What are the microbial breeding techniques?

Its methods usually include natural breeding and artificial breeding, which can be used alone or by hybridization.

DNA shuffling technique

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With the development and application of PCR technology, 1994, American stemmer proposed a brand-new artificial molecular evolution technology-DNA shuffling (also known as shuffling technology), which can simulate the molecular evolution process of organisms for hundreds of years, and can directionally screen out functional mutant genes with hundreds or even tens of thousands of times higher enzyme protein activity encoded by specific genes in a short experimental period. The basic principle is to digest a group of homologous genes with different sources but the same function with DNA nuclease I to produce random small fragments, which form a library and can be used as primers and templates for PCR amplification. Template conversion and recombination occur when a gene copy fragment is used as a primer for another gene copy. After introduction into vivo, positive mutants were selected for a new round of in vitro recombination. Generally, through 2-3 cycles, recombinant mutants with greatly improved products can be obtained.

2 natural reproduction

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The microorganisms in nature are separated and purified without artificial mutation or hybridization (see separation and purification of microorganisms), and then pure culture and determination are carried out (see microbial determination method). First, the strains of microorganisms are selected. This method is simple and feasible, but the probability of obtaining excellent strains is small, which is generally difficult to meet the needs of production.

3 artificial propagation

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There are two kinds of mutation breeding and cross breeding.

mutation breeding

Breeding technology of gene mutation microorganism. 1927, H. J. Mahler discovered that X-rays can increase the mutation rate. 1944, C. auerbach first discovered the mutagenic effect of nitrogen mustard gas; Subsequently, many physics (such as ultraviolet rays, gamma rays, fast neutrons, etc. ) and chemical mutagens were discovered one after another. There are three kinds of chemical mutagenic factors: ① Mutants undergo chemical changes with one or more nucleic acid bases, resulting in base substitution during DNA replication, such as hydroxylamine nitrite, diethyl sulfate, ethyl methanesulfonate, nitroguanidine, nitrosomethylurea, etc. ② Mutants are structural analogues of natural bases, which will cause variation when DNA molecules are added during replication, such as 5- bromouracil, 5- aminouracil, 8- azaguanine, 2- aminopurine, etc. ③ Mutants decrease or increase 1 ~ 2 bases on DNA molecules, which leads to errors in the transcription and translation of all genetic codes below the base mutation point, thus leading to the emergence of code group moving mutants, such as acridine substances and some nitrogen mustard derivatives (ICR). Mutation breeding has the advantages of simple operation, high mutation rate and wide mutation spectrum. It can not only increase the output and improve the quality, but also expand the product variety and simplify the process conditions. For example, the titer of penicillin-producing bacteria isolated from nature by 1943 is only 20 units /ml. After a series of mutation breeding, the titer has reached 40,000 units/ml. After mutagenesis, demethylaureomycin accumulated in the fermentation broth. Corynebacterium glutamicum 1299 can produce lysine and valine after ultraviolet mutagenesis, which increases the variety of products. After mutation, the mutant strain which can reduce foam was screened out, thus improving the utilization rate of fermentation tank. The deficiency of mutation breeding is the lack of directionality.

crossbreeding

Strains or genera with different genotypes form hybrids through mating or somatic cell fusion, or form recombinants through transformation and transduction, and then select excellent strains from these hybrids or recombinants or their descendants. By this method, the recombinant with new gene combination can be isolated, and new strains with vigorous growth, large biomass, strong adaptability and improved enzyme activity can be selected. The methods of hybrid breeding vary according to the propagation mode of experimental strains, such as sexual hybridization, quasi-recombination, protoplast fusion, transformation, transduction, hybrid plasmid transformation and so on. However, the process of genetic analysis of hybrids is basically the same when parents are selected, offspring of separated populations are cultivated, the best ones are selected and the worst ones are eliminated. Crossbreeding generally refers to the mating or joining of strains with mating reaction to form hybrids. This method has a wide range of applications, and has achieved success in breeding wine, bread, medicinal and feed yeast, increasing the antibiotic output of Streptomyces and Penicillium, and enhancing the activity of Aspergillus enzyme.

Somatic fusion is cell fusion and chromosome recombination between strains or species that have no sexual response. Firstly, the cell wall was dissolved by enzyme, and then the protoplast was treated with calcium chloride-polyethylene glycol to promote fusion and obtain hybrids. This method has played a positive role in the improvement of industrial microbial strains.

Transformation and transduction were first used in bacteria, and now they have been widely used in Streptomyces and yeast. With the development of recombinant DNA technology, the construction of recombinant plasmid and the establishment of transformation system, the target gene can be transferred into recipient cells and strains that can produce bioactive substances (such as vaccines, enzymes, etc.) can be produced. ) has important economic value.

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.

4 Mutation breeding

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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.

1. 1.7 atmospheric pressure room temperature plasma mutation breeding

Atmospheric pressure room temperature plasma (ARTP) refers to a plasma jet with high concentration of active particles (including excited helium atoms, oxygen atoms, nitrogen atoms, OH radicals, etc.). ) at atmospheric pressure. As a new physical method, ARTP technology has a broad application prospect in the field of microbial mutation breeding.

Appropriate dosage of active particles in plasma can change the structure and permeability of microbial cell wall/membrane and cause gene damage. After the strain was damaged by genetic material, microorganisms started SOS repair mechanism and induced DNA polymerases Ⅳ and V which did not have the function of exonuclease correction of 3-nucleic acid. Therefore, even if there are unpaired bases in the damaged part of DNA chain, replication can continue. Allowing mismatch in this case can increase the chances of survival. The damage caused by ARTP to genetic material is highly diverse; Moreover, SOS-induced repair itself is fault-tolerant, so the destruction of ARTP diversity in the repair process is likely to be included in the DNA chain, and it may bring the possibility of diversity mismatch in microbial replication and repair.

ARTP is applied to microbial mutation breeding with low cost and convenient operation. Physical mutation equipment (such as ion beam implantation) needs few auxiliary equipment such as ion or electron acceleration, vacuum and refrigeration. The damage mechanism of ARTP to genetic material is diverse, with high positive mutation rate and various mutation manifestations, which have an impact on fungi, bacteria, algae and so on. ARTP has no pollution to the environment and ensures the personal safety of operators. No matter what kind of gas is used to discharge, it will not produce harmful gas. [ 1]

5 chemical mutagenesis

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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.