Secondary metabolite production, yield

Since most organic solvents are harmful to plant cell growth, the selection of an adequate lipophilic phase is essential for successful in situ extraction. Elicitation in conjunction with in situ extraction could alter the pattern of secondary metabolite production and enhance the product yield. 

Plant cell and organ cultures can produce higher metabolite concentrations than found in the corresponding intact plant organs (6, 9). However, plant cells grown in culture may also produce lower quantities of the desired secondary metabolites which are commonly stored intracellularly. The challenges to increase product yield and to enhance the release of secondary metabolites can be met in various ways (7). These include immobilization (9), permeabilization (12, ), the use of precursors (12,13), and the induction of secondary metabolite production via elicitors (14). 

Secondary metabolite production has been increased by applying immobilization procedures alone (9 19). Development of immobilized cell reactors, where cells remain viable and fully active biosynthetically after permeabilization treatment, would allow the continuous production of valuable secondary metabolites from a combined immobilization/permeabilization system as previously attempted by us (K), 18, Beaumont, M.D. and Knorr, D., Univ. of Delaware, unpublished data). This would also give higher product yields as compared to utilizing freely-suspended non-permeabilized cells. 

To date, progress achieved clearly demonstrates the potential of cultured plant cells for secondary metabolite production. Use of concurrent immobilization/permeabilization procedures, as well as precursor and elicitor treatments, may open new avenues of increasing product yields and will consequently affect the economic aspects of plant cell culture in a positive manner. However, our understanding of the many biosynthetic pathways of desired secondary metabolites is incomplete and successful industrial scale plant cell culture processes are still limited. Results of research in the area of plant cell culture will increase our understanding of the biosynthesis of plant metabolites, enhance our knowledge of plant-microorganism or plant-plant interactions and can lead to entirely new products or product lines of desirable compounds currently not available to use. Such work can also lead to development of industrial scale production processes for products now produced and recovered by conventional methods. Also, the genetic variety of the 250,000 to 750,000 plant species available remains to be explored. Presently only 5 to 15% of these species have been subject to even... 

It will also be necessary to relate the structures of the conjugates in the precursor fractions with the conposition of the hydrolysates. This is particularly important where polyfunctional molecules are involved allowing more than one site of conjugation, because acid hydrolysis of one conjugate of a particular secondary metabolite may yield different products from that of an alternatively or polyconjugated species. 

A major requirement for the application of IPCs in secondary metabolite production and biotransformations is that the plant cells release the products to the culture medium. In Capsiaum cells, capsaicin released into the medium accounts for 99% of the total yield. Similarly, the cardiac glycosides and transformed by D. lanata and C. roseus cell released into the medium. An extreme example is provided by cell suspensions of ThaUatrum minus producing berberine, an isoquinoline alkaloid used as an intestinal antiseptic (Figure 1). Most of the berberine produced by these cells was released continuously into the liquid medium, an excess of which crystallized as the nitrate salt ( ). 

An additional strategy for improving alkaloid yield and productivities of in vitro cultures is by the appropriate media design [68]. It is important to consider that the biosynthetic pathways that lead to the accumulation of secondary metabolites are complex and sometimes are not fully elucidated [6, 23]. Also, the regulation of these pathways is frequently unknown and these features are needed in order to perform a rational media design [68]. However, it is generally accepted that sucrose is the carbon source preferentially used. The increase of carbon source up to certain levels favors growth and secondary metabolites production. However, further increases may derive in catabolite repression [68]. 

The possibility to introduce genes into plants using the Agrobacterium technology as well as particle bombardment has increased the potential for secondary metabolites production in plant cultures by the exploitation and improvement of their own biosynthetic capacities [9, 79]. It is well known that the yield of tropane alkaloids is low for a commercial production. For this reason, is desirable to increase production of these alkaloids in plant species [32]. Numerous works have focused on the genetic engineering of the tropane alkaloids pathway according to the interest in these compounds. 

Standardization of medium is an important parameter in in vitro tissue culture technology since the composition of the culture media influences both the biomass yield as well as secondary metabolite production. Composition of the medium, culture conditions, and exogenous phytohormone combinations together influence the metabolite accumulation in the cell. Composition of macro- and micronutrients, nature and amount of carbon source, and level of total nitrogen has been found to play a vital role in the production of secondary metabolites. 

Large-scale production of secondary metabolites by plant cell culture seems to be feasible and attractive to industrial production. It has two major advantages over traditional monoculture methods [48] (1) controlled production of fine natural chemicals independent of climatic, edaphic, and political conditions and (2) higher quality and yield of the final product in well-defined systems. In recent years, metabolic engineering has opened a new promising perspective for improved secondary metabolites production. This approach can be used to improve production not only in cell culture but also in the plant itself or in other plant species or even other organisms. 

A major control of secondary metabolite production would be the availability of substrates. At the end of primary growth, acetyl-CoA is likely to be in excess, because its main utilizer (the Kreb s cycle) will be slowing down. As primary growth ends, this acetyl-CoA is utilized along with Trp to yield cAATrp. This metabolite in turn deflects excess DMAPP (whose synthesis requires 3 molecules of acetyl-CoA) away from isoprenoid biosynthesis. Therefore, it appears that secondary metabolism is an interlock mechanism, linking in this case protein biosynthesis and energy metabolism, and perhaps polyisoprenoid biosynthesis, which is called into play when a restraint is placed on a specific area of primary metabolism. 

Some of these compounds could be considered as dietary additives, but various other terms, including pesticides, can also be used. They can have beneficial effects on the environment and this aspect will be discussed later. The ionophore monensin, which is an alicyclic polyether (Figure 1), is a secondary metabolite of Streptomyces and aids the prevention of coccidiosis in poultry. Monensin is used as a growth promoter in cattle and also to decrease methane production, but it is toxic to equine animals. " Its ability to act as an ionophore is dependent on its cyclic chelating effect on metal ions. ° The hormones bovine somatotropin (BST) and porcine somatotropin (PST), both of which are polypeptides, occur naturally in lactating cattle and pigs, respectively, but can also be produced synthetically using recombinant DNA methods and administered to such animals in order to increase milk yields and lean meat production. "... 

Where biosynthesis of a product requires the net input of energy, the theoretical yield will be influenced by the P/O quotient of the process organism. Furthermore, where the formation of a product is linked to the net production of ATP and/or NADH, the P/O quotient will influence the rate of product formation. It follows that to estimate the potential for yield improvement for a given primary or secondary metabolite, it is necessary to determine the P/O quotient of the producing organism. 

A. niger normally produces many useful secondary metabolites citric and oxalic acids are stated as the dominant products. Limitation of phosphate and certain metals such as copper, iron and manganese results in a predominant yield of citric acid. The additional iron may act as a cofactor for an enzyme that uses citric acid as a substrate in the TCA cycle as a result, intermediates of the TCA cycle are formed. 

Plant cell culture is useful in laboratory and in industry because it allows plant natural products to be produced in a relatively controlled manner, and provides a supply of plant material that is not affected by sourcing problems, such as environmental, seasonal, geographical, and political factors.Also, plant cell culture allows for the tweaking and rearrangement of secondary metabolite biochemical pathways in order to produce novel metabolites, and to increase target compound yields, as well as allowing derivatives to be formed by introduction of analogs of natural intermediates.Plant cell culture can be performed with callus and suspension cultures, as well as with shoot cultures and hairy root cultures. These latter two approaches are especially useful when a metabolite is found to be produced more readily in differentiated cells. 

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