BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 5. INDUSTRIAL BIOTECHNOLOGY

Industrial Protein Synthesis Using Recombinant Microorganisms

One of the global challenges facing humanity is the food shortage. At the end of the 20th century, the global population stood at 7.5 billion. According to scientific projections, the world population will reach over 8 billion by 2025 and increase to 10 billion by 2050. UN data indicate that more than half of the Earth's population suffers from food insecurity. The primary nutritional challenge is the deficit of dietary protein.

The Biological value of Proteins depends on their amino acid profile, specifically the presence of Essential Amino Acids that cannot be synthesized by The Human Body. For humans, there are 8 essential amino acids: leucine, isoleucine, Lysine, Methionine, Threonine, Tryptophan, valine, and phenylalanine. Unlike animal proteins, plant proteins lack a complete spectrum of essential amino acids. The most Common sources of plant proteins are soybeans, sunflower seeds, and peanuts. Animal-derived proteins are costly to produce: obtaining 1 kg of animal protein requires an input of 5 — 10 kg of plant proteins.

Microorganisms represent an alternative protein source for Human and Animal Nutrition, with protein content reaching 60 — 80% of their dry weight. Due to high levels of methionine, lysine, Vitamins, and minerals, microbial protein has a high nutritional value. The key advantage of microorganisms as protein producers is their exceptionally rapid growth rate. Microorganisms synthesize proteins 10 to 100,000 times faster than plants and animals. For instance, a 500 kg cow produces 0.5 kg of protein per day, 500 kg of soybean plants yield 5 kg of protein, whereas 500 kg of Yeast can produce 50 tons of protein.

In 1966, the term "Single-Cell Protein" was coined.

Single-cell protein refers to protein products synthesized by microbial monocultures and used as dietary supplements for humans and animals.

Various microorganisms and substrates are utilized to produce single-cell protein (Table 5.1).

Class="center">Table 5.1.

Substrates and microorganisms used in the production

of single-cell protein

In the biotech industry, Yeasts, fungi, bacteria, and microalgae serve as protein producers. Substrates used for single-cell protein production include petroleum Hydrocarbons, methane, hydrogen, carbon dioxide, methanol, ethanol, acetic acid, starch, agricultural lignocellulosic waste, as well as byproducts from the coal, chemical, food, wine and spirits, and woodworking industries. Calculations show that with microbial assistance, 1 kg of petroleum can yield 1 kg of protein, while 1 kg of CARBOHYDRATES produces 500 g of protein.

Certain microorganisms utilize whey as a substrate. Using whey directly as animal feed is an inefficient disposal method because the conversion rate of whey protein into animal protein is very low: synthesizing 1 kg of animal protein requires 1,700 kg of milk whey. Microbial bioconversion of whey allows for the recovery of significant quantities of single-cell protein.

Industrial protein production most frequently relies on the yeast Candida lipolytica. Yeast protein surpasses cereal grain protein in its essential amino acid content and exceeds all protein feedstuffs, including fishmeal, in vitamin content. In addition to abundant protein, yeast biomass contains vitamins, Trace Elements, and Lipids rich in higher Unsaturated Fatty acids. It is estimated that 1 ton of feed yeast replaces 7 — 8 tons of feed grains, yielding an additional 800 kg of pork or 5 tons of poultry meat. Incorporating 1 ton of feed yeast into the diets of calves and piglets saves 6 tons of milk. Supplementing poultry diets with 1 ton of yeast biomass generates an additional

1.5 — 2 tons of meat or 25 — 35 thousand eggs.

Besides yeast, green Algae serve as highly productive sources of feed protein. Biomass from single-cell algae of the genera Chlorella and Scenedesmus is utilized as an animal feed additive. Chlorella Cells consist of 50% protein, 40% carbohydrates, 7 — 10% lipids, vitamins (A, B₂, B₅, K), and trace elements. Cultivated on a 1-hectare area, Chlorella cells yield 20 — 30 tons of protein, compared to just 2 — 3.5 tons for alfalfa crops.

Nostoc and spirulina algae are consumed as food. In Japan, Nostoc grown on volcanic slopes is consumed and referred to as Tengu barley bread. Due to its high vitamin and mineral content, biomass of the alga Spirulina platensis is used as a base for general tonic and immunomodulatory medications. Spirulina contains 65% protein, 19% carbohydrates, 6% pigments, 4% lipids, 3% fiber, and 3% minerals. Spirulina proteins feature a well-balanced amino acid profile. The productivity of spirulina is remarkably high (20 g of dry algal mass per 1 m² daily), exceeding wheat yields by roughly tenfold annually.

Promising protein sources also include cyanobacteria capable of fixing atmospheric nitrogen. The protein content in bacterial cells accounts for 80% of dry weight. The quality of bacterial and microbial biomass is evaluated based on high protein content, low nucleic acid levels, and the absence of harmful substances.

Microbial Biomass Production represents the largest sector in industrial microbiology. Large-scale single-cell protein production was first implemented in Germany during World War I, where S. cerevisiae yeast was cultivated on molasses (carbon source) and ammonium salts (nitrogen source), and the resulting biomass was added to foodstuffs to boost protein content.

Interest in single-cell protein surged in connection with waste valorization and disposal. Developing biotechnological processes for protein recovery from waste requires studying growth kinetics, METABOLISM, genetic modification potential, and microbial safety.

In the 1960s, facilities were established to produce protein using petroleum-utilizing microorganisms. It was established that 1 ton of petroleum can yield 1 ton of Candida yeast containing approximately 600 kg of protein. The first enterprise producing yeast from petroleum alkanes began operations in the USSR in 1973. During the 1970s and 1980s, the USSR ranked first globally in microbiological protein production, exceeding 1 million tons of feed yeast annually. Since 1985, single-cell protein has been utilized in the food industry. Microbial protein is used to formulate artificial meat, milk, cheese, and other products, which have undergone animal and human safety testing. Such synthetic foods can be found on grocery shelves in the USA, England, India, and various Asian and African nations. However, the widespread adoption of single-cell protein in human diets is constrained by the following factors:

- the potential presence of toxic substances in the microbial biomass, either adsorbed from the substrate (heavy metals) or synthesized by the microorganisms (mycotoxins), which necessitates additional costly analytical testing;

- a high nucleic acid content in microbial biomass, which may pose health risks for humans under certain pathological conditions;

- a low rate of microbial cell destruction in the digestive tract can lead to digestive disorders or allergic reactions.

Microorganisms are capable of synthesizing not only food and feed proteins. By introducing cloned genes, various Proteins can be produced using microorganisms. Genes isolated from the mussel Mytilus edulis have been introduced into The Genome of E. coli and yeast. These genes encode a protein that acts as a biological adhesive.

In 1995, genes encoding spider silk proteins were isolated. The Introduction of these genes into the microbial genome enabled the synthesis of spider silk proteins within their cells. As is known, spider silk is 100 times thinner than a human Hair, softer than cotton, stronger than steel, elastic, and resistant to Temperature fluctuations. This material can be used for manufacturing parachutes, body armor, and other Applications.

Thus, microorganisms can be utilized to produce a wide range of proteins.