The principles, characteristics and successful cases of several common physical and chemical mutation breeding methods in China in recent years were analyzed, which provided a basis for microbial mutation breeding. The application progress of information technology in the breeding of high-yield strains such as enzyme preparation, antibiotics, amino acids, vitamins and pesticides was reviewed. The application prospect of this technology combined with ion beam technology and space technology in microbial strain breeding is prospected.

Keywords: mutation; Microbial reproduction; Application progress; prospect

Microbes are closely related to the brewing industry, food industry and biological products industry. The quality of their strains is directly related to the quality of many industrial products, and even affects the quality of people's daily life, so it is necessary to cultivate high-quality and high-yield microbial strains. The purpose of microbial breeding is to guide the metabolic pathway of biosynthesis to the required direction, or to promote the recombination of genes in cells to optimize genetic traits and artificially accumulate some metabolites in order to obtain the required strains with high yield, high quality and low consumption. As one of the methods, mutation breeding has been widely used. At present, domestic microbial breeding circles still mainly use conventional physical and chemical factors and other mutagenesis methods. In addition, protoplast mutagenesis technology has been widely used in the breeding of enzyme preparations, antibiotics, amino acids, vitamins and other strains, and has achieved many significant results.

1, mutation breeding

1. 1 physical mutation

1. 1. 1 ultraviolet irradiation

Ultraviolet irradiation is a common physical mutation method and a very useful tool to induce microbial mutation. The maximum absorption peak of purine and pyrimidine in DNA and RNA is at 260nm, so ultraviolet at 260nm is the most effective lethal agent. There are many explanations for the function of ultraviolet rays, but the definite function is to make DNA molecules form pyrimidine dimers [1]. The formation of dimer will hinder the normal pairing between bases, so it may lead to mutation or even death [2].

Ultraviolet irradiation mutagenesis is simple and economical, which can be realized under general laboratory conditions, and the probability of positive mutation is high. This method is mainly used for mutagenesis of yeast strains.

1. 1.2 ionizing radiation

Gamma ray is one of the most widely used ionizing rays in ionization biology. It has high energy and can produce ionization, which can directly or indirectly change the structure of DNA. The direct effect is that the base of deoxyribose can be oxidized, or the chemical bond of deoxyribose and the chemical bond between sugar and phosphoric acid. Its indirect effect is that water or organic molecules can produce free radicals, which can chemically change with solute molecules in cells, leading to DNA deletion and damage [2].

Besides gamma rays, ionizing radiation also includes X rays, beta rays and fast neutrons. Ionizing radiation has certain limitations, high operational requirements and certain risks, and is usually used in the mutation breeding process where other mutagens cannot be used.

1. 1.3 ion implantation

Ion implantation is a high-tech that appeared in the early 1980s. Mainly used for surface modification of metal materials. It has been gradually used in crop breeding since 1986, and microbial breeding has been gradually introduced in recent years [3].

During ion implantation, biomolecules absorb energy and cause complex physical and chemical changes. The intermediate products of these changes are various active free radicals. These free radicals will damage other normal biomolecules, destroy chromosomal mutation and DNA chains in cells, and also destroy plasmid DNA. Because the range of ion implantation is controllable, with the development of microbeam technology and precise positioning technology, localized mutagenesis will become possible [4].

Ion implantation for microbial mutation breeding is difficult to realize under general laboratory conditions, and its application is relatively rare at present.

1. 1.4 laser

Laser is a quantum flow of light, also known as optical particles. Laser radiation can directly or indirectly affect organisms through the comprehensive application of light, heat, pressure and electromagnetic field effects, causing chromosome aberration effect, enzyme activation or inactivation, cell division and changes in cell metabolic activities. Once light quantum acts on any substance in cell contents, it may lead to the variation of cytological and genetic characteristics of biological organisms. Different kinds of laser irradiation biological organisms show different cytological and genetic changes [5].

As a breeding method, laser has the advantages of simple operation and safe use, and has made a lot of progress in microbial breeding in recent years.

1. 1.5 microwave

Microwave radiation is a kind of low-energy electromagnetic radiation, which has strong biological effects in the frequency range of 300MHz~300GHz, and has thermal and non-thermal effects on organisms. Its thermal effect means that it can cause the local temperature of living things to rise. Thereby causing physiological and biochemical reactions; Non-thermal effect refers to various physiological and biochemical reactions unrelated to temperature under the action of microwave. Under the combined action of these two effects, organisms will produce a series of mutation effects [6].

Therefore, microwave has also been used in mutation breeding in many fields, such as crop breeding, animal breeding and industrial microbial breeding, and has achieved certain results.

1. 1.6 space breeding

Space breeding, also known as space mutation breeding, is a new crop breeding technology that uses high-altitude balloons, recoverable satellites, spaceships and other spacecraft to carry crop seeds, tissues, organs or living individuals into space, uses the special environment of space to mutate biological genes, and then returns to the ground for breeding and cultivating new varieties and materials. Space environmental factors mainly include microgravity, space radiation and other mutagenic factors such as alternating magnetic field and ultra-vacuum environment. The interaction of these factors leads to the damage of genetic material in biological system, which leads to the occurrence of biological phenomena such as mutation, chromosome aberration, cell inactivation and abnormal development.

Compared with other breeding methods, space breeding is an organic combination of space technology and microbial breeding technology, with high technical content and high cost, which is difficult for a single scientific researcher or general scientific research unit to achieve. It can only be combined with space technology and completed by the state.

2. 1 chemical mutation

2. 1. 1 alkylating agent

Alkylating agents can react with one or several nucleic acid bases, leading to the transformation of base pairing during DNA replication and genetic variation. Commonly used alkylating agents include ethyl methylsulfonate, nitrosoguanidine, ethylenimine, diethyl sulfate, etc.

Ethyl methanesulfonate (EMS) is the most commonly used alkylating agent with high mutagenicity. Most of the induced mutations are point mutations, which have strong carcinogenicity and volatility. 5% sodium thiosulfate can be used as a terminator and antidote.

N- methyl -N'- nitro -N- nitrosoguanidine (NTG) is a supermutagen, which is widely used, but it is toxic to some extent, so attention should be paid to its operation. Under alkaline conditions, NTG will form diazomethane (CH2N2), which is the main cause of death and mutation. Its effect may be caused by alkylation of DNA with CH2N2 [2].

Diethyl sulfate (DMS) is also commonly used, but it is rarely used at present because of its strong toxicity. Ethylene imine, the output is small and it is difficult to buy. The use concentration is 0.000 1%~0. 1%, which is highly carcinogenic and needs to be prepared with buffer.

2. 1.2 base analogues

The molecular structure of base analogues is similar to that of natural bases, which can be integrated into DNA molecules, leading to mismatch, mRNA transcription disorder, functional protein recombination and phenotypic changes in the process of DNA replication. The toxicity of these substances is relatively small, but the negative mutation rate is high, so it is often difficult to get good mutants. There are mainly 5- fluorouracil (5- FU), 5- bromouracil (5- BU) and 6- chloropurine. Cheng et al. [25] mutagenized the cells of pigment-producing bacteria (Mycobacterium T 17- 2- 39) with 5- BU, and the average biomass increased by 22.5%.

2. 1.3 inorganic compound

The mutagenic effect is general and the risk is small. Lithium chloride is commonly used and crystallized in white. When it is used, it is prepared into 0. 1%~0.5% solution, or it can be directly added to the mutagenic solid culture medium for 30 min ~ 2 days. Nitrite decomposes easily, so it is used now. Sodium nitrite and hydrochloric acid are commonly used to prepare sodium nitrite. The concentration of sodium nitrite is 0.0 1~0. 1mol/L, and hydrochloric acid with the same concentration and volume can be added when used.

2. 1.4 others

Reducing agent hydroxylamine hydrochloride acts on C to change G- C into A-T, which is also commonly used. The concentration is 0. 1% ~ 0.5%, and the action time is 60 min ~ 2 h.

In addition, when mutagenizing, two or more mutagenic factors are used in combination, or the same mutagenic factor is used repeatedly, and the effect is better. Gu et al. [7] took Corynebacterium glutamicum-1376 1 as the starting strain, and obtained an L- histidine-producing strain through DMS and repeated mutagenesis.

2, mutagen

2. 1 mutant selection

When selecting mutagens, it is necessary to pay attention to the specificity of mutagens, that is, a mutagen or mutation treatment preferentially mutates some parts of the genome, while other parts rarely mutate, if any. Although the molecular basis of mutagen specificity is not clear, although the related repair pathway will definitely affect it, the relationship between them is not so simple, and other factors, including the environmental conditions of mutagenic treatment, will also affect the mutation type.

It is difficult for industrial geneticists to correctly predict what kind of molecular mutation is needed to improve a strain. Therefore, in order to produce as many types of mutants as possible, the most suitable method is to adopt several complementary types of mutation treatment. Far ultraviolet ray is undoubtedly the most suitable mutagen, which seems to induce all known types of damage. It is also easy to take effective and safe preventive measures. Among chemical mutagens, liquid reagents are easier to operate safely than powder reagents. Another disadvantage is that it tends to produce closely linked mutant clusters, although this effect may be an advantageous condition in some systems. Finally, it must be recognized that certain strains may not be induced by certain mutagens. Of course, this can be easily verified by measuring the mutation kinetics of easily detected mutants, such as drug-resistant mutants or prototrophic restorers. [8]

2.2 Dosage of mutagen

From the best effect of random screening, the best dosage of mutagen is to obtain the highest proportion of needed mutants in the survival population used for screening, because this will make it more labor-saving in the titer determination stage.

Therefore, before strain improvement, in order to determine the optimal dosage of mutagen and lay the foundation for mutation enhancement technology, it is usually wise to determine the mutation kinetics of different mutagens when dealing with different strains. High-unit mutation itself can sometimes not determine the optimal dose, because it is difficult to detect this mutation. However, if we use markers that are easy to detect, such as drug resistance markers, we can still provide some valuable information as long as we estimate the limitations of the method. [9]

3. Application progress of protoplast mutation in industrial microbial breeding.

3. The application of1in the breeding of enzyme preparation strains

Enzyme preparation is a catalytic protein produced by biological body and an essential element in all metabolic processes. Using protoplast mutagenesis technology, many high-yield strains were obtained.

Hu Jie et al. [10] carried out UV-LiCl, N- methyl-N ′-nitro -N- nitrosoguanidine (N-method-N ′-nitro -N- nitroguanidine, Nt. Eight mutant strains with high neutral protease production were screened out, and the highest enzyme production was 1 162 times that of the original strain, which provided an excellent candidate library for cell fusion and genome recombination in the future.

3.2 Application of Antibiotic-producing Strain Breeding

Antibiotics are secondary metabolites of microbial cells. At present, microbial fermentation is mainly used for biosynthesis. Because the yield of producing strains is restricted by multi-step metabolic regulation, it is also difficult to select high-yield strains. Protoplast mutation, as a mutation technology, has been widely used in the breeding of high-yield strains of antibiotics.

Zhu et al. [1 1] mutagenized the protoplast of Streptomyces cerevisiae by ultraviolet, and obtained a high-yield mutant with a yield of 1 159g/L, which was10/65438 higher than the original strain.

3.3 Application in Breeding of Amino Acids, Production Solvents and Organic Acids

Amino acid is the basic unit of protein, and protein is a kind of biological functional polymer. Widely used in food, feed, medicine, chemical industry, agriculture and other industries, countries are vigorously developing amino acid production. Fermentation has become the main method of amino acid production. Therefore, breeding high-yield strains is an important direction for the development of amino acid industry.

Production solvents and organic acids are the primary metabolites of microorganisms, and protoplast mutagenesis technology has also achieved results in the breeding of production solvents and organic acid-producing bacteria.

3.4 Application of Biological Strain Breeding

Vitamins are a kind of organic substances that are necessary to maintain human and animal life activities, but cannot be synthesized by themselves, and play an important role in the process of growth, metabolism and development. Han et al. treated the protoplast of Penicillium PT95 with laser, and selected a mutant strain L05 with significantly increased sclerotium biomass and carotenoid content. The yield of mutant sclerotia increased by 98.6%, the carotenoid content in sclerotia increased by 28.3%, and the carotenoid yield increased by 154.0%.

3.5 Application of Insect Breeding

Bacillus thuringiensis is a bacterial microbial insecticide screened from nature, which is mainly used to control agricultural and forestry pests. Wang Leehom et al. [12] mutated the protoplast of Bacillus thuringiensis NU- 2 by UV-LiCl compound mutagenesis, and the fermentation period of the screened mutant was shortened from 44h to 40.3h, and the crystal protein content was increased by 10.03%.

Step 4 look to the future

In recent years, with the emergence of new mutagenesis sources, the application of protoplast mutagenesis technology will also make new progress. As a new mutation source, ion beam has its unique mechanism [13], which makes ion beam mutation have the advantages of wide mutation spectrum, large variation range and high mutation rate. Its application has also made many important achievements, especially the success of ion implantation in breeding Vc strains, which has added vitality to China's VC industry. Microbial strains carried in space can produce rare gene mutation in a short time through the transformation of microgravity, space radiation, ultra-vacuum and other comprehensive space environmental factors, thus conducting microbial breeding, which is an important application field of space technology breeding. There have been some gratifying achievements in using space technology to improve the yield of some antibiotics and study enzyme preparations. Combining ion implantation, space technology and microbial protoplast technology, microbial protoplast mutagenesis technology will have a broader application prospect.

5. Conclusion

With the rapid development of genetics and molecular biology, many new and complex technologies have been applied to strain breeding, such as protoplast fusion breeding technology and genetic engineering breeding technology, but mutation breeding technology is still an important and effective means to improve strain productivity. Its positive mutation rate is high, and many excellent mutants and new beneficial gene types can be obtained. On the other hand, mutation breeding has certain blindness and randomness. In practical application, researchers should choose the appropriate mutation method according to the specific conditions such as the starting strain and laboratory conditions. In our laboratory, a combination of physical factors and chemical factors was used to mutate a variety of yeast strains, and all the ideal strains were obtained. In addition, we are still trying to use a variety of mutagenic factors for multiple mutagenesis to obtain more ideal strains.

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