Hygrine, an alkaloid that is formally the product of an acetone condensation with N-methylpyrrolidine, was for many years considered to be an intermediate in the biosynthesis of scopolamine, but recent studies suggest that hygrine is not involved in tropane ring biosynthesis [16].
Two tropinone reductases responsible for tropane alcohol formation have been isolated and characterized from root cultures of several species see [17] and references therein. All the plant species studied so far possess two strictly stereospecific tropinone reductase activities, one TRI producing tropine and the other TRII producing pseudotropine see Figure 3.
Pseudotropine, as a product of tropinone reduction, undergoes a rapid conversion to calystegines, while tropine is esterified to yield hyoscyamine, the precursor of scopolamine [19]. Figure 2. Biosynthesis of N-methylputrescine from arginine and ornithine.
ADC: Arginine decarboxylase. AIH: Agmatine iminohydrolase. CPA: N-carmaboylputrescine amidohydrolase. ODC: Ornithine decarboxylase. PMT: putrescine N-methyltransferase. Isotopic labelling studies in transformed root cultures of Datura stramonium have demonstrated that littorine [ R -phenyllactoyl tropine] is the direct biosynthetic precursor of hyoscyamine by an intramolecular rearrangement [22] Figure 3. Thus, the tropate ester moiety of hyoscyamine is derived by isomerisation from the R -phenyllactate ester of littorine.
Recently, a multifunctional cytochrome P capable of littorine rearrangement via a hyoscyamine aldehyde has been identified from Hyoscyamus niger [24]. Scopolamine, the 6,7-epoxide of hyoscyamine, is formed by direct oxidation of hyoscyamine without the intermediacy of a double bond. Figure 3. Overview of the most important steps in the scopolamine biosynthetic pathway. The enzymes overexpressed in scopolamine-producing hairy root cultures are in red.
Unorganized plant tissue cultures are frequently unable to produce secondary metabolites at the same levels as the intact plant. This is also the case of scopolamine production in undifferentiated in vitro cultures of Solanaceae, probably due to the specific location of some of the key enzymes involved in this biosynthetic pathway [27].
Suzuki et al. In addition, Nakajima and Hashimoto [31] have observed that TR proteins accumulate in the lateral roots of Hyoscyamus niger.
Another possible reason for the low production of scopolamine in undifferentiated in vitro cultures could be that the auxin added to the callus and cell culture media for normal growth inhibits the activity of some of the key enzymes involved in scopolamine biosynthesis, such as PMT [32]. The hairy root phenotype is characterized by fast hormone-independent growth, lack of geotropism, lateral branching and genetic stability.
The secondary metabolites produced by hairy roots arising from the infection of plant material by A. This feature, together with genetic stability and generally rapid growth in simple media lacking phytohormones, makes them especially suitable for biochemical studies not easily undertaken with root cultures of an intact plant.
During the infection process A. Some A. The ags genes responsible for opine biosynthesis in the transformed tissues are also located in the TR region [36]. Opines are synthesized by plant transformed cells and are only used by Agrobacterium as a source of nitrogen and carbon. Due to the similarities of the A. Aux genes provide transformed cells with an additional source of auxin [], but they do not seem essential for developing hairy root disease [39].
However, rol genes have functions that are most likely other than that of producing mere alterations in plant hormone concentrations [40]. As has been previously reported, a correlation exists between the expression of the rolC gene and tropane alkaloids [], Catharanthus roseus alkaloids [44], and ginsenoside production [45].
No correlation between rolA and rolB expression and secondary metabolism was found in any of these studies. Moyano et al. In general, the obtained hairy roots presented two morphologies: typical hairy roots with a high capacity to produce alkaloids, and callus-like roots with faster growth and lower alkaloid production.
The aux1 gene of A. These results demonstrate a significant role of aux genes in the morphology of transformed roots and the importance of typical hairy root morphology in the production of scopolamine.
The studies with Panax ginseng hairy roots also support the effects of the genes located in the TR-DNA on root morphology and secondary metabolism [47]. The hairy roots are normally induced on aseptic, wounded parts of plants by inoculating them with A. In scopolamine-producing Solanaceae plants, roots usually emerge at the inoculation sites after weeks Figure 4 , but in the case of other plant species such as Taxus it can be more than 4 months before the roots emerge [48].
The next step for establishing hairy root cultures is to select and characterize the root clones according to their capacity for growth and production of the desired compounds. Sometimes these productions were achieved after a laborious process to optimize the growth conditions, such as the selection of the more productive clones, and optimization of the production conditions by testing different ionic concentrations as well as the carbon source and pH of the medium.
Figure 4. The main steps for the establishment and culture of hairy roots from Datura metel. Molecules , 13 The treatment with biotic or abiotic elicitors, such as methyl jasmonate, chitosan, salicylic acid or silver nitrate, can also improve the production of tropane alkaloids [11, and references therein], but in some cases the effect of the elicitor is due to an enhancement of cell permeability, which may increase the formation of secondary products by inhibiting operative feedback mechanisms or intracellular degradation of the products [52].
The main constraint for the commercial exploitation of hairy root cultures is scaling up to an industrial level, since it has become clear that standard bioreactors are not suitable vessels to achieve this. The uneven distribution of biomass in the vessel does not permit the growth of interconnected tissues, which results in cell necrosis. The growth behavior of the roots also hampers the inoculation, harvesting and sampling procedures.
Furthermore, in order to protect the root integrity, the design of mechanical stirred bioreactors should be modified by including stainless or nylon meshes to protect against mechanical shear [54]. Srivastava et al. In the case of tropane alkaloids, different types of bioreactors are used for scopolamine production Figure 5.
Wilson [56] describes the only large droplet bioreactor system with a volume of L designed for hairy root cultures of Datura stramonium. On a smaller scale, modified airlift and stirred tanks have been used for scopolamine production in hairy root cultures of D. One such advance is the development of disposable wave bioreactor systems, whose working principle is based on wave-induced agitation, which significantly reduces stress levels.
This type of bioreactor has been successfully used for H. Overexpression of the pmt gene to improve scopolamine production It is known that tropane alkaloids are derived from putrescine via N-methylputrescine, and that putrescine can also be metabolized into polyamines such as spermidine and spermine.
As previously mentioned, the formation of N-methylputrescine is catalyzed by putrescine N-methyltransferase, which is the first committed step in the biosynthesis of these alkaloids Figure 2. This suggests that scopolamine production by plant cell cultures can be improved by overexpressing the pmt gene. This reaction is common to both tropane and tobacco alkaloids. The cDNA of pmt has been cloned from tobacco and Nicotiana sylvestris and the enzyme PMT has been purified from tobacco plant roots, where its activity has been measured [61].
Root cultures of Datura stramonium, [12, ], Hyoscyamus albus [] and H. Figure 5. Detail of a hairy root culture of Duboisia hybrid showing the anchorage of the roots in the stainless mesh added to protect the culture in the stirred system. The key role of PMT in tropane alkaloid biosynthesis converts this enzyme into a prime target for metabolic engineering, and there have been many attempts to increase the scopolamine production by over-expressing the pmt gene.
In most cases, the plant material has been transformed with a heterologous gene, such as the pmt gene from tobacco, under the control of the CaMVS promoter, with the advantages of no feedback inhibition by downstream products, and a higher affinity for the substrate [71]. PMT-overexpressing plants of A. These authors report an opposite effect of the transgene expression, since no changes were observed in the Atropa alkaloid content, while the nicotine content in N. In order to increase the bioproduction of hyoscyamine and scopolamine alkaloids, we have been working with two scopolamine-rich phenotypes, D.
In order to overexpress the tobacco pmt gene, we developed a binary system, consisting of a disarmed Agrobacterium tumefaciens C58C1, which contained the plasmid pRiA4 together with the plasmid pBMI. The results of the agroinfection showed that the Agrobacterium used in the three plant species had a high virulence. In all cases we obtained root lines with a high capacity for growth, similar to the root lines obtained after infection with a wild strain of A.
The results obtained from engineered roots of H. As Moyano et al. In the engineered roots of Duboisia, alkaloid production was closely related to the presence of the 35S-pmt transgene, showing that ectopic expression of tobacco pmt increased the biosynthetic flux towards the tropane alkaloids and, consequently, the tropane alkaloid contents of transformed roots were significantly enhanced.
It is evident that the transcriptional control by the 35S promoter of the transgenic pmt gene in the Datura root lines is not cell type specific. Furthermore, it may also indicate that the transgene allows the bypassing of the endogenous control of the metabolic flux to the alkaloids, which would take place as the first committed enzymatic step in their biosynthesis.
Similar results have been obtained in H. On the contrary, as already mentioned, in Duboisia and A. These results, unlike those obtained by Moyano et al. More recently, the tobacco pmt gene has been overexpressed in scopolamine-producing H.
The engineered lines showed a significant increase in PMT activity and the contents of N- methylputrescine also increased more than five-fold, although the level of tropane alkaloids remained constant. It has also been demonstrated that the exposure of the roots to the elicitor methyl jasmonate increases the levels of polyamines as well as tropane alkaloids, and suggests that the C-flux for tropane alkaloid production could be restricted in later steps of the biosynthetic pathway.
Overexpression of tropinone reductases Another branching point in scopolamine biosynthesis is at the tropinone reductase level see Figure 2. Atropa, Datura, Hyoscyamus and Duboisia contain additional tropane alkaloids, calystegines, which are characterized by the loss of a methyl group on the nitrogen bridge and by the presence of three to five hydroxyl groups on the tropane heterocycle [17].
Two separate tropinone reductases have been isolated and characterized from root cultures of several species, D. Engineered root lines with strong overexpression of the trI or trII gene from D. Strong expression of the trI gene was accompanied by a significant enhancement of the contents of hyoscyamine and scopolamine.
On the contrary, calystegine levels were lower than in the control roots. These results show the effectiveness of the system for increasing scopolamine production in hairy roots and suggest that the tropane alkaloid and calystegine pathways compete for the C-flux. Discusses the current progress of metabolic engineering, focusing on systems biology and synthetic biology Covers introductory, regulatory, strategies, production and challenges in the field Written technically for synthetic biologists, researchers, students, industrialists, policymakers and stakeholders.
Integrating systems biology and biotechnology with new concepts from synthetic biology enables the global analysis and engineering of microorganisms and bioprocesses at super efficiency and versatility otherwise not accessible. Without doubt, systems metabolic engineering is a major driver towards bio-based production of chemicals, materials and fuels from renewables and thus one of the core technologies of global green growth.
In this book, Christoph Wittmann and Sang-Yup Lee have assembled the world leaders on systems metabolic engineering and cover the full story — from genomes and networks via discovery and design to industrial implementation practises.
This book is a comprehensive resource for students and researchers from academia and industry interested in systems metabolic engineering. It provides us with the fundaments to targeted engineering of microbial cells for sustainable bio-production and stimulates those who are interested to enter this exiting research field.
Systems biology and synthetic biology are emerging as two complementary approaches, which embody the breakthrough in biology and invite application of engineering principles. Systems Biology and Synthetic Biology emphasizes the similarity between biology and engineering at the system level, which is important for applying systems and engineering theories to biology problems.
This book demonstrates to students, researchers, and industry that systems biology relies on synthetic biology technologies to study biological systems, while synthetic biology depends on knowledge obtained from systems biology approaches. There continues to be rapid changes and discoveries in both fields with a small number of recent peer-reviewed reviews indicating some of the relationships between systems biology and synthetic biology.
This proposed SpringerBrief will cover core concepts of systems biology and synthetic biology and illustrate the implementation of associated research methodologies for an integrated approach to specifically address engineering microorganisms for biofuel production. Yeasts are one of the most attractive microbial cell factories for the production of a wide range of valuable products, including pharmaceuticals, nutraceuticals, cosmetics, agrochemicals and biofuels.
Synthetic biology tools have been developed to improve the metabolic engineering of yeasts in a faster and more reliable manner. Today, these tools are used to make synthetic pathways and rewiring metabolism even more efficient, producing products at high titer, rate, and yield.
Biotechnol Adv — Bioresour Technol Rep Article Google Scholar. Nat Biotechnol — Microb Biotechnol 14 1 — Nat Chem Biol — Lin PP et al Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum. Metab Eng — Nakashima N, Tamura T A new carbon catabolite repression mutation of Escherichia coli , mlc, and its use for producing isobutanol. J Biosci Bioeng — Noguchi S et al Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production.
Metabolomics Novak K, Baar J, Freitag P, Pflugl S Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material. Pontrelli S et al Directed strain evolution restructures metabolism for 1-butanol production in minimal media.
Seo HM et al Combinatorial application of two aldehyde oxidoreductases on isobutanol production in the presence of furfural. Sherkhanov S et al Isobutanol production freed from biological limits using synthetic biochemistry. Nat Commun Song HS et al a L-glycine alleviates furfural-induced growth inhibition during isobutanol production in Escherichia coli. The family of secondary metabolites derived reaction to form hydroxymethylglutaryl CoA, which from the aromatic amino acids are known generally is then reduced to mevalonic and MVA then as phenolics, polyphenols or phenylpropane derivates.
Two more condensation with IPP diverse family of compounds ranging from simple give rise first to the C15 intermediate farnesyl phenolic acids to very large and complex polymers pyrophosphate and then to the Cro geranyl geranyl such as tannins and lignin.
The biosynthesis of most pyrophosphate. FPP is a branch point that can give phenolics being with the tryptophan. The aromatic rise to C15 sesquiterpenes possibly including absci- amino acids are in turn, synthesized from sic acid. The vast majority of terpenoids are second- phosphoenolpyruvate and erythrosephosphate by ary metabolite many of which appear to act as toxins a sequence of reactions, known as the shikimic acid or feeding deterrents to herbivorous insects.
The latter is common to bacteria, fungi and plant but is not found in animals. Phenylalanine and Steroids and Sterols: Steroids are known as tryptophan are consequently among the ten amino triterpenoids. They are synthesized from the acylic acids. Considered essential to animals and represent triterpenoid and qualana. Steroids with an alcohol the principal source of all aromatic molecules in group are known as sterols. The most abundant animals. Sterols are planner molecule with one molecule of PEP from glycolysis.
Resulting and their packing tend to increase the viscosity and 7-carbon sugar is then cyclized and then reduced to stability of membranes. Some sterols may have a form shikimate. Phenolic acids are plant metabolites protective function, such as the phytoecolysones, widely spread throughout the plant kingdom. Recent which have structure similar to the insect molting interest in phenolic acids stems from their potential hormones when ingested by insect herbivores, phyto- protective role, through ingestion of fruits and ecdysores disrupt the insects molting cycle Fig.
Saponins: Saponins are terpene glycosides. They Phenolic compounds are essential for the growth and may be steroid glycosides, steroid alkaloids sides production of plants, and are produced as a response triterpene glycosides.
They may occur as aglycones for defending injured plants against pathogens. The combination Phenolic acid compounds seem to be universally of hydrophobic triterpene with a hydrophilic sugar distributed in plants. They have been the subject of a gives saponines the properties of a surfactant or great number of chemical, biological, agricultural, and detergent. The name saponine is derived from the medical studies. Phenolic acids form a diverse group soapwort or bouncing bet which at one time was that includes the widely distributed hydroxybenzoic employed as a soap substitute.
Commercially, and hydroxycinnamicacids Fig. Saponins from the bark of Quillaja saponaria have been used as surfactants in photographic film in Plant phenolic compounds are diverse in structure shampoo, liquid detergents and beverages Fig. Digitalis purpurea, Examples of phenolic compounds are capsaicin, the D. The two principal cardinolides in digitalis pungent compound of chilli peppers, tyrosine, an are digitoxin and its close analog digoxin. Digitalis is amino acid, the neurotransmitters serotonin, dopamine, also the source of a saponin, digitonin Fig.
Digitalis adrenaline, and noradrenaline. Eugenol, the main heart condition such as arteriosclerosis. Another constituent of the essential oil of clove, chavibetol common source of cardionlides are the milkweeds from betel, estradiol and other estrogens and alkaloid Asclepias and Calotropis.
Cell Tissue Research Coumarins: The Coumarins are a widespread group eye examinations to dilate the pupil. Tropicamide has of lactone formed by ring closure of hydroxycinnamic also recently shown promise as an early diagnostic acid.
A by fungi to a toxic product dicoumarol, that is typically tonic prepared from the bark of Cinchona officinalis found in moldy hay. Dicoumarel causes fatal that contains the antimalarial drug quinine greatly hemorrhaging in cattle by inhibiting vitamin K, an facilitated European exploration and inhabitation of essential cofactor in blood clotting Figs. Lignin: Lignin is a highly branched polymer of three The alkaloids are a very large and heterogeneous simple phenolic alcohols.
Lymnospermum lignin is family of secondary metabolites that are of interest comprised mainly of coniferyl alcohol sub units while primarily because of their pharmacological properties an comprised mainly of subunits while angiosperm and medical applications.
Alkaloids generate vary lignin is a mixture of coniferyl and sinapyl alcohol degrees of physiological and psychological response subunits. The alcohols are oxidised to free radical in human largely by interfering with neurotransmitters. Lignin is found in cell Alkaloids or alkaloid rich extract have been used for walls, especially the secondary walls of tracheary variety of pharmacological purpose such as muscle element.
The elements in the xylem, where it relaxant, tranquilizer pain killer and mind altering drug. Lignins and other phenolic derivatives amino acids tyrosin, tryptophan and lysine.
0コメント