Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1–5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm³ of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm⁻³ indolebutyric acid, 0.5 mg dm⁻³ kinetin and 30 g dm⁻³ sucrose. A 1 % CO₂ supply was found beneficial for the production of cell mass; however, increasing CO₂ concentration to 2.5 and 5 % decreased the biomass accumulation. CO₂ enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO₂ supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.     

刺激剂(elicitor)在植物细胞培养次生代谢物生产中的应用

刺激剂（ｅｌｉｃｉｔｏｒ）在植物细胞培养中被用来作为提高次生代谢物产量的手段。文中概括介绍了微生物、寡聚糖、蛋白质、第二信使及其他物质作为刺激剂在植物细胞培养中的应用及其研究成果。     

Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites

The development of plant tissue (including organ and cell) cultures for the production of secondary metabolites has been underway for more than three decades. Plant cell cultures with the production of high-value secondary metabolites are promising potential alternative sources for the production of pharmaceutical agents of industrial importance. Medicinal plant cell suspension cultures (MPCSC), which are characterized with the feature of fermentation with plant cell totipotency, could be a promising alternative “chemical factory”. However, low productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and the application to large-scale production is still limited to a few processes. This review generalizes and analyzes the recent progress of this bioproduction platform for the provision of medicinal chemicals and outlines a range of trials taken or underway to increase product yields from MPCSC. The scale-up of MPCSC, which could lead to an unlimited supply of pharmaceuticals, including strategies to overcome and solution of the associated challenges, is discussed.     

气体成分对植物细胞悬浮培养的影响

气体成分对植物悬浮培养细胞的生长和次生代谢物的产量有深刻的影响。就有关氧、二氧化碳、乙烯和一些未知成分作用的研究进行了综述。     

Design of bioreactors suitable for plant cell and tissue cultures

Plant cell suspension cultures and hairy roots are potential sources of secondary metabolites and recombinant proteins. In contrast to traditionally grown “whole wild plants” or “whole transgenic plants”, their production in bioreactors guarantees defined controlled process conditions and therefore minimizes or even prevents variations in product yield and quality, which simplifies process validation and product registration. Moreover, bioreactors and their configuration significantly affect cultivation results by accomplishing and controlling the optimum environment for effective cell growth and production of bioactive substances. This review highlights the main design criteria of the most widely used bioreactor types, both for plant cell suspension cultures and for hairy roots, and outlines suitable low-cost disposable bioreactors which have found increasing acceptance over the last 10 years. Plants for human health in the post-genome era, PSE congress 26.8.2007–29.8.2007, Helsinki.     

植物细胞培养技术提高次生代谢物产量的方法(综述)

介绍植物细胞培养技术提高次生代谢物产量的方法。     

Bioactive secondary metabolites produced by microorganisms associated with plants

In the past few decades groups of scientists have focused their study on relatively new microorganisms called endophytes. By definition these microorganisms, mostly fungi and bacteria, colonise the intercellular spaces of the plant tissues. The mutual relationship between endophytic microorganisms and their host plants, taxanomy and ecology of endophytes are being studied. Some of these microorganisms produce bioactive secondary metabolites that may be involved in a host-endophyte relationship. Recently, many endophytic bioactive metabolites, known as well as new substances, possesing a wide variety of biological activities as antibiotic, antitumor, antiinflammatory, antioxidant, etc. have been identified. The microorganisms such as endophytes may be very interesting for biotechnological production of bioactive substances as medicinally important agents. Therefore the aim of this review is to briefly characterize endophytes and summarize the structuraly different bioactive secondary metabolites produced by endophytic microorganisms as well as microbial sources of these metabolites and their host plants.     

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures

Elicitation is a possible aid to overcome various difficulties associated with the large‐scale production of most commercially important bioactive secondary metabolites from wild and cultivated plants, undifferentiated or differentiated cultures. Secondary metabolite accumulation in vitro or their efflux in culture medium has been elicited in the undifferentiated or differentiated tissue cultures of several plant species by the application of a low concentration of biotic and abiotic elicitors in the last three decades. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability, high growth rate in growth regulator‐free media, and production consistence in response to elicitor treatment. Elicitors act as signal, recognized by elicitor‐specific receptors on the plant cell membrane and stimulate defense responses during elicitation resulting in increased synthesis and accumulation of secondary metabolites. Optimization of various parameters, such as elicitor type, concentration, duration of exposure, and treatment schedule is essential for the effectiveness of the elicitation strategies. Combined application of different elicitors, integration of precursor feeding, or replenishment of medium or in situ product recovery from the roots/liquid medium with the elicitor treatment have showed improved accumulation of secondary metabolites due to their synergistic effect. This is a comprehensive review about the progress in the elicitation approach to hairy root cultures from 2010 to 2019 and the information provided is valuable and will be of interest for scientists working in this area of plant biotechnology.     

Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals

The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemicals. Due to low or no productivity of the desired compounds the economy is only in a few cases favorable. Various approaches are studied to increase yields, these encompass screening and selection of high producing cell lines, media optimization, elicitation, culturing of differentiated cells (organ cultures), immobilization. In recent years metabolic engineering has opened a new promising perspectives for improved production in a plant or plant cell culture.     

Production of anthocyanins by plant cell cultures

Anthocyanins, responsible for the various attractive colors in plants, are becoming important alternative to many synthetic colorants due to increased public concerns over the safety of artificial food colors. Production of anthocyanins by plant cell cultures has been suggested as a feasible technology that has attracted considerable industrial and academic interests in the past two decades. This paper is to provide an overview of the present status and the future prospects in the commercial development of plant cell cultures for production of anthocyanins. The focus is on the strategies for enhancement of anthocyanin biosynthesis to achieve an economically viable technology for commercial applications. Through strain improvement, optimization of media and culture conditions, and intelligent process strategies such as elicitation and two-stage system, significant enhancement in productivity has been achieved in a number of cultures. However the yield of anthocyanins obtained so far is still far away from the full potential of anthocyanin synthesis by plant cell cultures. Further improvements require the insights on the regulation of anthocyanin synthesis, accumulation, storage and breakdown that will eventually lead to genetic manipulation of anthocyanin biosynthesis. Many studies have elucidated the metabolic pathway of anthocyanin biosynthesis. Preliminary studies on the regulation of anthocyanin biosynthesis on the levels of genes and enzymes are reviewed, showing that it is feasible to clone genes from secondary metabolism with an improved yield of anthocyanins. There is currently no commercial-scale trial for production of anthocyanin by plant cell cultures, but an intelligent integration of those existing strategies could provide a technology for industrial application competitive to the current production methods.     

Biotechnology for the production of plant secondary metabolites

The production of plant secondary metabolites by means of large-scale culture of plant cells in bioreactors is technically feasible. The economy of such a production is the major bottleneck. For some costly products it is feasible, but unfortunately some of the most interesting products are only in very small amounts or not all produced in plant cell cultures. Screening, selection and medium optimization may lead to 20- to 30-fold increase in case one has producing cultures. In case of phytoalexins, elicitation will lead to high production. But for many of the compounds of interest the production is not inducible by elicitors. The culture of differentiated cells, such as (hairy) root or shoot cultures, is an alternative, but is hampered by problems in scaling up of such cultures. Metabolic engineering offers new perspectives for improving the production of compounds of interest. This approach can be used to improve production in the cell culture, in the plant itself or even production in other plant species or organisms. Studies on the production of terpenoid indole alkaloids have shown that the overexpression of single genes of the pathway may lead for some enzymes to an increased production of the direct product, but not necessarily to an increased alkaloid production. On the other hand feeding of such transgenic cultures with early precursors showed an enormous capacity for producing alkaloids, which is not utilized without feeding precursors. Overexpression of regulatory genes results in the upregulation of a series of enzymes in the alkaloid pathway, but not to an improved flux through the pathway, but feeding loganin does result in increased alkaloid production if compared with wild-type cells. Indole alkaloids could be produced in hairy root cultures of Weigelia by overexpression of tryptophan decarboxylase and strictosidine synthase. Alkaloids could be produced in transgenic yeast overexpressing strictosidine synthase and strictosidine glucosidase growing on medium made out the juice of Symphoricarpus albus berries to which tryptamine is added. Metabolic engineering thus seems a promising approach to improve the production of a cell factory.     

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.     

A new method for the production of fine plant cell suspension cultures

Fine, almost single cell, suspensions were produced from both existing suspension cultures containing large cell clumps and from chopped callus pieces by immobilizing the cells in 4–5 mm diameter calcium alginate beads. The immobilized cells continued to divide inside the beads and at the bead surface, and after 2–3 weeks' culture, fine cell suspensions were formed as a result of loss of the surface cells into the medium. After removal of the cell suspensions by filtration, subsequent culture of the beads in fresh medium resulted in the further production of homogeneous cell suspensions after 1–2 weeks. In this way an almost continuous supply of fine cell suspensions could be obtained from cultures containing large clumps of cells. The cells produced by this method remained in this state for at least one culture period, although in some instances repeated subculture resulted in an increase in the size of cell groups. The technique has been successfully applied to the production of fine cell suspensions ofCatharanthus roseus, Nicotiana tabacum andDaucus carota.     

两株地理隔离的桦褐孔菌深层发酵产生抗氧化次生代谢产物的代谢组学分析

采用基于核磁共振代谢组学的方法研究了两株地理隔离的桦褐孔菌中国牡丹江菌株(IOM)和芬兰万塔菌株(IOV)在深层培养过程中主要代谢产物组成的差异。IOM和IOV两菌株在培养过程中积累菌丝体生物量、菌丝体酚类(TMP)以及三萜化合物(TMT)的过程相似,但IOM菌株产生较高水平的TMP。代谢产物组成动力学比较结果表明,IOM在第3天含有较高水平的硬毛素类似物、桦褐孔菌二糖和TMT,但在第5天均有所下降。导致这些差异的代谢产物为fuscoparianolD,21,24-羊毛甾-7,9(11),23-三烯-3β,22,25-三醇,羊毛甾-8,23-二烯-3β,22,25-三醇,23,24-环戊羊毛甾-3β,21,25-三醇-8-烯,桦褐孔菌二糖,phelligridinsC,D及H,甲基地花菌素A和C,地花菌素B,davallialactone及methyl davallialactone。这些代谢产物在IOV的提取物中均参与清除自由基,而在IOM的提取物中主要是酚类化合物参与自由基的清除。两菌株的这些差异反应了环境条件对桦褐孔菌生长及代谢的影响,即不仅影响其次生代谢产物的合成,还影响代谢产物在防止细胞氧化损伤过程中所起的作用。     

Engineering the plant cell factory for secondary metabolite production

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks. This revised version was published online in August 2006 with corrections to the Cover Date.     

Statistical experimental designs for the production of secondary metabolites in plant cell suspension cultures

Statistical experimental designs, also known as the “design of experiments” (DoE) approach, are widely used to improve not only technical processes but also to answer questions in the agricultural, medical and social sciences. Although many articles have been published about the application of DoE in these fields, few studies have addressed the use of DoE in the plant sciences, particularly in the context of plant cell suspension cultures (PCSCs). Compounds derived from PCSCs can be developed as pharmaceuticals, chemical feedstocks and cosmetic ingredients, and statistical experimental designs can be used to improve the productivity of the cells and the yield and/or quality of the target compounds in a cost efficient manner. In this article, we summarize recent findings concerning the application of statistical approaches to improve the performance of PCSCs and discuss the potential future applications of this approach.     

Novel synthetic jasmonates as highly efficient elicitors for taxoid production by suspension cultures of Taxus chinensis

Suspension cultures of Taxus chinensis were used as a model plant cell system to evaluate novel synthetic jasmonates as elicitors for stimulating the biosynthesis of secondary metabolites. Significant increases in accumulation of taxuyunnanine C (Tc) were observed in the presence of newly synthesized 2-hydroxyethyl jasmonate (HEJA) and trifluoroethyl jasmonate (TFEJA) without their inhibition on cell growth. Addition of 100 microM HEJA or TFEJA on day 7 led to a high Tc content of 44.3 +/- 1.1mg/g or 39.7 +/- 1.1 mg/g (at day 21), while the Tc content was 14.0 +/- 0.1 mg/g and 32.4 +/- 1.6 mg/g for the control and that with addition of 100 microM methyl jasmonate (MJA), respectively. The superior stimulating ability of HEJA and TFEJA over MJA, which was generally considered as the best chemical for eliciting taxoid biosynthesis, suggests that the novel jasmonate analogues may have great potential in application to other cell culture systems for effcient elicitation of plant secondary metabolites.     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

刺激剂对植物细胞悬浮培养的影响

对刺激剂对植物细胞悬浮培养的影响及如何提高刺激剂的诱导率进行了综述。刺激剂对细胞悬浮培养的影响主要表现为酶活化、蛋白质含量改变、氧迸发、细胞程序性死亡、pH值改变、次生代谢产物积累等防御反应。提高刺激剂对悬浮培养细胞的诱导效率必须优化刺激剂的种类,刺激剂的浓度和加入时间,创建更好的诱导模式。