <i>Catharanthus roseus</i> and <i>Rauvolfia serpentina</i> transcriptomes were clustered using OrthoMCL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-Li1" target="_blank">[23]</a>. A) Number of clusters (c) and genes (g) for each orthologous group. B) Number of genes in the different clusters for each species. The number of clusters and genes for each OrthoMCL group are shown. Group 1: Clusters (blue) and genes shared among <i>C. acuminata</i> (red), <i>C. roseus</i> (green) and <i>R. serpentina</i> (purple). Group 2: Clusters (blue) and genes shared among <i>C. acuminata</i> (red) and <i>C. roseus</i> (green). Group 3: Clusters (blue) and genes shared among <i>C. roseus</i> (green) and <i>R. serpentina</i> (purple). Group 4: Clusters (blue) and genes shared among <i>C. acuminata</i> (red) and <i>R. serpentina</i> (purple).</p

Libraries and sequencing generated for <i>Camptotheca acuminata</i>.

1<p>Libraries used in the first, initial de novo assembly.</p>2<p>Libraries only sequenced for expression abundance estimates, reads were not included in de novo assembly.</p>3<p>Libraries used in the second, final de novo assembly.</p><p>M: Million.</p

Phylogenetic relationships.

<p><i>Camptotheca acuminata</i> (Nyssaceae) is in the order Cornales within the asterid superorder of core eudicots, and <i>Catharanthus roseus</i> and <i>Rauvolfia serpentina</i> (both Apocynaceae) are in Gentainales, also within the asterids. <i>Arabidopsis thaliana</i> is in the family Brassicales within the rosid superorder. Fabales and Astrales are shown for orientation. Redrawn and greatly simplified from APG III <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-APGIII1" target="_blank">[19]</a>.</p