Green bioreactors

Biofactory plants

Evgenia Mardanova, "Biomolecule"

The development of biotechnologies has opened up new opportunities for the use of living organisms for the benefit of mankind. Genetic engineering methods allow us to produce various substances in living objects, therefore, we can use these objects as natural "factories". The central dogma of molecular biology generally states: DNA – RNA – protein. Protein is often the final product of biotechnological production: it can be insulin, interferons, antibodies, enzymes, vaccines... We just need to set a program and "write" it into DNA, and a living object will do everything by itself. Yeast cells, bacteria, plants, as well as insect and mammalian cell cultures are used as "factories". In this article we will talk about plant biofactories.

What is a biofactory plant?

How to explain the concept of "biofactory plant"? We can say that this is a natural enterprise that will produce the bioproduct we need. Unlike a conventional factory, such an enterprise will employ not workers, but cell components: nucleic acid polymerases, ribosomes, tRNAs and many others. And they will produce protein.

Why plants?

Currently, bacteria, yeast, insect and mammalian cell cultures are most often used to develop proteins. Plants are also a very attractive system of synthesis and accumulation of recombinant proteins (expression system), and there are several reasons for this. First of all, in plant tissues there is no risk of contamination of recombinant protein by animal viruses and prions – infectious proteins [1]. Plant cells provide the correct modification of the recombinant protein characteristic of eukaryotic cells [2, 3]. Cost, simplicity and time are also of great importance. Figure 1 compares several major expression systems.

Figure 1. Comparison of recombinant protein production systems
(from the most attractive "+" to the least attractive "–").

The table shows that there is no ideal expression system. Bacterial systems with E. coli as the most popular "workhorse" are the most widespread in Russia today. Back in 2009, this microorganism accounted for 85% of all expression systems, despite a number of significant drawbacks.

Each biofactory has its pros and cons. But the plant system is the most attractive for many situations.

How to make a plant produce protein?

In order for a plant to produce the desired protein, it is necessary to introduce foreign genetic material into the cells of this plant – a DNA sequence encoding the amino acid sequence of the desired protein.

The first stage of plant modification using genetic engineering methods involves the search and isolation (or synthesis) of genes that will be transferred to the plant genome. Genes of interest to biotechnologists (target genes) can be "grown" chemically, as well as developed using PCR (polymerase chain reaction). Then the target gene is embedded in a suitable vector, which delivers it to the place of protein production – just as a wagon with raw materials is attached to a steam locomotive heading to the factory.

How to transfer the necessary DNA sequence into a plant cell?

Currently, two methods are used more often.

The first is associated with the use of natural genetic engineering "skills" of the soil Agrobacterium tumefaciens, which is able to transfer DNA fragments into a plant cell, that is, to modify it genetically. This process in nature occurs everywhere and regularly. In addition to the chromosome, the natural A.tumefaciens contains a Ti plasmid, which includes the so-called T-DNA (transferred DNA) with a length of 12-22 thousand nucleotide pairs, embedded in the DNA of the plant chromosome. It encodes enzymes for the synthesis of phytohormones and opines – derived amino acids, which are used by the bacterium as a source of carbon, nitrogen and energy.

Ti-plasmid T-DNA has two properties that make it an almost ideal vector for introducing foreign genes into plant cells. Firstly, the range of hosts of agrobacteria is very wide: they transform the cells of almost all dicotyledonous plants (and with some efforts it is possible to achieve infection of monocotyledons, including cereals). Secondly, the T-DNA integrated into the plant genome is inherited as a simple dominant trait in accordance with Mendel's laws. The simplest way to introduce T-DNA into plant cells is to infect it with A.tumefaciens containing a suitable Ti plasmid, and the rest is left to the natural course of events. It is only necessary to be able to embed the necessary genes into the T-segment of plasmid DNA.

After the penetration of agrobacteria into the intercellular space, T-DNA is transferred from the agrobacteria into the nucleus of a plant cell and embedded in chromosomal DNA. Then transcription and translation take place – the target protein is synthesized. The bacterium itself does not penetrate into the cell, but remains in the intercellular space.

The second method is ballistic transformation using a gene gun. Small gold or tungsten particles cover foreign DNA and "shoot" into young plant cells. This method allows you to embed the necessary genes not only in the chromosomes of plants, but also in the genome of their organelles – plastids. This is very useful primarily for obtaining plants that are protected from pests, but at the same time safe for pollinators, because transgenes are not expressed in flowers that do not have plastids. Recently, in this way, transgenic (namely, transplastomic) potatoes were created, in whose chloroplasts a double–stranded RNA is formed and preserved intact, blocking the synthesis of the vital protein of the Colorado potato beetle – beta-actin. Eating the leaves of such potatoes, beetle larvae die in a matter of days [6].

Less common, but still effective ways of transformation are electroporation and transformation with the help of viruses.

Permanent and temporary gene expression

In plant genetic engineering, the following concepts can be found: a genetically modified (GM, or transgenic) plant and a plant that provides transient (temporary) gene expression. What is the difference?

If we are talking about a transgenic plant, it means that the foreign DNA is integrated into the chromosome. To date, dozens of transgenic plant species have been obtained, in whose genome DNA encoding various medical proteins, such as antigens of various pathogens of infectious diseases, therapeutic proteins and monoclonal antibodies have been transferred [7, 8, 9]. However, the amount of synthesized target protein in such plants is usually small (less than 1% of the total soluble protein).

With transient expression, DNA is usually not included in the nuclear genome, is not replicated and is not inherited. This type of expression is not permanent, however, a large number of copies of foreign DNA may be present in one cell for some time, which ensures a high level of synthesis of the final product. When producing proteins in plants, this option is most effective. It's like we rent a factory, and it turns out to be more cost-effective than buying it. Figure 2 shows a model of DNA transfer to a plant cell for transient expression (Agrobacterium tumefaciens as a courier).

Figure 2. Transient expression of foreign genes in plants by agroinfiltration.
A – the general mechanism of the process, B – visualization of the reporter synthesized in the plant –
GFP (green fluorescent protein) – by illuminating the leaf with ultraviolet light.

What can a biofactory plant produce?

The use of plants in biotechnology is developing in several directions (Fig. 3).

Figure 3. Possibilities of using plants for biotechnological purposes.

The first direction includes the creation of plants with new properties. A lot has been said about the undoubted advantages of transgenic plants [10]. Thus, varieties are being developed that are resistant simultaneously to insect pests and diseases caused by viruses, mold fungi and bacteria. Studies are underway that will allow us to develop varieties of crops that tolerate unfavorable climatic and chemical conditions, for example, drought and soil salinity [11]. Products are created in which the proportion of useful and nutritious substances is significantly increased, the content of saturated fats and allergens is reduced. Particular attention is paid to multidirectional modifications of rice, a valuable and relatively inexpensive food product that could be produced in all the poorest regions of the world, including arid ones [12].

The same group includes transgenic plants that are used as model objects for studying fundamental problems of gene functioning.

Many GM plants are now in mass production. These are soybeans, corn, potatoes, oily plants (rapeseed and sunflower) and many others (Fig. 4). The leading countries in the production of such plants are the USA, Canada, Argentina and Brazil. China and Japan are catching up with them. Some EU countries and Australia work with a number of plants.

Figure 4. Examples of transgenic plants.

Among the companies developing transgenic plants, we can mention Calgen, Monsanto, Ciba Seeds. Despite the fact that GM plants are sold in many markets around the world, discussions about the safety of their use are not over yet. Most of the rumors and scandals unfold around the company "Monsanto". The main products of this company are genetically modified seeds of corn, soy, cotton, as well as the most common herbicide in the world "Roundup" (nonproprietary name – glyphosate).

Founded by John Francis Quiney in 1901 as a purely chemical company, Monsanto has evolved into a concern specializing in high technology in the field of agriculture. The key moment in this transformation was 1996, when Monsanto launched the first genetically modified crops on the market: soy "Roundup Ready" (Roundup Ready, RR), resistant to glyphosate, and cotton "Bollgard" (Bollgard), resistant to insect pests (caterpillars).

In March 2005, Monsanto acquired the largest seed–growing company Seminis, specializing in the production of seeds of vegetables and fruits, in 2007-2008 absorbed 50 seed-producing companies around the world, after which it was severely criticized by society. In protest against the genetic manipulation of the biotech giant, on May 25, 2013, a "March against Monsanto" was held, which was attended by more than 2 million people on six continents, in 52 countries of the world.

The Bioengineering Center of the Russian Academy of Sciences has been working on plant genetic engineering for both basic research and agriculture for two decades. Genetically modified potato varieties resistant to the Colorado potato beetle, beetroot varieties resistant to herbicides and viruses, etc. were created. These crops could solve a number of agricultural problems, but due to the legal restrictions still in force in Russia, they are not grown in the open ground. Of course, this ban is more than strange, because the import of GM products into the country is allowed.

The second direction is the creation of edible vaccines. In this case, a genetically modified plant synthesizing the vaccine is obtained. The idea seems so attractive: you lie under a palm tree, eat a banana, and no tropical infection takes you!

The concept of vaccine production in plants was first formulated by Hugh Mason and co-authors [13]. They attempted to obtain an edible vaccine against hepatitis B virus based on transgenic tobacco. At the next stage, a GM potato was created that produces a surface antigen of the hepatitis B virus. When mice were fed tubers of such potatoes, the development of a specific immune response was observed. In 1999, experiments were started on volunteers, and the formation of specific immunity was observed in people who ate raw potato tubers. Edible vaccines against hepatitis B virus based on lupin and lettuce were also obtained.

Transgenic potato and tobacco plants have been created that produce the nucleocapsid protein of the Norfolk virus, which causes acute gastroenteritis in humans and is resistant to alcohol antiseptics. Transgenic potatoes also appeared, synthesizing the polypeptide LT-B– a subunit of the thermolabile toxin E.coli, which causes diarrhea. However, despite active research in this area, there are no commercial drugs to date.

The third direction is connected with the production of certain products in plants, which are then isolated from plants and can be used, for example, as medicines. Biotech companies around the world have already created a large number of GM plants for the production of proteins, including for medical purposes [14].

Among the companies whose activities are based on the use of transgenic plants, it should be noted the firms Protalix (Israel), Medicago (Canada), LemnaGene (France), Planet Biotechnology (USA), ProgyGene (Luxembourg), Chlorogen Inc. (USA), SemBioSys Genetics (Canada) and Bayer AG (Germany).

Of the medical proteins, insulin, lysozyme, lactoferrin, collagen, lipase, antibodies, vaccines, etc. are the most popular among manufacturers.

Many of these drugs are already undergoing clinical trials. But trypsin (in the picture on the right) can already be bought from Sigma.

When developing medical products in plant cells, the method of agrobacterial transformation is also used, which ensures transient gene expression at a high level. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of the final product. In addition, in this case, the synthesis of complex proteins consisting of several subunits is possible. This method allows you to obtain protein in large quantities for several days (up to several grams of protein per kilogram of plant weight). The yield of the product begins after three hours (!) after the penetration of agrobacteria into the cell and DNA transfer, and expression persists for up to 10 days. The maximum operating time is determined for each protein individually, but on average it is 3-4 days. In total, it takes 2-3 weeks to obtain proteins in plants (Fig. 6).

Figure 6. Schematic diagram of gene expression of target proteins in plants.
The whole process of obtaining protein takes 2-3 weeks.

Vaccines against human papillomavirus, hepatitis B [15], influenza, bovine papillomavirus [16], African catarrhal fever, cattle herpes [17], foot-and-mouth disease [18], etc. are already being developed in plants.

The Bioengineering Center is also working on the expression of therapeutic proteins in plants. Thus, vaccine preparations against influenza virus were produced in Nicotiana benthamiana cells (a type of tobacco) [19, 20]. The basis of the drug is a highly conserved viral protein M2, which is attached to a carrier protein to increase immunogenicity. The carrier may be the bark protein of the hepatitis B virus or bacterial flagellin. In the case of flagellin, the vaccine drug is used intranasally, which is a clear advantage. And the use of a highly conserved M2 protein sequence makes the vaccine universal, which eliminates the need to manufacture more and more new variants of it every year. These vaccine preparations have shown good results in immunogenicity and protectivity in experiments with laboratory animals; the next stage should be clinical testing.

About success in the world

A personalized therapeutic vaccine for the treatment of lymphoma, obtained by transient expression in the plant Nicotiana benthamiana, has already passed the I and II phases of clinical trials [21]. At the moment (2015), the start of phase III is expected. The plant vaccine against pandemic H5N1 influenza is undergoing phase II clinical trials, the results will be published in June 2015 [22, 23]. The vaccine was developed by Medicago. The standard process of obtaining vaccine proteins in plants by this company is shown in the video.

Clinical trials of vaccine preparations take a long time (about 10 years). Involuntarily, the question arises with the flu vaccine, since new strains appear every year, and will the vaccine that has passed clinical trials be relevant? Here, the technology of obtaining the drug is of greater importance. A new type of vaccine is undergoing a full cycle of clinical trials, and then a vaccine can be obtained according to a proven method, taking into account the circulating strains of the virus. So, now seasonal preventive flu vaccinations are obtained in chicken eggs, and such vaccines are no longer undergoing clinical trials. Time will tell how things will be with the production of recombinant vaccines for mass use in plants.

* * *

Summing up, we can say that plants have made it possible to obtain vital proteins using biotechnology methods. Man has learned to take the best from nature and avoid the worst. How William Shakespeare wrote about a plant in Romeo and Juliet:

There is a healing fragrance in its flowers,
And in the leaves and roots – the strongest poison.

So humanity has learned to take a healing fragrance, but not a deadly poison from plants. Biofactory plants have a great future!

The work was supported by a grant from the President of the Russian Federation for state support of leading scientific schools of the Russian Federation NSH-6150.2014.4.

Literature

1. Biomolecule: "Prions: research on mysterious molecules continues";
2. Fischer R., Schilberg S., Helliwig S., Twyman R.M., Drossard J. (2012). GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnol. Adv. 30, 434–439;
3. Martinez C.A., Guilietti A.M., Talou R. (2012). Research advances in plant-made flavivirus antigens. Biotechnol. Adv. 30, 1493–505;
4. biomolecule: "Molecular cloning, or how to put alien genetic material into a cell";
5. Biomolecule: "And whether we should take a swing at... genome change?";
6. Biomolecule: "Double-stranded RNA protects transgenic plants only from unwanted insects";
7. Sourrouille C., Marshall B., Lienard D., Faye L. (2009). From Neanderthal to nanobiotech: from plant potions to pharming with plant factories. Methods Mol. Biol. 483, 1–23;
8. Rybiski E.P. (2008). Plant-produced vaccines: promise and reality. Drug Discov. Today. 14, 16–24;
9. Yusibov V., Rabindran S. (2008). Recent progress in the development of plant derived vaccines. Expert Rev. Vaccines. 7, 1173–1183;
10. biomolecule: "Are transgenic plants the saviors of the planet or time bombs?";
11. biomolecule: "A cure for hunger is a remedy for drought";
12. biomolecule: "Cooking GM rice together";
13. Mason H.S., Lam D.M., Arntzen C.J. (1992). Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA. 89, 11745–11749;
14. Paul M., Ma J.K-C. (2011). Plant-made pharmaceuticals: Leading products and production platforms. Biotechnol. Appl. Biochem. 58, 58–67;
15. Thuenemann E.C., Lenzi P., Love A.J., Taliansky M., Becares M., Zuniga S. et al. (2013). The use of transient expression systems for the rapid production of viruslike particles in plants. Curr. Pharm. Des. 19, 5564–5573;
16. Love A., Chapman S., Matic S., Noris E., Lomonosoff G., Taliansky M. (2012). In planta production of a candidate vaccine against bovine papillomavirus type 1. Planta. 236, 1305–1313;
17. Pérez Filgueira D.M., Zamorano P.I., Domínguez M.G., Taboga O., Del Médico Zajac M.P., Puntel M. et al. (2003). Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity. Vaccine. 21, 4201–4209;
18. Wigdorovitz A., Pérez Filgueira D.M., Robertson N., Carrillo C., Sadir A.M., Morris T.J., Borca M.V. (1999). Protection of mice against challenge with foot and mouth disease virus (FMDV) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the FMDV structural protein. Virology. 264, 85–91;
19. Ravin N.V., Kotlyarov R.Yu., Mardanova E.S., Kupriyanov V.V., Migunov A.I., Stepanova L.A. et al. (2012). Production of recombinant influenza vaccine in plants based on virus-like HBc particles carrying the extracellular domain of M2 protein. Biochemistry. 77, 33–40;
20. Mardanova E.S., Kotlyarov R.Y., Kuprianov V.V., Stepanova L.A., Tsybalova L.M., Lomonosoff G., Ravin N.V. (2015). Rapid high-yield expression of a candidate influenza vaccine based on M2 protein linked to flagellin in plants using viral vectors. BMC Biotechnology. In print;
21. McCormick A.A., Reddy S., Reinl S.J., Cameron T.I., Czerwinkski D.K., Vojdani F. et al. (2008). Plant-produced idiotype vaccines for the treatment of non-Hodgkin’s lymphoma: safety and immunogenicity in a Phase I clinical study. Proc. Natl. Acad. Sci. USA. 105, 10131–10136;
22. D’Aoust M.A., Couture M.M., Charland N., Trépanier S., Landry N., Ors F., Vézina L.P. (2010). The production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza. Plant Biotechnol. J. 8, 607–619;
23. Sheldon E., Seiden D.J. (2014). Immunogenicity, safety and tolerability of a plant-derived seasonal virus-like-particle quadrivalent influenza vaccine in adults. ClinicalTrials.gov . (service of the US National Institutes of Health).

Portal "Eternal youth" http://vechnayamolodost.ru 08.05.2015