KaiA gene mutant and application thereof

Abstract

The invention relates to a kaiA gene mutant capable of improving the growth speed of cyanobacteria, the 266th-273rd amino acids are shown as SEQ ID NO: 5, and the 274th amino acids and the amino acids behind the 274th amino acids are deleted; the invention also relates to application of the kaiA gene mutant in improving the growth speed of cyanobacteria and a constructed fast-growing engineering strain. According to the invention, the kaiA gene of synechococcus PCC 7942 is mutated, so that the 266th-273rd amino acids of the coded protein are changed, and the 274th and subsequent amino acids are deleted, so that the obtained mutant strain has a faster growth speed, and the increase of the growth speed has a synergistic effect with AtpA C252Y mutation. Therefore, the growth speed of the synechococcus can be greatly improved by performing corresponding genetic modification on the synechococcus, and the yield of the microalgae and the target compound can be improved by using the mutant strain as the chassis algae for metabolic modification and production of the target compound.

Description

Technical Field

[0001] The present invention relates to the field of cyanobacterial genetic engineering. More particularly, it relates to a kaiA gene mutant that can improve the growth rate of cyanobacteria, and also relates to the application of the above-mentioned kaiA gene mutant in improving the growth rate of cyanobacteria, as well as the constructed fast-growing engineering strain. Background Art

[0002] Cyanobacteria are a type of prokaryotic microorganism that performs plant-type oxygenic photosynthesis and can convert carbon dioxide into various carbohydrates using solar energy. Due to their advantages such as simple structure, rapid growth, and convenient genetic modification, cyanobacteria are considered to be a highly potential photosynthetic synthesis platform for bioenergy and biobased chemicals. In recent years, with the development of synthetic biology, through systematic genome editing, the deep remodeling of the photosynthetic metabolic network to construct a photosynthetic cell factory can achieve the directional conversion of solar-driven carbon dioxide and water into biofuels and biobased chemicals in a single process and a single platform, ultimately realizing the resource utilization and high-value utilization of carbon dioxide.

[0003] Growth rate is an important indicator for evaluating the efficiency of cyanobacterial chassis cells and cell factories. A faster cell growth rate means a shorter culture-harvest cycle, which can effectively reduce time and process costs; a faster growth rate usually also means a higher carbon fixation rate, which can more efficiently synthesize biofuels and biobased chemicals from fixed carbon dioxide; a faster growth rate also means that high-density culture can be achieved faster during large-scale and engineering culture, reducing the risk of invasion and pollution of the culture system by other microorganisms in the surrounding environment. Therefore, improving the photosynthetic carbon fixation efficiency of cyanobacteria and achieving rapid cell growth and proliferation are of great significance for optimizing the overall efficiency of photosynthetic cell factories and promoting the development and application of photosynthetic biomanufacturing technologies.

[0004] Existing studies have shown that introducing some mutations in cyanobacteria results in mutant strains with higher growth rates. For example, introducing the C252Y mutation on the α subunit of ATP synthase in PCC 7942 can increase the growth rate of the mutant strain, especially under normal and high-light conditions.

[0005] Therefore, finding gene loci that can affect the growth rate of strains can provide new tools for the transformation and industrial cultivation of cyanobacteria. Summary of the Invention

[0006] In this study, by combining a controllable hypermutation and a single-cell Raman sorting system, the organic coupling of the two processes of "continuously and rapidly generating genetic and phenotypic diversity" and "precisely and rapidly screening fast-growing phenotypes" was achieved, and a high-throughput and automated cyanobacteria mutant breeding platform was constructed. Through multiple rounds of "mutation - screening" processes, mutant algal strains with continuously increasing growth rates were obtained, thereby achieving stable and efficient synthesis and accumulation of biomass and target products under large-scale conditions. By studying the screened fast-growing strains, it was found that specific mutations in the circadian clock gene kaiA could increase the growth rate of the strains.

[0007] Based on the above findings, the present invention provides a kaiA gene mutant that can improve the growth rate of cyanobacteria. The amino acids at positions 266 - 273 are as shown in SEQ ID NO:5, and the amino acids at position 274 and those after it are deleted. The reference amino acid sequence encoded by the kaiA gene before mutation is as shown in SEQ ID NO:2.

[0008] In a specific embodiment, the amino acid sequence encoded by the kaiA gene mutant is as shown in SEQ ID NO:4.

[0009] The present invention also provides the application of the above kaiA gene mutant in improving the growth rate of cyanobacteria.

[0010] In a specific embodiment, the cyanobacteria is Synechococcus.

[0011] The present invention also provides a method for improving the growth rate of cyanobacteria, including the step of replacing the kaiA gene in the cyanobacterial genome with the kaiA gene mutant described in claim 1 or 2.

[0012] In a specific embodiment, the method further includes the step of introducing a mutation from cysteine to tyrosine at position 252 in the α subunit of the ATP synthase of the cyanobacteria. The reference amino acid sequence of the α subunit of the ATP synthase is as shown in SEQ ID NO:6.

[0013] In a specific embodiment, the cyanobacteria is Synechococcus.

[0014] The present invention also provides fast-growing engineered cyanobacteria constructed by the above method.

[0015] The present invention also provides the application of the above engineered cyanobacteria in metabolic engineering transformation.

[0016] By mutating the kaiA gene of Synechococcus PCC 7942, the amino acids at positions 266 - 273 of the encoded protein are altered, and the mutant strain obtained by deleting the amino acids at position 274 and those behind it has a faster growth rate. Moreover, this increase in growth rate has a synergistic effect with the AtpA C252Y mutation. Therefore, corresponding genetic modification of Synechococcus can significantly improve its growth rate. Using the mutant strain as a chassis alga for metabolic modification for the production of target compounds is conducive to increasing the yields of microalgae and target compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the screening process for fast - growing strains.

[0018] Figure 2 It is the plasmid map for constructing the control strain (A) and the engineered strain (B).

[0019] Figure 3 It is the growth curves of strains JC45, JC46, JC70, and JC71 under high light (A) and normal light (B). DETAILED DESCRIPTION OF THE INVENTION

[0020] 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 intended to limit the scope of the present invention.

[0021] 1. Integration of controllable hypermutation technology and single - cell high - throughput sorting technology

[0022] We integrated the controllable hypermutation technology with the single - cell high - throughput sorting technology to build an efficient sorting system ( Figure 1 ): 1) Subject the hypermutant strain HS84 (a strain with a high mutation rate constructed from Synechococcus PCC 7942, see Patent CN116855513A) to a short - term high - light treatment to accumulate the mutation rate; 2) Conduct 13 C NaHCO 3 labeling for 12 h under normal growth conditions, take a certain sample for high - throughput sorting; 3) Use a single - cell flow cytometry Raman sorting system to sort the labeled mutant cells. The cells with a fast labeling rate are the target cells. Collect the target cells for subsequent culture; 4) After repeating the sorting three rounds, sequence the genome of the bacterial population and conduct phenotypic analysis.

[0023] By the above method, we screened some fast-growing strains. Detection of mutation sites in the screened fast-growing strains revealed that there was a mutation in the biological clock gene kaiA in one fast-growing strain. This mutation was the insertion of 22 bp bases at positions 755 and 756 of the kaiA gene (the reference sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2), resulting in corresponding changes in the amino acids at positions 266 - 273 (changing from IDVIAHLC to AGLSADFN (SEQ ID NO: 5)), and translation was terminated prematurely. That is, the final sequence of the mutated biological clock gene kaiA is shown in SEQ ID NO: 3, and the encoded amino acid sequence is shown in SEQ ID NO: 4.

[0024] 2. Construction of engineered bacteria with mutated biological clock gene kaiA

[0025] Plasmids pJC125 (KaiA mutant) and pJC138 (KaiA WT) were constructed. The plasmid maps are as Figure 2 , with the mutated site part placed on the homologous arms. The mutated site was integrated into the genomes of 7942WT and HL7942 (containing the AtpA C252Y mutation site, and the reference amino acid sequence encoded by AtpA is shown in SEQ ID NO: 6) through homologous recombination resistance screening, obtaining engineered bacteria JC45 (integrating the plasmid carrying the kaiA mutation of pJC125 on the 7942WT strain), JC70 (integrating the plasmid carrying kaiA WT of pJC138 on the 7942WT strain), JC46 (integrating the plasmid carrying the kaiA mutation of pJC125 on the HL7942 strain), and JC71 (integrating the plasmid carrying kaiA WT of pJC138 on the HL7942 strain).

[0026] 3. Growth phenotype analysis of engineered strains

[0027] The constructed engineered strains were cultured under 500 μmol photons / m 2 / s and 1500 μmol photons / m 2 / s, and growth curves were measured. The results are as Figure 3 shown. Under normal conditions, the growth of strain JC70 was about 46% higher than that of the control strain JC45, and the growth of strain JC71 was about twice that of the control strain JC45. Under high-light conditions, the growth of strain JC70 was about 90% higher than that of the control strain (JC45), and the growth of strain JC71 was about 1.4 times that of the control strain JC45.

[0028] The above experiments prove that mutating the kaiA gene of Synechococcus PCC 7942, resulting in changes in the amino acids at positions 266-273 of the encoded protein and deletion of the amino acids at position 274 and those following it, produces a mutant strain with a faster growth rate. Moreover, this increase in growth rate exhibits a synergistic effect with the AtpA C252Y mutation.

[0029] It should be noted that for the purpose of clear illustration, the present invention uses a reference sequence to define the mutation sites. However, those skilled in the art should clearly understand that this reference sequence is only used to define the mutation sites, and the amino acids or nucleic acids at the mutation sites or other positions on the reference sequence should not be used to limit the present invention. Substitutions, additions, and deletions of amino acids or nucleic acids at the mutation sites or other positions on the reference sequence should all be covered within the protection scope of the present invention, as long as the mutation sites in the mutated sequence are the same as the corresponding sequences.

[0030] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall all be included within the protection scope of the present invention.

Claims

1. A kaiA gene mutant capable of increasing the growth rate of cyanobacteria, characterized in that: The amino acids at positions 266-273 are shown in SEQ ID NO:5, and the amino acids 274 and thereafter are deleted. The amino acid reference sequence encoded by the kaiA gene before mutation is shown in SEQ ID NO:

2.

2. The kaiA gene mutant according to claim 1, characterized in that The encoded amino acid sequence is shown in SEQ ID NO:

4.

3. Use of the kaiA gene mutant according to claim 1 or 2 in increasing the growth rate of cyanobacteria.

4. The use according to claim 3, characterized in that: The cyanobacteria is Synechococcus.

5. A method for increasing the growth rate of cyanobacteria, characterized in that: The method comprises the step of replacing the kaiA gene in the cyanobacterium genome with the kaiA gene mutant according to claim 1 or 2.

6. The method according to claim 5, characterized in that The method further comprises the step of introducing a mutation from cysteine ​​at position 252 to tyrosine in the ATP synthase α subunit of the cyanobacterium, wherein the reference amino acid sequence of the ATP synthase α subunit is shown in SEQ ID NO:

6.

7. The use according to claim 5 or 6, characterized in that: The cyanobacteria is Synechococcus.

8. A fast-growing engineered cyanobacterium, characterized in that: It is constructed by the method according to any one of claims 5 to 7.

9. Application in metabolic engineering of engineered cyanobacteria according to claim 8.

Images