Abdullah Makhzoum

Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming

Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing.

Hairy Roots: An Ideal Platform for Transgenic Plant Production and Other Promising Applications

The infection of plants by Agrobacterium rhizogenes results in a “hairy root”phenotype characterized by rapid growth in hormone-free medium, an unusual ageotropism and extensive lateral branching. The pathological rhizogenicity of A. rhizogenes arises from the stable insertion of a region of the A. rhizogenes Ri (root-inducing) plasmid into the plant nuclear genome. This plasmid can be engineered to contain foreign genes, which can also be stably inserted into the host genome. As such, A. rhizogenes represents a viable alternative for the genetic transformation of plant tissue not readily transformed by A. tumefaciens. However, to be effective as a genetic transformation system, the routine regeneration of full plants from hairy root cultures is essential. In this chapter, we report on some important features of hairy roots, describe recent progress in the regeneration of plants from A. rhizogenes-derived hairy roots and provide a summary of selected applications. These include the use of A. rhizogenes as an efficient system to boost rhizogenesis in recalcitrant plant species and to create new plant varieties and the use of hairy root cultures and A. rhizogenes-engineered plants for secondary metabolite production, in phytoremediation and for the production of recombinant proteins (i.e. molecular farming) for the healthcare industry.

Molecular farming on rescue of pharma industry for next generations

Abstract Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification methods. DNA technology and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant molecular pharming and outline some important challenges that hamper these ambitious biotechnological developments.

Taxol from Corylus avellana : paving the way for a new source of this anti-cancer drug

Taxol is amongst the most effective anti-cancer drugs available in market. The increasing demand of this compound due to its use in treating wide range of cancers, as well as its high cost have triggered efforts to find alternative ways to obtain this drug. Hazel (Corylus avellana), which is already cultivated for nutritional aspects, is now attracting attention for its phytochemical content. Notably the discovery of taxol and related taxanes in this plant species prompted extensive interest to explore biotechnological production of these compounds using in vitro cultures of C. avellana. This review emphasizes the potential of C. avellana cells for production of taxol and related taxanes. The botanical description of C. avellana, its pharmacological uses and various biotechnological approaches, such as micropropagation, cell culture and genetic engineering to increase the production of taxol and related taxanes are discussed. To present an overall overview, the experience of researchers working on these aspects is mentioned and major highlights or discoveries are presented. A review of the literature suggests that C. avellana may act as a commercial and alternative source for taxol production in an eco-friendly way, which will meet the ever-increasing demand, and also help reducing the cost of this anti-cancer compound.

Strigolactone biology: genes, functional genomics, epigenetics and applications

Abstract Strigolactones (SLs) represent an important new plant hormone class marked by their multifunctional role in plant and rhizosphere interactions. These compounds stimulate hyphal branching in arbuscular mycorrhizal fungi (AMF) and seed germination of root parasitic plants. In addition, they are involved in the control of plant architecture by inhibiting bud outgrowth as well as many other morphological and developmental processes together with other plant hormones such as auxins and cytokinins. The biosynthetic pathway of SLs that are derived from carotenoids was partially decrypted based on the identification of mutants from a variety of plant species. Only a few SL biosynthetic and regulated genes and related regulatory transcription factors have been identified. However, functional genomics and epigenetic studies started to give first elements on the modality of the regulation of SLs related genes. Since they control plant architecture and plant-rhizosphere interaction, SLs start to be used for agronomical and biotechnological applications. Furthermore, the genes involved in the SL biosynthetic pathway and genes regulated by SL constitute interesting targets for plant breeding. Therefore, it is necessary to decipher and better understand the genetic determinants of their regulation at different levels.

Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass in Catharanthus roseus hairy roots

Abstract Context Catharanthus roseus (L.) G. Don (Apocynaceae) is still one of the most important sources of terpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. Objective This study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methods Comparative analysis has been carried out on three selected lines of Catharanthus roseus hairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results Maximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusion Our results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli.

A simplified Protocol to Induce Callogenesis in Protoplasts of Date Palm (Phoenix dactylifera L.) Cultivars

BACKGROUND In Algeria, date palm is currently confronted to the Bayoud disease. Biotechnological tools such as protoplastsfusion can appear as an alternative to ensure rapid multiplication and improvement of this species. OBJECTIVES Callogenesis induction in protoplasts isolated from embryogenic callus of three date palm cultivars. MATERIALS AND METHODS Some factors influencing the isolation and culture of protoplasts segregated from the calli of three date palm (Phoenix dactylifera L.) cultivars (Deglet Nour, Akerbouch and Degla Beida) were studied. Protoplasts of each cultivar were cultured on a semi-solid medium supplemented with various hormonal balances. RESULTS Maceration with an enzymatic solution containing 1.5% cellulase and 1% macerozyme R10 in the presence of 0.5 M mannitol for more than 16 h with gentle agitation allows isolation of a great number of viable protoplasts. In addition, purification of protoplasts on a cushion of 21 or 25% sucrose was effective in cell debris removal and maximum recovery. The culture of isolated protoplasts on a semi-solidified Murashige and Skoog medium, with 0.3% agarose, 2 mg. L-1 2,4-D and 0.5 mg.L-1 BAP allowed good viable protoplast maintenance as well as cell wall regeneration. After more than two months of culture, cell divisions were still occurring and microcalli became visible to the naked eye, containing a large number of cells. CONCLUSIONS The developed protocol can be useful for application of somatic hybridization to improve date palm cultivars.

Bioengineering Hairy Roots: Phytoremediation, Secondary Metabolism, Molecular Pharming, Plant-Plant Interactions and Biofuels

Hairy root cultures are an efficient tool to understand plant biology, biotechnology and other applied sciences. In particular such cultures have revealed many cues of plant cells related to growth, development, physiological and biochemical processes. Thus, hairy roots are used to study secondary metabolism and production of bioactive compounds such as flavonoids. Transgenic roots are used as biofoactories to produce heterologous recombinant proteins for pharmaceutical purposes. Furthermore, they have shown promising applications in phytoremediation and restoring the environment because of advantages in the reduction of toxic organic and inorganic pollutants from soil, air, wastewater, groundwater and biowaste. This review focuses on the recent progress of bioengineering hairy root culture systems.

Recent Patents in Agricultural Biotechnology; Focus on Health

BACKGROUND Agricultural biotechnology, including the generation of genetically modified food crops, has been the subject of much controversy over the last few years. Initially serving the basic needs of farmers, Ag Biotech has more recently gained much appeal for its opportunities with respect to both the nutritional and pharmaceutical sciences. METHODS The following review describes a number of recently approved patents that could have direct implications for the field of medicine. Topics range from the development of pharmaceuticals in plants using hairy roots or virus expression vectors, to the role of epigenetics for improving the nutritional value of food crops. RESULTS Many of these patents were developed by smaller companies or publically funded research institutes, disproving the perception that intellectual property in Ag Biotech is restricted to only large multinational corporations. CONCLUSION The review concludes with a discussion of the future of these technologies in the face of the current negative political climate.