These living beings will never be able to live in freedom. Their genome has been repeatedly redrawn for the sake of only one task - to work tirelessly for humans. Millions of these biorobots produce in huge quantities what they themselves practically do not need. They resist, they would like to live differently, but who will allow it?
Written in a dystopian style, the introductory passage is in fact an everyday reality. These are microorganisms specially adapted to work in biotechnological production. In fact, microorganisms - bacteria and fungi - have been injecting humanity since time immemorial, and before Louis Pasteur's discoveries, people did not even realize that, kneading yeast dough, fermenting milk, making wine or beer, they were dealing with the work of living beings.
In search of superpowers
But be that as it may, intuitively, by the method of spontaneous selection over the millennia, people have managed to select from natural, "wild" forms of microorganisms high-quality crops for winemaking, cheese making, baking. Another thing is that already in the newest era, new applications have been found for working bacteria. Large-scale biotechnology enterprises have sprung up to produce, for example, important chemicals such as amino acids or organic acids.
The essence of biotechnological production is that microorganisms, absorbing raw materials, such as sugar, release a certain metabolite, a metabolic product. This metabolite is the final product. The only problem is that several thousand metabolites are present in the cell, and production needs one, but in very large quantities - for example, 100 g / l (despite the fact that under natural conditions the metabolite would be produced in quantities by two three orders of magnitude smaller). And of course, bacteria must work very quickly - to give out the required amount of product, say, in two days. Such indicators are no longer capable of wild forms - this "sweatshop" system requires supermutants, organisms with dozens of different genome modifications.
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Closer to nature
Here it is worth asking a question: why involve biotechnology at all - isn't the chemical industry able to cope with the production of the same amino acids? Copes. Chemistry can do a lot these days, but biotechnology has several major advantages. First, they operate on renewable resources. Now, starch and sugar-containing plants (wheat, corn, sugar beets) are mainly used as raw materials. In the future, it is believed that cellulose (wood, straw, cake) will be actively used. The chemical industry works primarily with fossil hydrocarbons.
Secondly, biotechnology is based on the enzymes of living cells that work at atmospheric pressure, normal temperature, in non-aggressive aqueous media. Chemical synthesis takes place, as a rule, under enormous pressure, high temperatures, using caustic, as well as explosive and fire hazardous substances.
Thirdly, modern chemistry is based on the use of catalytic processes, and metals, as a rule, act as catalysts. Metals are not a renewable raw material, and their use is risky from an environmental point of view. In biotechnology, the function of catalysts is performed by the cells themselves, and, if necessary, the cells are easy to utilize: they decompose into water, carbon dioxide, and a small amount of sulfur.
Finally, the fourth advantage lies in the properties of the resulting product. For example, amino acids are stereoisomers, that is, molecules have two forms that have the same structure, but are spatially organized as mirror images of each other. Since the L- and D-forms of amino acids refract light in different ways, such forms are called optical.
Chemistry versus biotechnology.
From the point of view of biology, there is a significant difference between the forms: only the L-forms are biologically active, only the L-form is used by the cell as a building material for protein. In chemical synthesis, a mixture of isomers is obtained; the extraction of the correct forms from it is a separate production process. The microorganism, as a biological structure, produces substances of only one optical form (in the case of amino acids, only in the L-form), which makes the product an ideal raw material for pharmaceuticals.
Cage battle
So, the problem of increasing productivity for biotechnological industries with natural strains cannot be solved. It is necessary to use genetic engineering techniques to actually change the cell's lifestyle. All her strength, all her energy, and all that she consumes should be directed towards lean growth and (mainly) the production of large quantities of the desired metabolite, be it an amino acid, organic acids or an antibiotic.
How are mutant bacteria created? In recent times it looked like this: they took a wild strain, then carried out mutagenesis (that is, treatment with special substances that increase the number of mutations). The treated cells were plated, and thousands of individual clones were obtained. And there were dozens of people who tested these clones and looked for those mutations that are most effective as producers.
The most promising clones were selected, and the next wave of mutagenesis followed, and again dispersal, and again selection. In fact, all this was not much different from the usual selection, which has long been used in animal husbandry and crop production, except for the use of mutagenesis. So for decades, scientists have selected the best of the many generations of mutant microorganisms.
A different approach is used today. Everything now starts with the analysis of metabolic pathways and the identification of the main pathway for the conversion of sugars to the target product (and this path may consist of a dozen intermediate reactions). Indeed, in the cell, as a rule, there are many side pathways, when the initial raw material goes to some metabolites that are not at all necessary for production. And first, all these paths need to be cut off so that the conversion is directed directly to the target product. How to do it? Change the genome of a microorganism. For this, special enzymes and small fragments of DNA - “primers” are used. With the help of the so-called polycyclic reaction in a test tube, a single gene can be pulled out of a cell, copied in large quantities, and altered.
The next task is to return the gene to the cell. The already changed gene is inserted into "vectors" - these are small circular DNA molecules. They are able to transfer the altered gene from the test tube back into the cell, where it replaces the previous, native gene. Thus, you can introduce either a mutation that completely disrupts the function of an unnecessary gene production, or a mutation that changes its function.
In the cell, there is a very complex system that prevents the production of an excessive amount of any metabolite, the same lysine, for example. It is produced naturally in an amount of about 100 mg / l. If there is more of it, then lysine itself begins to inhibit (slow down) the initial reactions leading to its production. A negative feedback arises, which can be eliminated only by introducing another gene mutation into the cell.
However, clearing the path of raw materials to the final product and removing the inhibitions built into the genome on the excessive production of the required metabolite is not all. Since, as already mentioned, the formation of the desired product takes place inside the cell a certain number of stages, at each of them a "bottleneck effect" can occur. For example, at one of the stages the enzyme works quickly and a lot of intermediate product is produced, and at the next stage the throughput drops and an unclaimed excess of the product threatens the vital activity of the cell. This means that it is necessary to strengthen the work of the gene that is responsible for the slow stage.
You can enhance the work of a gene by increasing its copy number - in other words, by inserting not one, but two, three or ten copies of the gene into the genome. Another approach is to "link" to a gene a strong "promoter", or a section of DNA responsible for the expression of a particular gene. But the “unsealing” of one “bottleneck” does not mean at all that it will not arise at the next stage. Moreover, there are a lot of factors affecting the course of each stage of obtaining a product - it is necessary to take into account their influence and make adjustments to the gene information.
Thus, the "competition" with the cage can last for many years. It took about 40 years to improve the biotechnology of lysine production, and during this time the strain was “taught” to produce 200 g of lysine per liter in 50 hours (for comparison: four decades ago this figure was 18 g / l). But the cell continues to resist, because such a mode of life for the microorganism is extremely difficult. She clearly does not want to work in production. And therefore, if the quality of cell cultures is not regularly monitored, mutations will inevitably arise in them that reduce productivity, which will be readily picked up by selection. All this suggests that biotechnology is not such a thing that can be developed once, and then it will act on its own. And the need to increase the economic efficiency and competitiveness of biotechnological industries, and the prevention of degradation of the created high-performance strains - all require constant work, including fundamental research in the field of gene functions and cellular processes.
One question remains: are not mutant organisms dangerous for humans? What if they end up in the environment from bioreactors? Fortunately, there is no danger. These cells are flawed, they are absolutely not adapted to life in natural conditions and will inevitably die. Everything in the mutant cell has changed so much that it can grow only under artificial conditions, in a certain environment, with a certain type of nutrition. There is no way back to the wild state for these living beings.
The author is Deputy Director of the State Research Institute of Genetics, Doctor of Biological Sciences, Professor Alexander Yanenko.