DNA Shuffling Technology
Editor
With the development and application of PCR technology, in 1994, stemmer of the United States proposed a brand-new artificial molecular evolution technology-DNA Shuffling (also known as shuffling technology), which can simulate the molecular evolution process of organisms in hundreds of years, and can directionally screen functional mutant genes with hundreds or even tens of thousands of times higher enzyme protein activity encoded by specific genes in a short experimental cycle. The basic principle is that a group of homologous genes with different sources but the same function are digested with DNA nuclease I to generate random small fragments, and these small fragments form a library, which can be used as primers and templates for PCR amplification. When one gene copy fragment is used as the primer of another gene copy, the template conversion and recombination occur. After being introduced into the body, the positive mutant is 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 breeding
editor
The microorganisms in nature are separated and purified without artificial mutagenesis or hybridization (see separation and purification of microorganisms), and then pure culture and determination (see microbial determination method) are carried out to select the best strains of microorganisms. 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 breeding
editing
there are two kinds of mutation breeding and hybrid breeding.
mutation breeding
microbial breeding technology by inducing gene mutation. In 1927, H.J. Mahler discovered that X-rays could increase the mutation rate. In 1944, C. auerbach first discovered the mutagenic effect of nitrogen mustard gas; Subsequently, many physical (such as ultraviolet rays, gamma rays, fast neutrons, etc.) and chemical mutagenesis factors were discovered one after another. There are three kinds of chemical mutagenesis factors: ① Mutants change chemically with one or more nucleic acid bases, which causes base replacement during DNA replication, such as hydroxylamine nitrite, diethyl sulfate, ethyl methanesulfonate, nitroguanidine, nitrosomethylurea, etc. ② Mutants are structural analogues of natural bases, which cause variation when they are added to DNA molecules during replication, such as 5- bromouracil, 5- aminouracil, 8- azaguanine and 2- aminopurine. 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 appearance 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 yield and improve quality, but also expand product varieties and simplify technological conditions. For example, the titer of penicillin-producing bacteria isolated from nature in 1943 was only 2 units/ml, and after a series of mutation breeding, the titer had reached 4 units/ml; After mutagenesis, demethylaureomycin was accumulated in the fermentation broth. After ultraviolet mutagenesis, Corynebacterium glutamicum 1299 can produce lysine and valine, which increases the variety of products. After mutagenesis, the mutant strain which can reduce foam was selected, thus improving the utilization rate of fermentation tank. The deficiency of mutation breeding is the lack of orientation.
cross breeding
between strains or genera of different genotypes, hybrids are formed by means of mating or somatic cell fusion, or recombinants are formed by transformation and transduction, and then excellent strains are selected from these hybrids or recombinants or their descendants. By this method, recombinant with new gene combinations can be isolated, and new strains with vigorous growth, large biomass, strong adaptability and improved enzyme activities can also be selected. The methods of hybrid breeding vary with the reproductive modes of experimental strains, such as sexual hybridization, quasi-recombination, protoplast fusion, transformation, transduction, hybrid plasmid transformation, etc. However, the process of selecting parent plants, cultivating offspring of separated populations, selecting the best and eliminating the worst and genetic analysis of hybrids are basically the same. Hybridization 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 been successful in the breeding of wine, bread, medicinal and feed yeast, the improvement of antibiotic production of Streptomyces and Penicillium, and the enhancement of enzyme activity of Aspergillus.
Somatic cell fusion is cell fusion and chromosome recombination between strains or species that have no sexual response. First, cell walls are dissolved with enzymes, and then protoplasts are treated with calcium chloride-polyethylene glycol to promote fusion and obtain hybrids. This method plays an active role in improving the strains of industrial microorganisms.
Transformation and transduction were first applied to bacteria, and now they have been widely used in Streptomyces and yeasts. 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.) with important economic value can be obtained.
Microorganisms are closely related to the brewing industry, food industry and biological products industry, and the quality of their strains is directly related to the quality of various 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 in the desired direction, or to promote the recombination of genes in cells to optimize genetic traits, and artificially accumulate some metabolites to obtain the required strains with high yield, high quality and low consumption. Mutation breeding, as one of the ways, has been widely used. At present, the domestic microbial breeding community still mainly adopts 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
editor
1.1 physical mutation
1.1.1 ultraviolet irradiation
ultraviolet irradiation is one of the commonly used physical mutation methods and a very useful tool to induce microbial mutation. The maximum absorption peak of purine and pyrimidine in DNA and RNA is at 26nm, so ultraviolet radiation at 26nm is the most effective lethal agent. There are many explanations for the function of ultraviolet radiation, 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 mutation is simple and economical, which can be achieved under general laboratory conditions, and the probability of positive mutation is high. This method is mostly used for mutation of yeast strains.
1.1.2 ionizing radiation
γ -ray is one of the most widely used ionizing rays in ionization biology, with high energy, which can produce ionization and directly or indirectly change DNA structure. 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 phosphate. 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 γ-rays, ionizing radiation includes X- rays, β-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 rose in the early 198s, and it is mainly used to modify the surface of metal materials. It has been gradually used in crop breeding since 1986, and it has been gradually introduced into microbial breeding in recent years [3].
during ion implantation, biomolecules absorb energy and cause complex physical and chemical changes, and the intermediates of these changes are all kinds of active free radicals. These free radicals can cause damage to other normal biomolecules, break chromosomal mutation and DNA chains in cells, and also break 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 achieve in general laboratory conditions, and it is relatively rarely used at present.
1.1.4 laser
laser is a kind of optical quantum flow, 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 irradiated 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 a strong biological effect in the frequency range of 3MHz~3GHz, and has thermal and non-thermal effects on organisms. Its thermal effect means that it can cause the local temperature of organisms to rise. Thereby causing physiological and biochemical reactions; Non-thermal effect refers to various physiological and biochemical reactions that are not related to temperature under the action of microwave. Under the combined effect 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 mutation 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, and 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 materials in biological systems, which leads to biological occurrence such as mutation, chromosome aberration, cell inactivation and abnormal development.
compared with other breeding methods, aerospace breeding is an organic combination of aerospace technology and microbial breeding technology, which is high in technology content and high in cost, which is difficult for individual researchers or general research units to achieve, and can only be combined with aerospace technology and completed by the state.
1.1.7 atmospheric pressure room temperature plasma mutation breeding
atmospheric and room temperature plasma (ARTP for short) refers to a plasma jet with high active particle concentration (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 dose of active particles in plasma can change the structure and permeability of microbial cell wall/membrane, and cause gene damage. After the genetic material damage of the strain, the microorganism starts SOS repair mechanism, which induces the production of DNA polymerases Ⅳ and V, which do not have the exonuclease correction function of 3ˊ nucleic acid. Therefore, even if there are unpaired bases at the damaged site 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 of high diversity; Moreover, SOS-induced repair itself is fault-tolerant, so the damage of ARTP diversity will probably be contained in the DNA chain during the repair process, and it may bring the possibility of diversity mismatch when microorganisms replicate and repair.
ARTP is applied to microbial mutation breeding, with low cost and convenient operation, without many auxiliary equipment such as ion or electron acceleration, vacuum and refrigeration required by physical mutation equipment (such as ion beam implantation); The damage mechanism of ARTP to genetic material is diverse, with high positive mutation rate and diverse mutation performance, and it has effects 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 for discharge, it does not produce harmful gas. [1]
5 Chemical mutagenesis
Editor
2.1.1 Alkylating agent
Alkylating agent can react with one or several nucleic acid bases, causing base pairing conversion during DNA replication and genetic variation. Commonly used alkylating agents include ethyl methylsulfonate, nitrosoguanidine, ethylenimine and diethyl sulfate.
Ethyl methanesulfonate (EMS) is the most commonly used alkylating agent, with high mutation rate. Most of the mutants induced by it are point mutations, which have strong carcinogenicity and volatility, and 5% sodium thiosulfate can be used as terminator and antidote.
N- methyl-N'- nitro-N- nitrosoguanidine (NTG) is a kind of supermutagen, which is widely used, but it has certain toxicity, 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 is probably 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, produced less, is difficult to buy. The use concentration is .1%~.1%, which is highly carcinogenic and requires the use of buffer solution.
2.1.2 base analogues
the molecular structure of base analogues is similar to that of natural bases, and they can be mixed into DNA molecules, resulting in mismatches in DNA replication, disordered mRNA transcription, functional protein recombination and phenotypic changes. 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 Shiqing et al. [25] used 5- BU to mutate the cells of pigment-producing bacteria (Mycobacterium T17- 2- 39), and the biomass increased by 22.5% on average. < P > 2.1.3 inorganic compound < P > has a general mutagenic effect and less risk. Commonly used are lithium chloride, white crystals, which are prepared into .1%~.5% solution when used, or can be directly added to the mutagenic solid culture medium for 3 min ~ 2 days. Nitrite is easy to decompose, so it is now used. Sodium nitrite and hydrochloric acid are commonly used to prepare sodium nitrite, and the concentration of sodium nitrite is .1~.1mol/L, and hydrochloric acid with the same concentration and volume can be added when it is used.
2.1.4 other
hydroxylamine hydrochloride, a reducing agent, acts on C to change G- C into A-T. It is also commonly used, the concentration is .1% ~ .5%, and the action time is 6 min ~ 2 h.
In addition, it is better to use two or more mutagenic factors in combination or reuse the same mutagenic factor. Gu Zhenghua