Biodiesel fuel production by transesterification of oils.

Hydrogen production by biological processes: a survey of literature

Plant cell cultures: Chemical factories of secondary metabolites

Application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway distributions and rates. Recruitment of heterologous proteins enables extension of existing pathways to obtain new chemical products, alter posttranslational protein processing, and degrade recalcitrant wastes. Although some of the experimental and mathematical tools required for rational metabolic engineering are available, complex cellular responses to genetic perturbations can complicate predictive design.

https://science.sciencemag.org/content/sci/252/5013/1668.full.pdf

Toward a science of metabolic engineering

Production of plant secondary metabolites: a historical perspective

For some years, there has been great interest in the exploitation of plant cell cultures to produce fine chemicals. With a few exceptions, progress in commercialization has been slow, largely due to the low and/or unstable productivity of many undifferentiated cultures. Recent developments leading to the production of rapidly growing, organized, ‘hairy’ root cultures following the genetic transformation of plants with Agrobacterium rhizogenes may revolutionize certain areas of plant cell biotechnology. We discuss the application of hairy root technology to the production of plant secondary metabolites.

New Routes to Plant Secondary Products

Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation.

Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation

‘Hairy root’ cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of ‘in vitro’ plant tissue culture for the production of valuable plant secondary products is identified and confirmed.

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes.

The factors by which the endogenous regulation of tropane-alkaloid biosynthesis may be effected have been examined in a transformed root culture of Datura stramonium. Pools of intermediates showed a subculture-related maximal accumulation, as did the enzyme activities by which they are synthesised and/or metabolised. The end-products, principally hyoscyamine and apohyoscyamine, in contrast, accumulated steadily in growing cultures. Feeding putrescine, agmatine or tropine did not enhance alkaloid accumulation, but rather may even have resulted in a lowering of hyoscyamine levels. Similarly, feeding precursors for the tropate moiety of hyoscyamine either had no influence or had a detrimental effect on hyoscyamine accumulation. Under some feeding conditions, intermediates in the pathway from N-methylputrescine up to and including tropine accumulated up to 40-fold. Little effect on early intermediates was found, however, when tropinone or tropine were fed. The expression of the enzyme arginine decarboxylase (EC 4.1.1.19) was particularly sensitive to feed-back repression, both by its product agmatine and by more distant pathway intermediates, notably putrescine and tropine. Some diminution of the levels of putrescine N-methyltransferase (EC 2.1.1.53) and N-methylputrescine oxidase, the first committed enzymes of alkaloid biosynthesis, was also seen with tropine, although only at rather high levels. It is concluded that the pathway is not regulated in a simple manner and that (i) the early enzymes of the pathway are at near rate-limiting levels, (ii) there is a major limitation to flux at the level of the esterification of tropine, and (iii) the level of free tropine may be important in determining the flux into and through the tropane pathway.

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures : 1. The kinetics of alkaloid production and the influence of feeding intermediate metabolites.

Richard J. Robins

Papers

New Routes to Plant Secondary Products

Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes.

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures : 1. The kinetics of alkaloid production and the influence of feeding intermediate metabolites.

Variation in tropane alkaloid accumulation within the solanaceae and strategies for its exploitation