General Biotechnology: Lecture Course, Part II - Blinov, V. A. 2004

Microbiological Production of Antibiotics and Vitamins

♦ General Information on Antibiotics. Selection of antibiotics. Basics of technology.

♦ Microbiological production of Vitamins.

♦ Biotransformation of Organic compounds. Cell-free synthesis.

General information on antibiotics. Antibiotics, Alkaloids, plant growth Hormones, and toxins belong to secondary metabolites (idiolites). They are produced by a limited number of taxonomic groups. Microorganisms that produce secondary metabolites first undergo a rapid growth stage—the trophophase—during which the Synthesis of Secondary metabolites is negligible. Then, as one or more essential nutrients in the culture medium are depleted, the microorganisms transition into the idiophase. It is during this period that the bulk of secondary metabolites is synthesized. As for antibiotics, most microorganisms are sensitive to their own antibiotics during the trophophase. Therefore, the process should be shifted to the idiophase as quickly as possible, and antibiotic-producing microorganisms should be cultivated in this phase.

In 1940, A. Fleming and others first isolated penicillin from the culture broth in an amorphous and subsequently in a crystalline form. Today, antibiotics constitute the largest Class of pharmaceutical compounds synthesized by microbial Cells. This same class includes antifungal agents, antitumor drugs, and alkaloids. Six genera of filamentous Fungi produce about 1,000 different antibiotics, including Cephalosporins and Penicillins. Two genera of non-filamentous Bacteria synthesize 500 antibiotics, and three genera of actinomycetes produce about 3,000 antibiotics. Currently, approximately 6,000 antibiotics are known. Moreover, There is a trend toward obtaining new forms of antibiotics not through The Biosynthesis of novel substances, but via the chemical or biotechnological transformation of already known antibiotics.

Based on their biological action, antibiotics are divided into antibacterial, antifungal, and anticancer agents. Antibacterial antibiotics include those that inhibit the growth of gram-positive bacteria (penicillin, cephalosporin, erythromycin, oleandomycin, carbomycin, etc.); those that inhibit the growth of gram-negative bacteria (tetracycline, neomycin, streptomycin, polymyxin, gramicidin, etc.); and those with antitubercular activity (streptomycin, biomycin, cycloserine, etc.). The antifungal group includes nystatin, griseofulvin, levorin, candidicidin, etc. Introduction/45.html">Antitumor Antibiotics include actinomycin, mitomycin C, and others.

From a commercial standpoint, penicillin, cephalosporin, and tetracycline have proven to be the most widespread. The value of global antibiotic production in 1985 reached $8 billion. In 1980, worldwide antibiotic production was approximately 25,000 tons, of which 17,000 tons were penicillins, 5,000 tons Tetracyclines, 1,200 tons cephalosporins, and 800 tons erythromycins.

The core Structure of penicillins is 6-aminopenicillanic acid (6-APA), which is used to produce semi-synthetic penicillins.

Typically, 6-APA is obtained from benzylpenicillin (penicillin G) using penicillin amidase (penicillin acylase). This is a microbiological process utilizing either E. coli or B. megatherium. The enzyme is adsorbed from the culture broth onto bentonite, and the reaction is carried out in a batch mode. Immobilized penicillin amidase is also used to produce new antibiotics from the cephalosporin group.

If one of the hydrogens on the amino group of 6-APA is replaced with a specific radical, the antimicrobial spectrum of such a semi-synthetic antibiotic can be enhanced or broadened. For example, The addition of a methoxy group (CH3O-) to the ß-lactam ring led to The Development of cephamycins. Currently, biosynthetic antibiotics are produced (using Penicillium notatum or P. chrysogenum), along with semi-synthetic antibiotics (ß-lactams) derived from them.

The core of another major group of extracellular ß-lactam antibiotics—the cephalosporins—is 7-aminocephalosporanic acid (7-ACA), isolated during the purification of cephalosporin C (produced by Cephalosporium acremonium).

Resistance to penicillins and cephalosporins is associated with ß-lactamase Enzymes, which are widespread among bacteria, actinomycetes, cyanobacteria, and Yeasts. The genes encoding these enzymes are part of Plasmids, meaning resistance can be transmitted via plasmid transfer from one bacterial strain to another. ß-Lactamase enzymes inhibit the synthesis of ß-lactam antibiotics, including thienamycins produced by Streptomyces cattleya.

Selection of antibiotics. Antibiotics are produced As a result of the combined action of 10–30 genes. Their number makes it impossible to detect individual Mutations that could increase the yield of the target product. Therefore, high-yielding strains of penicillin and tetracycline (Penicillium chrysogenum and Streptomyces aureofaciens) were obtained through successive cycles of mutagenesis and selection. If a mutant producing a large amount of antibiotic was discovered, it served as the Starting Material for new cycles of mutagenesis and screening. Thus, at The First stage, a spontaneous mutant producing 60 mg/L of penicillin was discovered, and subsequently, a super-producer with a penicillin yield of 150 mg/L was isolated. After X-ray irradiation, a strain producing 300 mg/L of penicillin was obtained; it was then subjected to ultraviolet irradiation, yielding a strain that produced 550 mg/L of penicillin. This final strain served as the starting material for several cycles of mutagenesis and selection, in which mustard gas was applied In addition to UV irradiation. A productive strain capable of producing 7 g/L of penicillin was thus obtained. In this way, over two decades across 21 cycles of mutagenesis and selection, the penicillin yield was increased 55-fold. Later, a producer yielding 20 g/L of penicillin was identified, which is 10,000 times higher than the antibiotic yield obtained in 1941.

As a result of mutations, new secondary metabolites appeared, such as 6-demethylchlortetracycline and 6-demethyltetracycline, which possess antibiotic activity. Furthermore, certain idiotrophic mutants were able to synthesize only half of the antibiotic molecule, requiring the medium to be enriched with the other half. This led to the discovery of new antibiotic derivatives, including those belonging to the aminocyclitol group. It was subsequently discovered that the synthesis of certain antibiotics—for example, those produced by various Streptomyces species—is determined not by chromosomal genes, but by a plasmid. It is believed that introducing several plasmids of this type into a single Streptomyces cell can help combine different metabolic pathways and lead to the biosynthesis of novel antibiotics.

Basics of technology. Currently, many antibiotics are produced by submerged Fermentation. For example, intensive penicillin synthesis begins with a high biomass of mycelium, the complete utilization of glucose and lactic acid in the medium, and a near-neutral pH. It has been established that benzylpenicillanic acid, as one of the precursors of penicillin, is synthesized from L-cystine, phenylacetic acid, and dimethylpyruvic acid.

To obtain penicillin, spores are first propagated, typically on Agar media. Their composition includes (in %): 0.5 molasses, 0.5 peptone, 0.4 sodium chloride, 0.01 monopotassium phosphate, and 0.05 magnesium sulfate. Under industrial conditions, spores are grown on millet in flasks at 25–27 °C for 4–5 days. Dried spores can be stored at room Temperature. Inoculators are then seeded with the spores (1–3 flasks per apparatus), where the mycelium is propagated to 5–10% of the seed fermenter volume. In these fermenters, the mycelium is grown for 12–18 hours, and 15–20% of the culture broth volume is used for the main fermentation.

Nutrient media for both mycelium growth and penicillin biosynthesis are usually prepared from corn steep liquor (2–3%), lactose (5%), glucose (1.5%), ammonium sulfate and phosphates (0.5–1.0%), as well as phenylacetic or phenoxyacetic acid derivatives as antibiotic precursors (0.3–0.6%). Chalk is used to stabilize the pH. Fermentation is carried out at 22–26 °C, pH 5.0–7.5, with intensive aeration of the medium [(1 m³ O₂ / (m³·min)] and a power input of 1–4 kW/m³. Most commonly, bioreactors with volumes ranging from 30 to 200 m³ equipped with a mechanical stirrer, cooling systems, and defoamers are used. Over 4 days, The amount of penicillin reaches a maximum (up to 10,000 conventional units/mL). The mycelium is separated by filtration, usually using vacuum filters, and is subsequently used in animal husbandry as a source of Proteins and vitamins.

After separating the mycelium, the culture broth contains 3–6% dry matter (DM), of which 30–40% consists of Mineral Substances and 15–30% of penicillin. The filtrate contains protein—sometimes up to 700 mg per 100 mL—which complicates penicillin isolation. Protein impurities are removed by precipitation with polyvalent metal salts (Al, Fe, Zn), coagulation with tannin, or Denaturation at 65–70 °C and a medium pH of 5.5–6.0. Penicillin losses during this process amount to 5–15%.

Penicillin is extracted using organic Solvents (butyl acetate or amyl acetate) at a pH of 1.9–2.0. As a result of extraction, product purity increases 4- to 6-fold. Then, penicillin is transferred from the butyl acetate extract into Water using a sodium bicarbonate solution (medium pH 6.6–7.2), yielding a liquid with a dry matter content of 5–7% and an activity of 30,000–50,000 conventional units/mL. For further purification, penicillin is re-extracted with butyl acetate. The phase ratio during extraction is 1:0.5–1:1, and the extract activity is 50,000–70,000 conventional units/mL. The penicillin yield is approximately 86% of its amount in the culture broth.

Antibiotic production costs are distributed as follows (% of total costs): fermentation—50–70; filtration—3–5; extraction—15–30; crystallization—5–10; drying—5–10. In turn, the fermentation process includes the following costs: Materials—45–65%; labor—5–10; steam—5–10; electricity—15–30; inoculum preparation—2–8% of the total fermentation process costs.

When formulating nutrient media, it must be kept in mind that glucose is the limiting factor for penicillin synthesis, whereas for Other Antibiotics produced by Streptomyces, growth is limited by phosphates. Recently, the extraction and chemical purification of penicillin have been carried out using a continuous scheme. If penicillin is intended for medical purposes, special attention is paid to the purification of the drug.

Actinomycetes as antibiotic producers are "omnivorous," meaning they are capable of growing and producing the product on many media containing proteins (soy flour, fish meal, wheat gluten protein, etc.) or starch. However, each producer has its own specific features. For example, the inoculum of Streptomyces kanamyceticus is obtained on a soy-starch medium, which is also used for the main fermentation conducted at 27–28 °C for 4–5 days while maintaining the pH at 7.1–7.6. For the fermentation of Str. floridae (the violamycin producer), a medium containing glucose or hydrol, soybean meal, corn steep liquor, nitrates, and chalk is used; the temperature should be 27–29 °C, and the pH 7.0–7.3. When cultivating Str. erythreus, propyl alcohol is added to the nutrient medium as a precursor for the antibiotic erythromycin. The nystatin producer Str. noursei readily assimilates ammonium nitrogen rather than nitrate nitrogen.

Microbiological production of vitamins. As is known, vitamins are supplied to the body through food or prescribed for certain medical conditions. Vitamins A, D, B₂, B₁₂, C, and others are produced biotechnologically.

Carotenoids (precursors of vitamin A) are synthesized by numerous pigmented microorganisms belonging to the genera Aleuria, Blakeslea, Corynebacterium, Fusarium, Halobacterium, Pseudomonas, Sarcina, Sporobolomyces, and others. A total of about 500 carotenoids produced by bacteria, yeasts, and filamentous fungi are currently known. They occur as complex esters and Glycosides, either embedded in The Cell membrane of microorganisms or in a free state within cytoplasmic lipid droplets. Light promotes the biosynthesis of carotenoids, which primarily perform protective Functions. By cultivating certain species of microorganisms, it is possible to achieve a yield of 3–4 g of carotene per 1 liter of medium.

Nutrient media for carotenoid cultivation are complex and must include sources of carbon, nitrogen, vitamins, Trace Elements, and growth stimulants (such as hydrol, corn-soybean flour, plant oils, kerosene, ß-ionone, and isoprene dimers). Stimulants are usually added to the nutrient medium at the end of the trophophase, i.e., when the producer begins to transition into the idiophase.

Initially, the strains are grown separately and then coinoculated at 26 °C with enhanced aeration before being transferred to the main fermenter. The fermentation period lasts 6–7 days. Carotenoids are extracted using a polar solvent, frequently acetone, and then transferred to a nonpolar solvent. If the Extraction of Protein-carotenoid complexes is required, Surfactants are used at a concentration of 1–2 %. Subsequent Hydrolysis of ß-carotene yields vitamin A₁.

Vitamin D is based on the ergosterol Skeleton, which is located in the cell membranes of eukaryotes. Accordingly, baker's or brewer's yeasts are widely used, in which the content of the provitamin (ergosterol) ranges from 0.2 to 11 %. Under METABOLISM/18.html">The Influence of UV radiation, ergosterol is transformed into vitamin D₂, which easily converts into vitamin D₃. The physiological activities of vitamins D₂ and D₃ are identical. In addition to yeasts, producers of ergosterol include Aspergillus and Penicillium Molds, which may contain from 1.2 to 2.2 % ergosterol. As is well known, a deficiency of Vitamin D Group leads to Rickets in children and Osteomalacia in adults.

The production of ergosterol requires the following stages: Propagation of the initial culture and accumulation of the inoculum, fermentation, cell Separation, ultraviolet irradiation of the cells, drying, and packaging of the target product.

Irradiated dry Yeast is used in livestock farming. It is marketed under the name "hydrolytic feed yeast enriched with vitamin D₂." Such a preparation contains at least 46 % crude protein, essential amino acid^s (Lysine, Methionine, Tryptophan), and 5,000 IU of vitamin D₂/g.

To obtain crystalline vitamin D₂, producer cells are hydrolyzed with Hydrochloric acid at 110 °C, after which the temperature is lowered to 75–78 °C and ethanol is added. The mixture is filtered at 10–15 °C, the filter cake is washed with water, dried, pulverized, heated to 78 °C, and treated twice with three volumes of ethanol. The alcoholic extracts are combined and evaporated to a dry matter content of 70 %. This "lipid concentrate" is then treated with a sodium hydroxide solution.

Ergosterol crystallizes from the unsaponifiable fraction of the concentrate at 0 °C. It is purified by repeated recrystallizations. The crystals are dried, dissolved in diethyl ether, irradiated with ultraviolet rays, the ether is distilled off, and the vitamin D₂ solution is concentrated and crystallized. The "acid filtrate" is evaporated to a 50 % dry matter content and used as a vitamin concentrate. An oil concentrate of vitamin D₂ is also manufactured.

Vitamin B₂ (riboflavin) is produced by bacteria, yeasts, and filamentous fungi. Currently, strains have been selected that yield 0.5 g or more of riboflavin per 1 liter of liquid medium. These include: Ashbya gossypii, Eremothecium ashbyii, and Candida guilliermondii. Using Genetic Engineering Methods, a strain of hay bacillus has been obtained that produces about 6 g of riboflavin per 1 liter of medium containing molasses, protein-vitamin concentrate, and its hydrolysate.

Glucose and sucrose, yeast and corn extracts, soybean meal, and oils are typically used as carbon sources. For instance, a medium of the following composition is known for producing seed culture: sucrose, peptone, corn extract, potassium dihydrogen phosphate, magnesium sulfate, and sunflower oil. The growth time of the producer on this medium is 2 days at 27–30 °C. Fermentation is most often carried out for 5 days at pH 5.5–7.7. The resulting biomass is dried, and the product—with a residual moisture content of 8 %, containing 1.5–2.5 % riboflavin, 20 % protein, thiamine, nicotinic acid, pyridoxine, cyanocobalamin, and trace elements—is used for animal feed. If riboflavin production is sufficiently high, vitamin B₂ can be isolated separately and used in medicine along with synthetic vitamin.

Vitamin C (ascorbic acid) is synthesized by all plants and animals except primates and guinea pigs. Humans are also unable to synthesize ascorbic acid, making it an essential nutrient for them. Microorganisms do not synthesize vitamin C, nor do they require it. However, certain species of acetic acid bacteria produce an intermediate of ascorbic acid—L-sorbose. This biological stage of the process is catalyzed by membrane-bound polyol dehydrogenase. This is followed by a multi-step chemical stage, ultimately yielding 2-keto-L-gulonic acid. It undergoes enolization and is transformed into L-اسcorbic acid. Consequently, The process of obtaining ascorbic acid is mixed, i.e., chemo-enzymatic.

Fermentation by the producer (Gluconobacter oxydans) is carried out on media containing sorbitol (20%), corn or yeast extract, requiring intensive aeration (8–10 g O₂ /L/h). The yield of L-sorbose can reach 98 % within 1–2 days. Cultivation is carried out in batch or continuous mode. Ascorbic acid is used as an antioxidant in healthcare and the food industry.

Vitamin B₁₂ (cyanocobalamin) is obtained exclusively via microbiological synthesis. It is produced by prokaryotes, primarily propionic acid bacteria. In Russia, Propionibacterium var. shermanii, cultivated in batch mode without oxygen access, is used for vitamin B₁₂ production. The fermentation medium contains glucose, corn extract, ammonium and cobalt salts, with a pH of about 7.0 maintained by adding NH₄OH. The duration of fermentation is 6 ^days. After 3 days, 5,6-dimethylbenzimidazole (a precursor of vitamin B₁₂) is added to the medium, and fermentation is continued for another 3 days.

Cyanocobalamin accumulates inside bacterial cells. Therefore, its isolation involves: cell separation, extraction with water at pH 4.5–5.0 and a temperature of 85–90 °C in the presence of a stabilizer (0.25 % sodium nitrate solution). Extraction lasts one hour, after which the aqueous solution is cooled, neutralized with sodium hydroxide solution, protein coagulants (ferric chloride, ammonium sulfate) are added, and the mixture is filtered. The filtrate is evaporated and purified using Ion Exchange and Chromatography. Then, the vitamin is crystallized at 3–4 °C from an aqueous-acetone solution. Sometimes resorcinol or phenol is used, which form adducts with vitamin B₁₂ that are subsequently easily decomposed.

In Russia, a feed preparation (KMB₁₂) containing vitamin B₁₂ and other growth factors is produced from acetobutyl and alcohol stillage with the addition of cobalt and methanol. Here, the biological agent is a mixed culture of methanogenic bacteria. Vitamin B₁₂ is highly photosensitive, so all operations must be performed in dark conditions or under red light.

Vitamin B₁₂ is an anti-anemic factor and is absolutely essential for the normal functioning of living organisms. Global production of cyanocobalamin reaches 10 tons per year, of which 6.5 tons are used for medical purposes and 3.5 tons in animal husbandry.

Biotransformation of organic compounds. Many microorganisms are capable of converting various substances into new compounds. For example, Gluconobacter suboxydans transforms sorbitol, glycerol, glucose, and mannitol into sorbose, dihydroxyacetone, 5-ketogluconic acid, and gluconic acid, respectively. Pseudomonas miyamiryu converts D-phenylalanine into L-phenylalanine, and so on.

Biotransformation of organic substances into new compounds differs in that it involves a single enzyme that catalyzes oxidation, decarboxylation, methylation, etc. In contrast, biosynthesis and fermentation processes, as is well known, involve A large number of different enzymes.

To obtain the desired compound, the culture of the corresponding microorganism is first propagated to an amount equal to 5–10 % of the volume of the solution to be transformed. This approach is based on two premises. First, the maximum possible amount of the substance to be transformed—usually 10–25 %—should be dissolved in the nutrient medium. Second, a minimum of nutrient salts necessary for the growth of the culture must be used, and the Isolation of the target product should not be hindered. Transformation is carried out under sterile conditions while maintaining optimal pH, temperature, and other factors. The process typically lasts 0.5–2 days. Microbiological transformation is followed by the chemical isolation of the substance from the solution.

Technological methods of transformation can include the following:

♦ under batch conditions using a growing culture;

♦ using non-multiplying cells;

♦ using spores;

♦ employing disintegrated cells;

♦ using Immobilized Microbial Cells;

♦ using enzymes isolated from microorganisms, including immobilized ones;

♦ continuous methods.

The transformation of organic compounds is carried out with a high conversion rate of 0.9–1.0. Two-phase systems (water – organic solvent) are highly promising as they provide favorable thermodynamic reaction conditions. Cell membrane permeability is critical for biotransformation and sometimes needs to be regulated. For instance, when converting Purines or Pyrimidines into their corresponding NUCLEOTIDES using Brevibacterium ammoniogenes, membrane permeability is controlled using manganese ions. In some cases, to achieve more efficient conversion of certain organic compounds into others, the culture is grown on one substrate while the conversion itself is performed on another. However, it should be kept in mind that the transforming properties of strains may change as a result of mutation.

Microbial transformation gained widespread use after 1934, when it was discovered that Acetobacter suboxydans converts D-sorbitol into L-sorbose, an essential precursor for ascorbic acid synthesis. Today, biotransformation is employed to produce and modify Cholesterol, ergosterol, Sex Hormones, Adrenocortical Hormones, Prostaglandins, thromboxanes, Leukotrienes, prostacyclin, and others. These physiologically active substances are widely used in medicine.

Cell-free synthesis. Academician A.S. Spirin developed a cell-free Technology for Obtaining target products, utilizing special bioreactors instead of living cells. In these systems, product synthesis is driven by a set of purified cellular components. This protects the synthesis process from cellular limiting factors and places it entirely under operator control. A cell-free synthesis system consists of Ribosomes, specific protein factors, Amino Acids, tRNA, as well as ATP and GTP. Cell-free biotechnology enables the Replication of genetic material and Protein Biosynthesis (Translation).

Extracellular cloning of genetic material eliminates The Need for complementary DNA Synthesis, vector construction, host cell selection, isolation of amplified DNA, and generation of mRNA transcripts from it. Cell-free synthesis reduces the time required for cloning genetic material tenfold and makes it possible to yield large quantities of mRNA for large-scale cell-free translation systems. Utilizing continuous-flow setups along with selective inlet and outlet membranes significantly increases the productivity of the cell-free system.

This approach is used in laboratory setups to produce Peptide Hormones, diagnostic Antigens, antitoxins, protein toxins, antiviral protective proteins, enzymes, and more.