CN116855513A - Method for obtaining genetically stable microalgae with target properties and ultra-mutant strain - Google Patents

Method for obtaining genetically stable microalgae with target properties and ultra-mutant strain Download PDF

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CN116855513A
CN116855513A CN202310163031.8A CN202310163031A CN116855513A CN 116855513 A CN116855513 A CN 116855513A CN 202310163031 A CN202310163031 A CN 202310163031A CN 116855513 A CN116855513 A CN 116855513A
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吕雪峰
孙绘梨
栾国栋
马依凡
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

The invention relates to a method for obtaining genetically stable microalgae with target characters, which comprises the following steps: obtaining a replication fidelity mechanism defect strain; culturing and passaging the replication fidelity mechanism defective strain by using a high-temperature high-light stress condition to obtain a neutral hypermutant strain library; applying selection pressure, and screening to obtain a hypermutation evolutionary strain with target characters; recovering the fidelity mechanism of the hypermutant strain with the target property to obtain the microalgae with the target property and stable heredity. The invention also provides a replication fidelity mechanism defect strain with the mutation rate of the Synechococcus PCC7942 increased. The method overcomes the defect that the conventional physicochemical mutagenesis factors are adopted in the conventional evolution engineering strategy, and the hyper-mutant library with more abundant genetic diversity is obtained in a short period, but the activity of the mutant in the library is not influenced. In addition, the supermutant library obtained in this aspect is progressive, and as subculturing continues, a more abundant library of supermutants can be obtained.

Description

Method for obtaining genetically stable microalgae with target properties and ultra-mutant strain
Technical Field
The invention belongs to the field of metabolic engineering, and more particularly relates to a method for obtaining genetically stable microalgae with target properties and a replication fidelity mechanism defect strain with increased mutation rate of Synechococcus PCC 7942.
Background
Cyanobacteria is a prokaryotic microorganism for plant type oxygen-releasing photosynthesis, and can convert carbon dioxide into various carbohydrates by utilizing solar energy. Cyanobacteria is considered as a very potential photosynthetic platform for bioenergy and bio-based chemicals because of its advantages of simple structure, rapid growth, convenient genetic engineering, etc. Through the introduction of exogenous metabolic pathways and the modification of the background metabolic network, targeted photosynthetic synthesis of dozens of natural and unnatural metabolites such as hydrocarbons, sugars, alcohols, ketones, acids, terpenes, and aliphatics has been achieved in cyanobacteria.
Based on economic and environmental considerations, the large-scale cultivation of cyanobacteria photosynthetic cell factories needs to be performed in an open outdoor environment. Cyanobacteria engineering algae strains are subjected to severe high temperature, high light stress and dynamic change of illumination-temperature-carbon source under the round-the-clock cycle condition far more than that under the laboratory condition; in addition, engineering algal strain cultivation is often required to be carried out under high-salt, high-acid/alkali conditions in order to control potential mixed bacterial contamination.
The natural physiological tolerance of cyanobacteria chassis cells commonly used for constructing photosynthetic cell factories at present is generally difficult to meet the requirement, and in a harsher large-scale culture system, biomass accumulation and product synthesis of engineering algae strains are greatly influenced, so that the large-scale application and popularization prospect of photosynthetic organism manufacturing technology are influenced.
The evolution engineering strategy is a method for modifying the physiological tolerance phenotype of microorganisms, and the characteristics of the optimized microorganisms are improved and optimized on a genome level by simulating a mutation-selection process in natural evolution. Currently, there are two factors that limit the biggest evolution engineering strategy, 1) how libraries accumulating large numbers of mutations are obtained; 2) How to screen libraries for mutants with stable genetic traits of interest.
Traditional laboratory adaptive evolution methods require long periods of time (typically months or even years) because of the extremely low spontaneous mutation rates of cells, which require long serial passages to accumulate genetic diversity. Mutation rates can be increased by combining physical or chemical means, however, the mutagens used can cause significant damage to cells, and the chemical mutagens used are often toxic to humans and the environment. In addition, physical or chemical mutagenesis requires repeated manual manipulations during long-term serial passages. Thus, a new approach is needed to construct mutant libraries.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for obtaining genetically stable microalgae with target traits, comprising the following steps:
s1: using genetic operation means to destroy the DNA replication fidelity mechanism of the starting microalgae, and obtaining replication fidelity mechanism defect strains with improved mutation rate;
s2: culturing and passaging the replication fidelity mechanism defect strain with the increased mutation rate under the high-temperature high-light stress condition to obtain a neutral super mutant strain library;
s3: applying selection pressure to the neutral hypermutant strain library, and screening to obtain a hypermutant evolutionary strain with target characters;
s4: recovering the fidelity mechanism with the target character to obtain the genetically stable microalgae with the target character.
The method overcomes the defect that the conventional physicochemical mutagenesis factors are adopted in the traditional evolution engineering strategy, and can obtain the super mutant strain library with more abundant genetic diversity in a short period, but the activity of the mutant strain in the library is not affected excessively severely. In addition, the supermutant library obtained in this aspect is progressive, and as subculturing continues, a more abundant library of supermutants can be obtained. Therefore, the method can shorten the period of obtaining the target character, and simultaneously does not need manual repeated mutagenesis operation, thereby saving manpower and material resources and being more environment-friendly (without chemical mutagens).
In a specific embodiment, the fidelity mechanism of the starting microalgae is disrupted by inactivating genes involved in the DNA replication fidelity process, or by overexpressing genes that make DNA replication error-prone.
In a specific embodiment, the gene involved in the DNA replication fidelity process is selected from the group consisting of mutS, mutL, dam, xth, uvrB, uvrC, mutY and mutM;
the gene that makes the DNA replication process error-prone is selected from the group consisting of one or more of umuC, umuD, recN and recA.
In a specific embodiment, the starting microalgae is synechocystis PCC7942 (Synechococcus elongatus PCC 7942) or synechocystis PCC 6803 (synechocystis sp. PCC 6803).
In a specific embodiment, the mutS gene is inactivated in the supermutant and comprises a recA gene overexpression cassette. The mutation rate of the mutant strain is obviously improved relative to the wild type, the background mutation rate is nearly 200 times of that of the wild type PCC7942, and the mutant strain can be used for constructing a large super mutant library with rich genetic diversity.
In a specific embodiment, mutagenesis is performed in S2 using non-lethal high temperature and/or high light stress to obtain a library of hypermutant strains with a greater genetic diversity. For example, 500-1500. Mu. Mol of photons/m are combined at 30-35 DEG C 2 Illumination of/s; or at 35-45deg.C, 100-500 μmol photons/m 2 Illumination of/s; a combination of high temperature (35-45 ℃) and high light conditions (500-1500. Mu. Mol photons/m) can also be used 2 S) these conditions can induce a significant increase in mutation rate of the hypermutant.
In one embodiment, the mutagenesis conditions are a temperature of 35-45℃and a light intensity of 500-1500. Mu. Mol photons/m 2 And/s. Mutation rate of the super mutant strain can be greatly improved by using high temperature and high light conditions in combination, and the mutation rate can reach tens to thousands times of wild mutation rate. By combining proper induction conditions such as temperature, light intensity and the like, the mutation rate of the super mutant strain can be improved to more than 8000 times of that of a wild type.
In a specific embodiment, the selection pressure employed in S3 is a stress condition sufficient to cause death of the starting microalgae. For example, the temperature, the light intensity, the pH, the ionic strength, etc. that cause death of the starting microalgae.
The invention also provides a replication fidelity mechanism defect strain with increased mutation rate of Synechococcus PCC7942, wherein the mutS gene is inactivated and comprises a recA gene overexpression frame. The mutation rate of the mutant strain is obviously improved relative to the wild type, the background mutation rate is approximately 200 times of that of the wild type PCC7942, and the mutation rate of the super mutant strain can be improved to more than 8000 times of that of the wild type by combining proper induction conditions such as temperature, light intensity and the like, so that the mutant strain can be used for constructing a large super mutant library with rich genetic diversity.
Drawings
FIG. 1 is a statistical plot of mutation rates of Synechococcus PCC7942 with a fidelity-related gene knocked out or overexpressed, wherein A is a single mutant and B is a multiple mutant.
FIG. 2 is a statistical plot of mutation rates of wild-type PCC7942 (A) and hypermutant HS84 (B) at different temperatures and light intensities.
FIG. 3 shows a photograph (A) of a plate coated with a library of hypermutant strains prepared from wild-type and hypermutant strains HS84, which plate has been cultured at high temperature and high light (a condition of high temperature and high light lethal to the wild-type) and a photograph (B) of an algal strain grown from the hypermutant strain, which plate has been streaked thereon, which plate has been cultured at high temperature and high light.
FIG. 4 shows a process (a) for obtaining a desired trait using hypermutant HS84, and growth curves of three obtained high temperature resistant and high light resistant algal strains cultured under different conditions.
FIG. 5 is a statistical graph of mutation rates of three high temperature resistant and high light resistant algal strains.
FIG. 6 is a photograph (A) of a plate coated with a library of hypermutant strains prepared from wild-type and hypermutant strains HS84 after culturing at high pH, and a photograph (B) of an algal strain grown from the hypermutant strain after streaking on the plate after culturing at high pH.
FIG. 7 is a statistical plot of the survival of wild-type cells treated with different doses of MMS (a), and a comparative statistical plot of the relative mutation rates of HS84 and 0.2% of MMS treated wild-type (b).
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
1. Presentation of hypermutation platform concept
Existing studies show that there is a fidelity mechanism for genome replication in microbial cells. These fidelity mechanisms have been widely studied, including: base selection, exo-correction, methylation-mediated DNA mismatch repair (MMR), DNA damage repair, and DNA cross-damage repair, among others. The efficient intracellular replication fidelity mechanism maintains the genetic stability of the cell, while also resulting in an extremely low mutation rate of the cell.
Therefore, we propose a new mutation-screening mode to obtain microorganisms with the desired trait, i.e. to construct hypermutation platforms.
The hypermutation platform refers to a chassis microorganism obtained by genetic manipulation and having a mutation of a fidelity mechanism related gene. Because of the problem of the fidelity mechanism in the chassis microorganism cells, the replication errors of the genome cannot be repaired in time, so that a large number of mutations are randomly introduced. On this basis, we propose a novel method for preparing genetically stable microorganisms with the desired trait. The method comprises the following steps:
s1: using genetic operation means to destroy the fidelity mechanism of the starting microorganism to obtain a super mutant;
s2: culturing and passaging the super mutant strain to obtain a neutral super mutant strain library;
s3: applying selection pressure to the neutral hypermutant library, and screening to obtain a hypermutant with the target property;
s4: recovering the fidelity mechanism of the strain with the target property to obtain the genetically stable microorganism with the target property.
In one embodiment, the fidelity mechanism of the starting microorganism is disrupted by knocking out genes involved in the fidelity of DNA replication, or by overexpressing genes that make DNA replication error-prone.
In the method, the super mutant strain is obtained by destroying the fidelity mechanism of microorganisms, and the super mutant strain cannot perform high fidelity on the replication of the genome in the culture and passage process, so that a large number of neutral mutations are introduced into the genome of offspring, and a super mutant strain library with a large number of accumulated neutral mutations can be obtained in a short period. After screening the super mutant strain with the target character, the genes related to the fidelity mechanism of the mutation inactivation in S1 are complemented back, the fidelity mechanism of the super mutant strain is recovered, and the cells with stable inheritance are obtained, so that the target character is inherited stably.
2. Construction of a hypermutant Strain Using Synechococcus elongatus PCC7942 (PCC 7942) as the starting algal Strain
The genome of PCC7942 was analyzed to find 8 genes involved in DNA replication fidelity (mutS (DNA mismatch repair protein mutS), mutL (DNA mismatch repair protein mutL), dam (DNA adenine methylase), xthA (deoxyribonuclease xha), uvrB (ABC exonuclease B subunit), uvrC (ABC exonuclease C subunit), mutY (a/G specific DNA adenine glycosylase) and mutM (formyl pyrimidine DNA glycosylase)), 4 genes that make DNA replication process error prone (umuC (DNA polymerase V), umuD (DNA polymerase V), recN (DNA repair protein recN) and recA (recombinant protein recA)). We have attempted to construct hypermutant strains by knocking out genes involved in fidelity, or over-expressing genes that make DNA replication error-prone, and combining these mutations.
The obtained hypermutant strain was cultured to obtain a library of hypermutant strains, and mutation rates of the control strain and the hypermutant strain were calculated using the rifampicin resistant colony generation frequency.
As a result, as shown in FIG. 1A, among the single mutant mutants, the relative mutation rates of HS1 (DeltauvrB), HS2 (DeltauvrC), HS10 (DeltamutS), HS7 (OE-recA; OE, over-expression) and HS25 (OE-umuDC) were increased as compared to WT, and the mutation rate of HS10 strain in which mutS gene was knocked out was highest (about 10-fold improvement over the wild type).
We selected and combined on the basis of the single mutant strains to construct multiple strainsHeavy mutant strains. Since the HS10 strain has the highest mutation rate, the ΔmutS mutation was used in combination with the other four mutations to produce double mutant strains HS83 (ΔmutS-. DELTA.uvrB), HS84 (ΔmutS:: P) cpcB560 -recA)、HS88(ΔmutS::P cpcB560 umuDC) and HS96 (Δmuts- Δuvrc). A double mutant strain HS91 (ΔmutS-. DELTA.dam) was also constructed. In addition, both umuDC and knockout of either uvrB or uvrC were also overexpressed, producing strain HS89 (DeltauvrB:: P), respectively cpcB560 umuDC) and HS90 (. DELTA.uvrC:: P cpcB560 -umuDC)。
As shown in FIG. 1B, HS84 carrying the ΔmutS+recA overexpression cassette showed a 130-fold increase in mutation rate. Overexpression of umuDC in synechococcus strains knocked out of uvrB (HS 89) or uvrC (HS 90) also further improved the mutagenesis effect, resulting in a 12-fold and 36-fold increase in mutation rate, respectively. At DeltatS:: P cpcB560 Combinations of further gene manipulations based on recA do not further increase mutation rate. Wherein the mutS has an amino acid sequence shown in SEQ ID NO. 1, a nucleic acid sequence shown in SEQ ID NO. 2, a recA amino acid sequence shown in SEQ ID NO. 3, and a nucleic acid sequence shown in SEQ ID NO. 4.
Since the mutation rate of HS84 is increased by 130 times or more relative to the wild type, and the mutant has a hypermutation property, HS84 was selected as a hypermutant.
3. Environmental induction factor
WT and hypermutant HS84 strains were set at initial OD 730 0.3 was inoculated into a column incubator containing 65mL of BG11 medium and aerated incubation was performed in an MC1000 incubator under controlled temperature and light conditions. As shown in FIG. 2, the mutation rates of both WT and HS84 strains were regulated by temperature and light intensity. It was observed that by increasing the illumination intensity from 400 to 1500. Mu. Mol photons/m 2 The WT mutation rate at 30℃was increased by 40% while at the same time increasing the temperature to 42℃further increased the mutation rate to 3-fold the initial level (FIG. 2A). A trend was also observed in the hypermutant HS84 showing a higher mutation rate with increasing temperature and light intensity, but this increase was more pronounced. 1000. Mu. Mol photons/m at 42 DEG C 2 After culturing HS84 at/s, mutation rate was increased by 1200-fold compared to WT (FIG. 2B).
We further optimized under the conditions of temperature and light, and can increase mutation rate of the hypermutant HS84 to be higher than 30 ℃ and 400 mu mol photons/m 2 The WT cultured per second was 8200 times higher.
4. Obtaining high-temperature high-light-tolerance type algae strain by using hypermutation platform
First, the strain of algae was subjected to mutagenesis culture (fig. 4 a): firstly, colonies on BG11 solid medium are scraped into a 50mL conical flask containing 30mL BG11 liquid medium (required antibiotics are added), and the flask is placed at a temperature of 30 ℃ and an illumination intensity of 30 mu mol photons/m 2 And/s (LED three-color light source), and culturing at 150rpm in a constant-temperature illumination shaking table.
After 4-5 days, the seed solution is inoculated into a 100mL column reactor containing 65mL BG11 liquid culture medium and expanded by air to be cultured as zero-order seed solution, wherein the culture condition is 30 ℃ and 250 mu mol photons/m 2 S (LED trichromatic lamp source). OD of seed liquid to be zero level 730 When reaching 2.0-3.0, the initial OD is used 730 Air was introduced into a 100mL column reactor containing 65mL BG11 liquid medium for expanded culture at 30℃and 250. Mu. Mol photons/m 2 /s (LED trichromatic light source), when OD 730 When reaching 2.0-3.0, the seed liquid is used as first-stage seed liquid. Centrifuging the primary seed solution to collect algae cell precipitate and re-suspending with fresh BG11 medium to initial OD 730 Inoculating 0.3 to column reactor, placing in MC1000-8 channel algae culture system for culturing at 42deg.C under 1000 μmol photons/m 2 /s (LED cool white light), when OD 730 When reaching 2.0-3.0, the seed liquid is used as secondary seed liquid for subsequent screening.
Centrifuging and concentrating the secondary seed solution 10 9 The individual algal cells were plated on non-anti BG11 solid plates and three runs were set up in parallel. The plate was placed at a temperature of 44℃and 510. Mu. Mol photons/m 2 Culturing under light intensity above/s (LED warm white light).
As shown in FIG. 3A, after treatment under the same conditions, the WT-coated plates did not survive the algae cells, but the hypermutant HS 84-coated plates formed a large number of single colonies and the single algae colonies were streaked to new BG11Further under the same high temperature and high light conditions as the screening conditions (44 ℃ C., 510. Mu. Mol photons/m) 2 S (LED warm white)), and as a result, as shown in fig. 3B, algal strains can still grow. The hypermutant library prepared by HS84 can be seen to contain more abundant genetic diversity than the wild type, which provides abundant genetic resources for screening algae strains with target characters.
Three algal strains were selected from the above colonies, the mutated mutS gene was repaired, and the recA overexpression cassette was removed to obtain HS121, HS122 and HS123, which were subjected to growth evaluation. The results are shown in FIG. 4, under normal conditions (30 ℃ C. And 500. Mu. Mol photons/m 2 S) a slight growth advantage was detected for the three clades compared to WT (fig. 4 b); furthermore, the temperature was raised to 42 ℃ at the same light intensity, and the growth of all strains was improved, whereas the evolved strain showed a significant growth advantage compared to the wild type (fig. 4 c). At high temperature (45 ℃ C. And 500. Mu. Mol photons/m) 2 S) and highlights (42 ℃ C. And 1500. Mu. Mol photons/m 2 /s) the strain can still grow rapidly, whereas the WT strain can hardly survive (fig. 4d and e). Under more severe conditions (FIG. 4f,42 ℃ C. And 2500. Mu. Mol photons/m) 2 S; FIG. 4g,45℃and 1500. Mu. Mol photons/m 2 S) the clades can still survive and grow, showing a significantly improved high temperature and high light adaptability. Furthermore, the improved high temperature and high light tolerance of the three clades remained stable throughout long term storage and culture.
The mutation rates of the three algae strains are detected, and as shown in figure 5, the relative mutation rates of the evolution strains are restored to normal level, which shows that the repair experiment successfully repairs the fidelity mechanism in the Synechococcus cells, and ensures the stability of phenotype and genotype.
The hypermutation platform is very efficient in obtaining libraries with high-abundance genetic diversity, which is proved by only two weeks from mutagenesis to screening to obtain the high-temperature and high-light resistant algae strain.
5. Obtaining high pH tolerant algal strains using a hypermutation platform
Firstly, the WT and HS84 algal strains were cultured according to the mutagenesis culture flow described in 4. Centrifuging and concentrating the secondary seed solution 10 9 Individual algal cells were plated on non-anti-BG 11 solid plates at pH 12.75. As a result, as shown in FIG. 6, all wild type strains died, while HS84 strain developed some single colonies. The strain was streaked onto a non-anti-BG 11 solid plate at pH 11.2 with WT as a control. HS124, HS125, HS126, HS127, HS128, HS129 are evolved algal strains selected from HS84 algal strains under high pH stress conditions, which can survive under high pH conditions while the WT dies.
And (3) carrying out complementation on the strains, respectively, carrying out complementation on the knocked mutS genes, and removing recA overexpression frames to obtain HS130, HS131, HS132, HS133 and HS134 (respectively, carrying out complementation on the strains HS124, HS125, HS126, HS127 and HS 129). The strain was streaked on a high pH plate (pH 11.2) and WT was used as a control, which indicated that the strain of Hui-Bulgaria was still tolerant to high pH conditions.
6. Evolution of high temperature and high light tolerant algal strains under the same procedure using MMS chemical mutagenesis method
We have evolved the high temperature, high light tolerance phenotype of algal strains using Methyl Methylsulfonate (MMS) as a chemical mutagen and compared the hypermutation approach with classical chemical mutagenesis approaches.
We first determined the MMS dose used for 90% mortality of the algal strains. PCC7942 wild-type cells were treated with MMS at different doses (0,0.2% (v/v), 0.4% (v/v), 0.6% (v/v), 8% (v/v), 1% (v/v), 2% (v/v), 3% (v/v). The specific treatment method is to collect cells in mid-log phase, re-suspend the cells in fresh BG11, and adjust the concentration to 10 per milliliter 8 Individual cells. To this, MMS was added at the corresponding concentration, and after incubation for 1min, the cells were washed twice with fresh BG 11. Then diluting the cells to obtain 10 4 Individual cells were plated on anti-solid BG11 free medium. The number of colonies grown on the plates was counted after about one week, and the survival rate (mortality=1-survival rate) was obtained by dividing the cell amount of the coated plates. The results indicated that 90% mortality was observed in samples treated with 2% (v/v) MMS (fig. 7 a) (the dose of chemical mutagen that resulted in 90% mortality was typically applied to random mutagenesis of microorganisms).
Will 2% (v-v) MMS-treated cells at initial OD 730 0.2 was inoculated into fresh BG11 medium at 30℃with 50. Mu. Mol photons/m 2 Under/s conditions (shaking culture) and at 42℃800. Mu. Mol photons/m 2 Recovery culture was performed under the conditions of/s (aeration culture in MC1000 incubator). After culturing to the logarithmic phase, the mutation rate was evaluated in the same manner as described above. The results showed that after recovery culture in fresh BG11, the mutation rate of the strain treated with 2% (v/v) MMS was about 30-fold higher than that of the untreated wild-type strain (FIG. 7 b), whereas the mutation rate of the strain of HS84 strain, which was not mutagenized by high temperature high light stress culture, was nearly 200-fold higher than that of the wild-type strain, which was far higher than that of the former strain.
We also used the same evolution protocol and screening method as described in 4 above to screen for high temperature and high light tolerant algal strains using MMS mutagenesis. The culture methods of the primary seed liquid and the secondary seed liquid in the earlier stage are the same as those described in 4. Subjecting the mid-log secondary seed solution to mutagenesis treatment with 2% (v/v) MMS, and concentrating by centrifugation as well for 10 9 The individual algal cells were plated on non-anti BG11 solid plates and three runs were set up in parallel. The plate was placed at a temperature of 44℃and 510. Mu. Mol photons/m 2 Culture was performed under light intensity conditions above/s (LED warm white), but high temperature and high light tolerance colonies were not obtained. Therefore, in view of the advantages of genome mutagenesis intensity and biosafety (chemical mutagens are generally considered to be highly oncogenic), the hypermutation system developed in this study is a beneficial complement to the existing cyanobacteria mutagenesis method and may improve the convenience and environmental friendliness of the strain improvement process.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (10)

1. A method for rapidly obtaining genetically stable microalgae with a target trait, comprising the steps of:
s1: using genetic operation means to destroy the fidelity mechanism of the starting microalgae, and obtaining a replication fidelity mechanism defect strain with improved mutation rate;
s2: culturing and passaging the replication fidelity mechanism defect strain with the increased mutation rate under the high-temperature high-light stress condition to obtain a neutral super mutant strain library;
s3: applying selection pressure to the neutral hypermutant strain library, and screening to obtain a hypermutant evolutionary strain with target characters;
s4: recovering the fidelity mechanism of the evolution strain with the target character to obtain the genetically stable microalgae with the target character.
2. The method of claim 1, wherein the fidelity mechanism of the starting microalgae is disrupted by inactivating genes involved in DNA replication fidelity or overexpressing genes that make DNA replication error prone.
3. The method of claim 2, wherein the gene involved in DNA replication fidelity is selected from the group consisting of mutS, mutL, dam, xth, uvrB, uvrC, mutY and mutM;
the gene that makes the DNA replication process error-prone is selected from the group consisting of one or more of umuC, umuD, recN and recA.
4. The method of claim 1, wherein the starting microalgae is cyanobacteria.
5. The method of claim 4, wherein the starting microalgae is synechococcus PCC7942 (Synechococcus elongatus PCC 7942) or Synechocystis PCC 6803 (Synechocystis sp. PCC 6803).
6. The method of claim 5, wherein the mutS gene is inactivated in the replication fidelity machinery deficient strain and comprises a recA gene overexpression box.
7. The method of claim 5, wherein the mutagenesis is performed in S2 using non-lethal high temperature and/or high light conditions to obtain a neutral hypermutant library with a greater genetic diversity.
8. The method according to claim 7, wherein the mutagenesis is carried out at a temperature of 30-45℃and a light intensity of 100-1500. Mu. Mol photons/m 2 /s。
9. The method of any one of claims 1-8, wherein the selection pressure employed in S3 is a stress condition sufficient to cause death of the starting microalgae.
10. A replication fidelity mechanism defective strain with increased mutation rate of synechococcus PCC7942, wherein the mutS gene is inactivated and comprises a recA gene overexpression box in the replication fidelity mechanism defective strain with increased mutation rate.
CN202310163031.8A 2023-02-24 2023-02-24 Method for obtaining genetically stable microalgae with target properties and ultra-mutant strain Pending CN116855513A (en)

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