A high temperature and high light tolerant engineered cyanobacteria based on shikimate kinase and preparation method thereof

Abstract

The present invention relates to a method for constructing an engineered cyanobacteria that tolerates high temperature and high light, including the step of increasing the expression amount of shikimate kinase in the cyanobacteria; and also relates to an engineered cyanobacteria that grows rapidly under high temperature and high light conditions constructed on the basis of the engineered cyanobacteria that tolerates high temperature and high light and a construction method thereof. The engineered cyanobacteria constructed by the present invention can grow rapidly and stably under high temperature and high light conditions that the wild type cannot tolerate, and solves the problem of decreased or unstable target product yield caused by high temperature and high light stress in outdoor large-scale cultivation. In addition, whether under normal temperature and normal light conditions or under high temperature and high light stress conditions, the engineered cyanobacteria of the present invention has a significantly higher growth rate than its wild type, can accumulate more biomass, synthesize more glycogen, and can accumulate more protein under high temperature and high light conditions, so the chassis bacteria as a "cell factory" have the potential to produce target products more efficiently.

Description

Technical Field

[0001] The invention belongs to the field of cyanobacteria genetic engineering, and more particularly, relates to an engineered cyanobacteria tolerant to high temperature and high light and a preparation method thereof. Background Art

[0002] Cyanobacteria are a type of prokaryotic microorganism that performs plant-type oxygenic photosynthesis and can use solar energy to convert carbon dioxide into various carbohydrates. Cyanobacteria are considered to be a promising platform for photosynthetic synthesis of bioenergy and bio-based chemicals due to their simple structure, rapid growth, and convenient genetic modification. Through the introduction of exogenous metabolic pathways and the modification of background metabolic networks, the directional photosynthetic synthesis of dozens of natural and non-natural metabolites such as hydrocarbons, sugars, alcohols, ketones, acids, terpenes, and aliphatic compounds has been achieved in cyanobacteria.

[0003] Based on overall economic and environmental considerations, large-scale cultivation of cyanobacterial photosynthetic cell factories needs to be carried out in an outdoor open environment. In outdoor environments, light intensity and temperature change in a periodic rhythm, and their peak values ​​far exceed the suitable temperature for cyanobacterial culture; moreover, the dynamic changes of temperature and light in outdoor environments have a high degree of overlap, and the peak stage of ambient temperature is often the strongest natural light. The excessive light energy brought by strong light will disrupt the redox balance in cells, and the accumulated reactive oxygen will destroy key cellular components, such as nucleic acids, lipids, pigments and proteins, leading to decreased growth and productivity, and even cell death. High temperature will strongly affect the chemical composition of cells and the fluidity of cell membranes. The stability of proteins and enzymes will be destroyed, leading to cellular metabolic disorders.

[0004] Therefore, improving the adaptability of chassis cells to high light and high temperature is of great significance for achieving stable and efficient synthesis and accumulation of biomass and target products under large-scale conditions. Previous reports have shown that the introduction of the C252Y mutation on the α subunit of ATP synthase in PCC7942 can make the algae tolerant to high temperature and high light. The method of this patent can improve the high temperature and high light tolerance of the algae by upregulating the expression of shikimate kinase in the cells. At the method level, a simple operation such as integrating the shikimate kinase encoding gene expression cassette controlled by a strong promoter into the genome or a free plasmid can improve the high temperature and high light tolerance of the algae, which is simpler and more adaptable than introducing point mutations in the genome. In addition, this method can be applied not only to Synechococcus PCC 7942, but also to Synechocystis PCC 6803. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for preparing engineered cyanobacteria that tolerates high temperature and high light, comprising the step of increasing the expression level of shikimate kinase in the cyanobacteria.

[0006] In a specific embodiment, the cyanobacterium is Synechococcus (eg, Synechococcus sp. PCC 7942) or Synechocystis sp. (eg, Synechocystis sp. PCC 6803).

[0007] Increasing the expression of shikimate kinase can be achieved in a variety of ways. In the embodiments of the present invention, the expression of shikimate kinase is increased by introducing a shikimate kinase gene overexpression cassette into the cyanobacteria, and by mutating the non-coding region of the shikimate kinase in the cyanobacteria. However, the above method is not for the purpose of exhaustive enumeration. Using other methods that are currently available or will be available in the art to increase the expression of shikimate kinase can also achieve the purpose of improving the high temperature and high light tolerance of cyanobacteria, which should be included in the protection scope of the present invention.

[0008] In a specific embodiment, the cyanobacterium is Synechococcus PCC 7942, and the non-coding region of the shikimate kinase is mutated: the G at position -28 upstream of the synpcc7942_0894 gene is mutated to A (C905470T), or the -35 position upstream of the synpcc7942_0894 gene is mutated from C to T (G905452A,), or the base at position -53 is mutated from G to A (C905445T).

[0009] In a specific embodiment, the cyanobacteria is Synechococcus sp. PCC 7942, and the shikimate kinase supergene expression cassette includes a shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO: 2 and a strong promoter regulating the shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO: 2. The strong promoter can use a promoter that can be strongly expressed constitutively in the target strain, or a promoter that can be induced to strongly express in the target strain. In Synechococcus sp. PCC 7942, such promoters include but are not limited to constitutive strong promoters such as cpcB1 promoter, cpcB560 promoter, rbcL promoter, or inducible strong promoters such as lac promoter, trc promoter, etc.

[0010] In a specific embodiment, the cyanobacteria is Synechocystis PCC 6803, and the shikimate kinase gene overexpression cassette includes a shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO: 4 and a strong promoter regulating the shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO: 4. The strong promoter can use a promoter that can be constitutively strongly expressed in the target strain, or a promoter that can be induced to strongly express in the target strain. In Synechococcus PCC6803, such promoters include but are not limited to constitutive strong promoters such as cpcB1 promoter, cpcB560 promoter, rbcL promoter, or inducible strong promoters such as lac promoter, trc promoter, etc.

[0011] The present invention also provides an engineered cyanobacteria that tolerates high temperature and high light constructed by the above method. The engineered cyanobacteria not only has a faster growth rate than the wild type under normal temperature and normal light conditions, but can also grow at a faster rate under high temperature and high light stress conditions that the wild type cannot tolerate, while accumulating more biomass, and the cell has a stronger ability to synthesize glycogen, and the protein synthesis ability is also enhanced. This makes the algae strain with upregulated shikimate kinase become a photosynthetic cell factory with more potential.

[0012] The present invention also provides a method for preparing cyanobacteria that grows rapidly under high temperature and high light, comprising introducing a point mutation of lysine at position 287 of IMP dehydrogenase into arginine, or a point mutation of valine at position 385 of GMP synthase into alanine, or a point mutation of proline at position 164 of translation elongation factor EF-Tu into leucine into the above-mentioned engineered cyanobacteria that tolerates high temperature and high light.

[0013] In a specific embodiment, the starting cyanobacterium is Synechococcus PCC 7942, the amino acid sequence encoded by the gene encoding IMP dehydrogenase is shown in SEQ ID NO:6, the amino acid sequence encoded by the gene encoding GMP synthase is shown in SEQ ID NO:8, and the amino acid sequence encoded by the gene encoding translation elongation factor EF-Tu is shown in SEQ ID NO:10.

[0014] The present invention also provides an engineered cyanobacteria constructed by the above method and capable of growing rapidly under high temperature and high light.

[0015] The present invention also provides the use of the above-mentioned engineered cyanobacteria in biosynthesis.

[0016] The engineering cyanobacteria of the present invention structure can grow rapidly and stably under the condition of high temperature and high light, and has solved the problem of target product output decline or unstable caused by high temperature and high light stress in outdoor large-scale cultivation. In addition, the engineering cyanobacteria of the present invention all has significantly higher growth rate and faster biomass accumulation speed than its wild type under high temperature and high light and conventional culture conditions, therefore has the potentiality of producing target product more efficiently as the chassis bacteria of "cell factory". For example, the engineering cyanobacteria of the present invention can be used for producing materials such as carbohydrates, proteins, alcohols, organic acids, hydrocarbons needed directly or after further genetic manipulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the structure of plasmids pHS58, pHS59 and pHS60.

[0018] Figure 2The growth curves of strain HS199 at different temperatures and light intensities, where A is the growth curve of strain HS199 at 30-45℃ / 500μmol l photons m -2 s -1 and 45℃ / 1500μmol l photons m -2 s -1 B is the growth curve of strain HS199 at 42℃ / 500-2500μmol l photons m -2 s -1 The growth curve below.

[0019] Figure 3 The immunoblot images are of shikimate kinase in cells of wild-type PCC 7942 (WT) and strain HS199 cultured at normal temperature and light intensity (NLT) and high temperature and high light intensity (HLT).

[0020] Figure 4 Schematic diagram of the structure of plasmid pHS67.

[0021] Figure 5 For strains HS199 and HS222, 500 μmol photons m at 30 °C -2 s -1 and 42℃ / 1500μmo lphotons m -2 s -1 The growth curve below.

[0022] Figure 6 The photos are of cultures of wild-type PCC 6803 (WT6803) and strain HS237 after 48 h of cultivation at different temperatures and light intensities.

[0023] Figure 7 These are the test results of dry weight, biomass content, glycogen content and protein content of HS199 algae strain.

[0024] Figure 8 Figure 2 shows the growth curves of strains HS228, HS230 and HS241 cultured under NLT, where HS229 / HS231 / HS199 are control strains.

[0025] Fig. 9 Figure 2 shows the growth curves of strains HS228, HS230 and HS241 cultured under HLT, where HS229 / HS231 / HS199 are control strains. DETAILED DESCRIPTION

[0026] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0027] In our work, our team constructed a hypermutant strain of Synechococcus PCC 7942 (a strain with higher mutation ability than the wild-type Synechococcus PCC 7942), and used the hypermutant strain to construct a mutant library, from which we screened some mutant strains with high temperature and high light resistance traits. Through research on these mutant strains, we found that some gene or site mutations can lead to high temperature and high light resistance traits.

[0028] 1. Mutation of non-coding region upstream of shikimate kinase coding frame

[0029] Our research found that the mutation sites of three mutant strains with high temperature and high light tolerance were located in the non-coding region upstream of the coding frame of shikimate kinase (the coding gene is synpcc7942_0894, the nucleic acid sequence is shown in SEQ ID NO:1, and the encoded amino acid sequence is shown in SEQ ID NO:2). The bases at positions -28, -35, and -53 were mutated from G to A (C905470T, NC2-2), from C to T (G905452A, referred to as NC2-1), and from G to A (C905445T, NC2-3). Since the gene is on the negative sense strand, the bases outside the above brackets are the bases in the gene coding direction, and the bases in the brackets are marked with their positions in the genome and the corresponding bases and mutations.

[0030] In order to verify that the above mutations can lead to tolerance to high temperature and high light, we constructed point mutation plasmids of NC2-1, NC2-2 and NC2-3 (without resistance fragment screening). The construction method is as follows: PCC 7942 genome was used as a template to amplify the upstream non-coding region synpcc7942_0894 UP and synpcc7942_0894 gene fragments, and the fragments were connected to the backbone plasmid pUC19 by seamless cloning technology. Then, the bases at positions -35, -28, and -53 upstream of synpcc7942_0894 were mutated from C to T, G to A, and G to A in the plasmid by quick change, respectively, to obtain plasmids pHS58, pHS59 and pHS60 (plasmid maps are shown in Figure 2). Figure 1 shown).

[0031] pHS58, pHS59 and pHS60 were introduced into PCC 7942, and point mutation strains HS199, HS200 and HS201 were screened from the transformants. Sequencing confirmed that the target point mutations occurred at the corresponding positions of the three strains. The three strains were cultured at 42℃ / 1500μmol l photons m -2 s -1 The results showed that the three strains grew well.

[0032] Take HS199 as an example. Figure 2 As shown in the figure, under normal temperature and light conditions, HS199 grows faster than the wild type. It can also grow rapidly under conditions that are lethal to the wild type PCC 7942 or even higher temperatures and light intensities. This shows that HS199 not only grows faster than PCC 7942, but is also extremely tolerant to high temperatures and high light.

[0033] It can be seen from this that the bases at positions -35, -28, and -53 upstream of synpcc7942_0894 mutated from C to T, G to A, and G to A, respectively, which can indeed cause the strain to have high temperature and high light tolerance.

[0034] 2. Mechanism of HLT resistance caused by point mutations

[0035] Since the three mutation sites are located upstream of the coding frame of shikimate kinase (coding gene synpcc7942_0894) and very close to the coding frame, located in the promoter region, we speculate that the above mutations may have caused changes in the expression level of shikimate kinase, thereby affecting the strain's tolerance to high temperature and high light. By comparing the transcription level and protein level of shikimate kinase in the three strains with the wild type, it was found that the expression of shikimate kinase in the three strains was upregulated. Figure 3 As shown, at normal temperature (NLT, 30℃ / 500μmol l photons m -2 s -1 ) cultured in HS199 strain (lane NC2-1), the expression level of shikimate kinase was much higher than that of wild type, and the expression level of shikimate kinase was significantly higher in high temperature and high light (HLT, 42℃ / 1500-2000μmo lphotons m -2 s -1 ), the expression of shikimate kinase was similar to that of NLT, indicating that the mutation led to constitutive upregulation of shikimate kinase expression. Similar results were observed at the other two mutation sites.

[0036] We constructed a shikimate kinase overexpression plasmid: synpcc7942_0894 was linked to the constitutive strong promoter P cpcB560 Downstream, a shikimate kinase superexpression cassette was constructed, and the shikimate kinase superexpression cassette was inserted between the upstream and downstream homology arms of the neutral site NS2, and a chloramphenicol resistance fragment was used as a screening marker. Then, the above fragment was integrated into the pUC19 backbone to obtain the plasmid pHS67. The plasmid map is shown in Figure 4 pHS67 was transferred into wild-type PCC 7942, and homozygous positive transformants were selected to obtain strain HS222. HS222 was cultured under NLT and HLT, respectively, and the results were shown in Figure 5As shown in the figure, the growth curves of HS222 under NLT and HLT were similar to those of HS199. Under HLT, the growth rate of HS222 was slightly better than that of HS199. This indicates that overexpression of shikimate kinase in situ by point mutation or introduction of shikimate kinase overexpression cassette can make Synechococcus PCC 7942 tolerant to high temperature and high light.

[0037] To determine whether the strategy of overexpressing shikimate kinase to improve the high temperature and high light tolerance and growth rate of algae can be extended to other cyanobacteria, we amplified the shikimate kinase gene (sll1669, nucleic acid sequence as shown in SEQ ID NO:3, encoded amino acid sequence as shown in SEQ ID NO:4) from the genome of Synechocystis PCC 6803 and linked it to the constitutive strong promoter P cpcB560 Downstream, the shikimate kinase superexpression cassette from PCC 6803 was constructed, and the superexpression cassette was inserted between the upstream and downstream homologous arms of the neutral site slr0168, and the chloramphenicol resistance fragment was used as a selection marker. Then the above fragment was integrated into the pUC19 backbone to obtain the plasmid pHS84. pHS84 was transferred into the wild-type PCC 6803, and the homozygous positive transformants were selected to obtain the strain HS237. HS237 was cultured at different temperatures and light intensities. The results are shown in Figure 2. Figure 6 As shown in the figure, HS237 has better tolerance to high temperature and high light than the wild type PCC 6803, and HS237 grows well under conditions that are lethal to the wild type. This shows that in cyanobacteria, overexpression of shikimate kinase has a certain universality in improving the tolerance of microalgae to high temperature and high light.

[0038] 3. Physiological characteristics of shikimate kinase overexpression strain

[0039] The above results show that the introduction of NC2-1, NC2-2, NC2-3 mutations or the introduction of shikimate kinase overexpression cassettes into Synechococcus PCC 7942 cells can make the algae strain tolerant to high temperature and high light by upregulating the expression of shikimate kinase. We further characterized the biomass content, glycogen content and protein content of the shikimate kinase overexpression strain. 730 0.05 inoculation, at 30℃ / 500μmol photons m -2 s -1 and 41°C / 1500 μmol photons m -2 s -1 The cells were cultured to mid-logarithmic phase under the same conditions (3% carbon dioxide aerated in MC1000 incubator).

[0040] First, the biomass content of the algae strain was detected and calculated. After centrifugation at 10000g for 15 minutes, the cells were collected, washed with ddH2O, and freeze-dried in a vacuum freeze dryer. After freeze-drying, the weight was subtracted from the weight of the centrifuge tube (weighed and recorded in advance) to obtain the dry powder weight. The weight of the algae powder was divided by the volume of the sampled algae liquid to obtain the biomass content (mg / mL), and the biomass content was divided by the cell concentration of the algae liquid at the time of sampling to obtain the cell dry weight (mg / mL / OD 730 The results show that under high temperature and high light conditions (41℃ / 1500μmo lphotons m -2 s -1 ) and normal temperature and light conditions (30℃ / 500μmol l photons m -2 s -1 ), the biomass content and accumulation rate of the shikimate kinase overexpression strain HS199 were significantly higher than those of the wild type, and the cell dry weight of HS199 under high temperature and high light conditions was also higher than that of WT ( Figure 7 A and B).

[0041] We also tested the glycogen content according to the following process. The cells were collected after centrifugation at 10,000 g for 15 minutes. They were washed three times with ddH2O, resuspended in 30% (w / v) KOH, heated at 95°C for 2 hours, and finally ice-cold ethanol was added to a final concentration of 70%-75% (v / v). The mixture was cooled at -20°C overnight and then centrifuged at 10,000 g for 15 minutes to collect the glycogen particles. The glycogen particles were washed twice in succession with 70% ethanol (v / v) and 98% ethanol (v / v), respectively, and then dried using vacuum centrifugation. The resulting glycogen was suspended in 100 mM sodium acetate and digested with amyloglucosidase (Novozymes). The amount of glucose produced in the glycogen solution was then measured using a sucrose / D-glucose assay kit (Megazyme, Ireland). The results showed that regardless of the high temperature and high light conditions (41°C / 1500 μmol lphotons m -2 s -1 ) or normal temperature and light conditions (30℃ / 500μmol l photons m -2 s -1 ),the glycogen content of shikimate kinase overexpression strain HS199 was significantly higher than that of wild type,9 times and 3 times that of wild type respectively. Figure 7 C).

[0042] In addition to dry weight and glycogen content, we also tested the protein content of the algae. 730 0.05 inoculum, at 30°C / 500 μmol photons m -2 s -1After culturing to the mid-logarithmic phase under the same conditions, samples were collected for testing and the results were labeled as NLNT-WT or NLNT-HS199. 730 0.05 inoculum, at 42°C / 2000 μmol photons m -2 s -1 After culturing to the mid-logarithmic phase under the same conditions, samples were taken for testing and the results were labeled as HLHT-HS199. 730 0.05 inoculum, at 30°C / 500 μmol photons m -2 s -1 After culturing to mid-logarithmic phase, the cells were transferred to 42°C / 2000 μmol l photons m -2 s -1 The samples were taken for testing after 12 hours of treatment under the conditions, and the results were marked as HLHT-WT. The protein content was determined by the Kjeldahl method. 0.2 g of freeze-dried sample powder, 1.0 g of CuSO4, 3.0 g of K2SO4, and 10.0 mL of H2SO4 were added to the digestion tube. In a graphite digestion apparatus, the mixture was heated in the following order: 200°C for 20 minutes, 300°C for 20 minutes, and 420°C for 90 minutes. After cooling to 25-30°C, the mixture was analyzed using an automatic Kjeldahl nitrogen analyzer. Finally, the protein content was calculated by multiplying the total N content by a conversion factor of 6.25. The results showed that the protein content of HS199 under high light culture was significantly higher than that of the wild type ( Figure 7 D).

[0043] 3. Double mutant algae strains that grow rapidly under high temperature and high light

[0044] We performed some mutations based on the shikimate kinase overexpression mutant and found that the double mutant had significantly better tolerance to high temperature and high light. We constructed the following point mutation plasmids:

[0045] NC2-1 resistant point mutation plasmid pHS50: PCC 7942 genome was used as a template to amplify the upstream non-coding region of synpcc7942_0894 and the synpcc7942_0894 gene fragment (synpcc7942_0894UP-synpcc7942_0894), as well as the downstream fragment of synpcc7942_0894. The above fragments and the spectinomycin-resistant fragment were connected to the backbone plasmid pUC19 by seamless cloning technology, and then the NC2-1 mutation was generated in the plasmid by quick change.

[0046] Point mutation plasmid pHS64 of IMP dehydrogenase: The coding gene of IMP dehydrogenase in PCC 7942 is synpcc7942_1831 (the nucleic acid sequence is shown in SEQ ID NO:5, and the encoded amino acid sequence is shown in SEQ ID NO:6). In order to mutate the lysine at position 272 of IMP dehydrogenase into arginine, we constructed a point mutation plasmid pHS64: the synpcc7942_1831 gene (1000bp before the stop codon) was amplified using the PCC 7942 genome as a template, synpcc7942_1831 DOWN, the above fragment and the chloramphenicol resistance fragment were connected to the backbone plasmid pUC19 by seamless cloning technology, and then the 815th base in synpcc7942_1831 was mutated from A to G (A815G) in the plasmid by quick change.

[0047] GMP synthase point mutation plasmid: The coding gene of GMP synthase in PCC 7942 is synpcc7942_0189 (the nucleic acid sequence is shown in SEQ ID NO:7, and the encoded amino acid sequence is shown in SEQ ID NO:8). In order to mutate the valine at position 385 of GMP synthase into alanine, we constructed a point mutation plasmid pHS62: the synpcc7942_0189 gene (1000bp before the stop codon) was amplified using the PCC 7942 genome as a template, synpcc7942_0189 DOWN, the above fragment and the chloramphenicol resistance fragment were connected to the backbone plasmid pUC19 by seamless cloning technology, and then the 1154th base in synpcc7942_0189 was mutated from T to C (T1154C) in the plasmid by quick change.

[0048] pHS50 was transformed into wild-type PCC 7942, and strain HS190 with the NC2-1 mutation was obtained by sequencing screening.

[0049] Plasmid pHS64 was transferred into strain HS190 to screen strains with dual resistance to spectinomycin and chloramphenicol. Since the point mutation requires the double exchange region to cover the point mutation position, if the point mutation position is not in the double exchange region, it is possible to obtain a strain in which only the resistance fragment is inserted into the genome. Therefore, through sequencing, we obtained two strains with dual resistance, strain HS228 with NC2-1 mutation and synpcc7942_1831 intragenic mutation (A815G) and strain HS229 with only NC2-1 mutation (which can be used as a control strain for HS228).

[0050] Plasmid pHS62 was transferred into strain HS190 to screen for strains with dual resistance to spectinomycin and chloramphenicol. Similarly, we obtained two strains with dual resistance, strain HS230 with NC2-1 mutation and intragenic mutation (T1154C) of synpcc7942_0189, and strain HS231 with only NC2-1 mutation (which can be used as a control strain for HS230).

[0051] In addition, during the research process, our team obtained a strain HS241 with high temperature and high light tolerance by evolutionary screening of the PCC 7942 super mutant strain. Whole genome sequencing found that there were only two mutations between the genome of this strain and the wild-type PCC7942: the NC2-1 mutation and the C491 T mutation in the translation elongation factor EF-Tu encoding gene synpcc7942_0884 (the nucleic acid sequence is shown in SEQ ID NO:9, and the encoded amino acid sequence is shown in SEQ ID NO:10). This mutation causes the proline 164 in the coding frame to mutate to leucine.

[0052] The above strains were cultured under NLT, and the growth curves did not show significant differences ( Figure 8 ). However, when cultured under HLT, the above double mutant strains not only survived, but also had significantly faster growth rates than HS199 ( Fig. 9 Similar results were obtained by introducing the above three point mutations into other shikimate kinase overexpression strains (including NC2-2, NC2-3 point mutants and mutants with shikimate kinase overexpression cassette inserted in the neutral site).

[0053] The above experiments show that the tolerance of cyanobacteria, including Synechococcus and Synechocystis, to high temperature and high light can be improved by increasing the expression of shikimate kinase. Introducing some point mutations on the basis of increasing the expression of shikimate kinase can greatly increase the growth rate of the strain under high temperature and high light.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an engineered cyanobacterium that grows fast and tolerates high temperature and high light, characterized in that: Comprising the steps of increasing the expression of shikimate kinase in cyanobacteria; The cyanobacteria are Synechococcus or Synechocystis.

2. The method according to claim 1, characterized in that The cyanobacteria are Synechococcus PCC 7942 or Synechocystis PCC 6803.

3. The method according to claim 2, characterized in that The shikimate kinase gene overexpression cassette is introduced into the cyanobacterium.

4. The method according to claim 3, characterized in that The cyanobacterium is Synechococcus PCC 7942, and the shikimate kinase gene overexpression frame includes a shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO: 2 and a strong promoter regulating the shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO:

2.

5. The method according to claim 3, characterized in that: The cyanobacterium is Synechocystis PCC 6803, and the shikimate kinase gene overexpression cassette includes a shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO: 4 and a strong promoter regulating the shikimate kinase gene encoding an amino acid sequence as shown in SEQ ID NO:

4.

6. The method according to claim 2, characterized in that The expression level of shikimate kinase is increased by mutating the non-coding region of shikimate kinase in the cyanobacterium, wherein the cyanobacterium is Synechococcus PCC 7942, and the non-coding region of shikimate kinase is mutated to synpcc7942_0894 The G at position -28 upstream of the gene mutates to A, or synpcc7942_0894 The -35th position upstream of the gene mutates from C to T, or the -53rd base mutates from G to A.

7. An engineered cyanobacterium that grows fast and tolerates high temperature and high light, characterized in that: It is prepared by the method according to any one of claims 1 to 6.

8. A method for preparing an engineered cyanobacterium that grows rapidly under high temperature and high light, characterized in that: The invention comprises introducing a point mutation of lysine at position 272 of IMP dehydrogenase into arginine, or a point mutation of valine at position 385 of GMP synthase into alanine, or a point mutation of proline at position 164 of translation elongation factor EF-Tu into leucine into the engineered cyanobacteria tolerant to high temperature and high light as described in claim 7, wherein the starting cyanobacteria is Synechococcus PCC 7942, the amino acid sequence encoded by the gene encoding IMP dehydrogenase is shown in SEQ ID NO:6, the amino acid sequence encoded by the gene encoding GMP synthase is shown in SEQ ID NO:8, and the amino acid sequence encoded by the gene encoding translation elongation factor EF-Tu is shown in SEQ ID NO:

10.

9. An engineered cyanobacterium that can grow rapidly under high temperature and high light, characterized in that: It is prepared by the method according to claim 8.

10. Use of the engineered cyanobacteria according to claim 7 or 9 in the preparation of biosynthetic products.

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