Bioreactor considerations for secondary metabolite production from plant cell tissue culture: Indole alkaloids from Catharanthus roseus

Batch shake flask studies with Catharanthus roseus demonstrated that alkaloid production commenced only after growth had slowed or ceased. To obtain high alkaloid productivities for extended periods, a hormone-free production medium was used. To develop a readily scalable process, both immobilized and suspended cell systems were studied. In the immobilized cell systems, growth, glucose utilization, and alkaloid production were suppressed; for the case of membrane entrapped cells this suppression was observed to be reversible. Based on the oxygen requirements of the cells, and the oxygen transfer capabilities of a pneumatically agitated bubble column, conditions were established that allowed the growth and production dynamics observed in shake flasks to be reproduced in the air sparged column reactor. The requirement for the aseptic exchange of growth for production medium was satisfied by using a coarse cotton filter and coupling filtration to aeration. With this filtration system, media can be rapidly and completely exchanged and the filter can be quickly and effectively backwashed. By coupling filtration to aeration, a two-stage batch operation can be employed while requiring only a single bioreactor. Studies with this system demonstrated its capabilities for alkaloid production.     

Indole alkaloid formation by Catharanthus roseus cells in a biofilm reactor.

Catharanthus roseus cells producing indole alkaloids were grown in the form of a biofilm. Production medium was circulated through the reactor parallel to the upper surface of the horizontal biofilm. Sugar consumption and indole alkaloid formation were followed to compare the performance of cultures with different biofilm thicknesses. Dissolved oxygen concentrations gradients within the biofilms were determined at the end of each run. RNA and protein content of the cells in the upper and lower layers of the the biofilms were compared. Results obtained in the biofilm experiments were compared to those obtained with suspension cultures. At optimized biofilm thicknesses, the biofilm reactor was more effective than suspension cultures in maximizing indole alkaloid titers. This is thought to be due to better cell-cell contact within the biofilm and nutrient concentration gradients, which resulted in low growth rates.     

Pravastatin: A tool for investigating the availability of mevalonate metabolites for primary and secondary metabolism in Catharanthus roseus cell suspensions

In the C20 strain of Catharanthus roseus , 2,4-dichlorophenoxyacetic acid (2,4-D) reduces alkaloid accumulation by inhibiting the synthesis of precursors of alkaloid terpenoids. However, the presence of this growth regulator is necessary to promote growth, as measured in terms of dry weight and sterol content. The terpenoid metabolism implicated in the accumulation of alkaloids would therefore be the target of 2,4-D inhibition and not the metabolism leading to sterol biosynthesis. The specific inhibition by pravastatin of 3-hydroxy-3-methylglutaryl CoA reductase (EC 1.1.1.34), the enzyme that catalyses mevalonate synthesis, enables the limitation of mevalonate on sterol content and on alkaloid accumulation to be studied. In presence of pravastatin cells are supplied with labelled mevalonate. Under these conditions the mevalonate is incorporated into sterols but not into alkaloids accumulated in the absence of 2,4-D. The inhibition of sterol biosynthesis induced by pravastatin is not overcome by zeatin or a cytokinin-like compound, whereas the inhibition of alkaloid accumulation can be partially overcome. The use of pravastatin shows that the availability of mevalonate for primary and secondary metabolism is differently regulated in Catharanthus roseus cells.     

Effects of alkaloid precursor feeding and elicitation on the accumulation of secologanin in a Catharanthus roseus cell suspension culture

In a Catharanthus roseus cell line accumulating secologanin, time-course studies on the uptake of loganin and the in vivo conversion to secologanin were performed. Four-day-old cells converted 100% of the fed loganin to secologanin within 24 hours, showing that this step is unlikely to be limiting for alkaloid accumulation. Thirteen-day-old cells also took up loganin, but only about 25% was recovered as secologanin. A saturation in the uptake of loganin and in the conversion of loganin into secologanin was observed after feeding increasing amounts of loganin. Elicitation by cellulase and pectinase decreased the cellular contents of secologanin and strictosidine whereas it increased the tryptamine content. In addition, the uptake of loganin in elicited cells was blocked. In vitro assays with protein extracts of elicited Catharanthus roseus cells indicated the activation of secologanin degrading enzyme(s). Feeding of tryptophan did not result in any increase in alkaloid contents, despite its complete uptake. Tryptamine feeding led to increased strictosidine contents, but ajmalicine levels remained unchanged. This revised version was published online in June 2006 with corrections to the Cover Date.     

Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.     

Enhancement of vincristine under in vitro culture of Catharanthus roseus supplemented with Alternaria sesami endophytic fungal extract as a biotic elicitor

International Microbiology - Vincristine, one of the major vinca alkaloid of Catharanthus roseus (L.) G. Don. (Apocynaceae), was enhanced under in vitro callus culture of C. roseus using fungal...     

The effect of inoculum density and conditioned medium on the production of ajmalicine and catharanthine from immobilized Catharanthus roseus cells

The effect of the cell-inoculum size and the addition of conditioned medium on ajmalicine and catharanthine production were studied using immobilized Catharanthus roseus cells. Higher specific-uptake rates of ammonium, nitrate, and sugars were observed in the low-inoculum-density cultures (50 g FW/L) compared to the high-inoculum-density cultures (100 g FW/L). Alkaloid production was not correlated with the exhaustion of a particular nutrient from the medium. The high-inoculum-density cultures produced higher ajmalicine concentrations throughout the experiment. Catharanthine production was similar between the two inoculum-density cultures. The addition of conditioned medium to MS-production medium dramatically improved the production of ajmalicine and catharanthine. The addition of conditioned medium enhanced ajmalicine production from immobilized Catharanthus roseus cultures on day 15 by at least two- to fourfold compared to media without the conditioning factors. Catharanthine production was increased by nearly fivefold in cultures with conditioned medium compared to those without conditioned medium. The enhancing effects of conditioned medium on alkaloid production were attributed to an unidentified factor produced and secreted by suspension cultures of C. roseus. The presence of conditioned medium also decreased the sucrose hydrolysis rate. The ajmalicine concentration in these immobilized cell cultures was found to be a function of the fresh-weight concentration, irrespective of the inoculum density or the culture medium. The medium choice and the inoculum density determined how rapidly fresh weight was accumulated and thus, how quickly ajmalicine was produced. Ajmalicine production correlated positively with fresh-weight concentration, but catharanthine production was not correlated with fresh-weight concentration.     

药用植物长春花WRKY转录因子的鉴定及表达谱分析

WRKY是调控植物生长发育和逆境胁迫反应等生命活动的一个转录因子大家族.然而,有关药用植物长春花CrWRKY转录因子的种类和功能却知之甚少.从26 009个长春花蛋白中鉴定出47个CrWRKY转录因子.依据WRKY结构域和系统进化,将CrWRKY分为G1、G2和G3三大类群.表达谱数据分析表明,长春花CrWRKY基因的表达具有器官特异性.47个CrWRKY基因的表达谱可分为3种表达模式:第1类型主要在花、甲基茉莉酸甲酯(MeJA)或酵母提取物(YE)处理的原生质体中高表达;第2类型主要在茎和毛状根中高表达;第3类型在根、茎、叶、幼苗和MeJA处理的毛状根中高表达.进一步用实时定量PCR检测了16个代表性CrWRKY基因在不同器官、MeJA处理原生质体和毛状根中的表达模式,检测结果与上述数字基因表达谱数据基本一致.约1/3以上CrWRKY基因的表达受MeJA和YE的调控,暗示它们可能参与萜类吲哚生物碱的合成和逆境胁迫反应.为进一步解析长春花WRKY转录因子的功能和萜类吲哚生物碱合成调控的网络奠定了基础.     

刈割对栽培长春花(Catharanthus roseus)生活史型转变的影响

为了提高栽培长春花（Catharanthus roseus）的生物碱产量,应用植物生活史型的理论和方法,研究了刈割对栽培长春花生活史型转变及其生物碱代谢的影响。运用主成分分析法（Principal Component Analysis, PCA）对刈割后的长春花后生活史型变化进行定量和定性划分,发现在对照栽培环境下生长的长春花处于DE生境,定性划分结果为SV生活史型,定量划分结果为V_0.3638S_0.6174C_0.0187,属于SV型。刈割使长春花的生活史型转变为V0.2847S0.6684C0.0469,属于SC型。同时,对两种生活史型的长春花中长春碱及其前体文朵灵和长春质碱的含量进行了检测分析,发现刈割后的SC型长春花不同叶位叶片中的生物碱含量均显著提高（p<0.05）,可以为提高栽培长春花生物碱含量提供科学指导,也进一步验证了生活史型理论。     

刈割对栽培长春花(Catharanthus roseus)生活史型转变的影响

为了提高栽培长春花（Catharanthus roseus）的生物碱产量,应用植物生活史型的理论和方法,研究了刈割对栽培长春花生活史型转变及其生物碱代谢的影响。运用主成分分析法（Principal Component Analysis,PCA）对刈割后的长春花后生活史型变化进行定量和定性划分,发现在对照栽培环境下生长的长春花处于DE生境,定性划分结果为SV生活史型,定量划分结果为V0.3638,S0.6174C0.0187,属于SV型。刈割使长春花的生活史型转变为V0．2847S0.6544C0.0469,属于SC型。同时,对两种生活史型的长春花中长春碱及其前体文朵灵和长春质碱的含量进行了检测分析,发现刈割后的SC型长春花不同叶位叶片中的生物碱含量均显著提高（P〈O．05）,可以为提高栽培长春花生物碱含量提供科学指导,也进一步验证了生活史型理论。     

Comparison of Cell Growth and Alkaloid Production of Catharanthus roseus Cells Cultured in Shake Flask and in Bioreactor

The cell growth and alkaloid production of Catharanthus roseus (L.) G. Don cells cultured in the shake flasks with different volumes and in the stirred tank bioreactor (10 L) were compared. Cell dry weight and alkaloid production showed no significant difference in the small volume scale-up shake flasks. When more broths were added to a certain volume in the shake flask, both cell weight and alkaloid production were decreased. The maximum cell dry weight was similar between the cell cultures in the shake flask and the bioreactor, but the alkaloid production of cells was much less in the bioreactor. Gas regime and shear stress were recognized to be the main factors contributing the important effect on alkaloid production during the scale-up processes.     

The effect of ajmalicine spiking and resin addition timing on the production of indole alkaloids from Catharanthus roseus cell cultures

The potential for the feedback inhibition of indole alkaloid synthesis was investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or 18 mg/L ajmalicine on day 0. The production of ajmalicine, catharanthine, and serpentine were inhibited in a dose-dependent manner. The inhibition was transient as the exogenous ajmalicine was ultimately either metabolized in the medium or within the cell. The addition of neutral resin has previously been shown to enhance ajmalicine production. To minimize product inhibition and product metabolism, Amberlite XAD-7 resin was added to immobilized cultures of C. roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for spent resin every 5 days. The addition of resin did not decrease the viability of the culture. Growth was reduced only in cultures with resin added on day 0. Alkaloid production was enhanced to different extents by the timing of resin addition, suggesting that feedback inhibition or product metabolism was present throughout the culture period. Ajmalicine recovery was nearly 100% when the resin was added initially either on day 0 or day 5. Ajmalicine recovery was reduced to 55% when the resin was added later in the culture period starting on day 15, presumably because of resin saturation or the inaccessibility of alkaloids trapped in the vacuole. Delaying the addition of XAD-7 resin until 5 days after the start of the culture resulted in the highest improvement in ajmalicine production, i.e approximately 70% and also resulted in the complete recovery of ajmalicine from the cell.     

Cloning of a cDNA encoding an E2 ubiquitin-conjugating enzyme from Catharanthus roseus: expression analysis in plant organs and in response to hormones in cell suspensions.

A novel cDNA (Crubie2) encoding ubiquitin-conjugating enzyme E2 was isolated from a Catharanthus roseus cDNA library. Sequence comparison with Arabidopsis thaliana E2 sequences revealed that CrUBIE2 is a member of a new plant E2 sub-family. Expression of Crubie2 is repressed in developing organs and down-regulated by cytokinin suggesting that a decrease in the ubiquitin-dependent proteolytic pathway may take part in the regulation of alkaloid biosynthesis in C. roseus cell suspensions.     

Opium poppy and Madagascar periwinkle: model non-model systems to investigate alkaloid biosynthesis in plants

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy ( Papaver somniferum ), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle ( Catharanthus roseus ), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.     

Localization of tabersonine 16-hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in Catharanthus roseus

The Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex anticancer dimeric alkaloids vinblastine and vincristine, which are derived by the coupling of vindoline and catharanthine monomers. Recent data from in situ RNA hybridization and immunolocalization suggest that combinatorial cell factories within the leaf are involved in vindoline biosynthesis. In this study, the cell types responsible for vindoline biosynthesis were identified by laser-capture microdissection/RNA isolation/RT-PCR to show that geraniol hydroxylase, secologanin synthase, tryptophan decarboxylase, strictosidine synthase, strictosidine ss-glucosidase and tabersonine 16-hydroxylase can be detected preferentially in epidermal cells. A new and complementary application of the carborundum abrasion (CA) technique was developed to obtain epidermis-enriched leaf extracts that can be used to measure alkaloid metabolite levels, enzyme activities and gene expression. The CA technique showed that tabersonine and 16-methoxytabersonine, together with 16-hydroxytabersonine-16-O-methyltransferase, are found predominantly in Catharanthus leaf epidermis, in contrast to vindoline, catharanthine and later enzymatic steps in vindoline biosynthesis. The results show that leaf epidermal cells are biosynthetically competent to produce tryptamine and secologanin precursors that are converted via many enzymatic transformations to make 16-methoxytabersonine. This alkaloid or its 2,3 dihydro-derivative is then transported to cells (mesophyll/idioblast/laticifer) within Catharanthus leaves to complete the last three or four enzymatic transformations to make vindoline.     

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.     

植物生长调节物质对长春花细胞中吲哚生物碱积累的影响

适当浓度的脱落酸(ABA)、乙烯利和乙酰水杨酸(ASA)均对长春花悬浮细胞中吲哚生物碱的积累有较明显的促进作用.2mg·L-1ABA对吲哚生物碱的诱导效果最好,乙烯利的最适剂量为1 g·L-1,1 mg·L-1ASA有利于阿玛碱积累,而2 mg·L-1ASA则有利于长春质碱积累.