A method for constructing an engineered cyanobacterial strain for improving photosynthetic efficiency based on NoxE oxygen scavenging module and application thereof

Abstract

The application relates to a blue-green algae engineering strain construction method and application for improving photosynthetic efficiency based on a NoxE oxygen scavenging module. NoxE The application relates to a blue-green algae engineering strain construction method and application for improving photosynthetic efficiency based on a NoxE oxygen scavenging module. The application relates to a blue-green algae engineering strain construction method and application for improving photosynthetic efficiency based on a NoxE oxygen scavenging module. The application relates to a blue-green algae engineering strain construction method and application for improving photosynthetic efficiency based on a NoxE oxygen scavenging module. The application relates to a blue-green algae engineering strain construction method and application for improving photosynthetic efficiency based on a NoxE oxygen scavenging module.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method and application for constructing cyanobacterial engineered strains that improve photosynthetic efficiency based on the NoxE oxygen scavenging module. Background Technology

[0002] Photosynthesis is the core physiological process by which photosynthetic organisms convert light energy into chemical energy and fix carbon dioxide. Its carbon assimilation reaction mainly relies on the catalysis of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO). RuBisCO catalyzes the carboxylation of RuBP with CO2 to produce 3-phosphoglycerate (3-PGA), a crucial step in carbon fixation in nature. However, RuBisCO also possesses oxygenase activity, which binds with O2 to generate photorespiratory metabolic intermediates such as 2-phosphoglycolic acid, leading to the re-loss of fixed carbon and additional consumption of ATP and reducing power, thus reducing the overall efficiency of photosynthesis. Although photosynthetic microorganisms such as cyanobacteria have evolved carboxysomes and carbon concentrating mechanisms (CCMs) to increase the CO2 concentration around RuBisCO and reduce oxygen competition, the large amount of oxygen continuously produced by photosystem II will still accumulate locally inside the cell under conditions of high light, high density culture, or limited gas exchange. This will enhance the oxygenation reaction of RuBisCO and thus limit carbon fixation efficiency and biomass accumulation.

[0003] Currently, research on improving photosynthetic efficiency mainly focuses on the following directions: first, protein engineering the RuBisCO enzyme itself to improve its carboxylation specificity; second, enhancing the CO2 concentration mechanism to increase intracellular inorganic carbon supply; and third, constructing or optimizing photorespiration bypasses to reduce carbon loss caused by photorespiration. However, the above methods usually suffer from problems such as complex enzyme structures, delicate regulatory mechanisms, difficulties in cross-component coordination, or high metabolic burden, which still limit their practical application. Therefore, developing a novel metabolic engineering strategy that reduces RuBisCO oxygenation by regulating intracellular oxygen levels without modifying the RuBisCO structure itself is of great significance for improving photosynthetic carbon fixation efficiency and enhancing the production performance of photosynthetic organisms. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one objective of this invention is to provide a method and application for constructing engineered cyanobacterial strains that enhance photosynthetic efficiency based on a NoxE oxygen scavenging module. This method, by introducing a NoxE oxygen scavenging module, directly catalyzes the reaction of intracellular NADH with oxygen to produce water. Without modifying RuBisCO itself, it regulates intracellular gas homeostasis, enhances RuBisCO carboxylation efficiency, and achieves stable growth in photosynthetic efficiency and biomass; filling a gap in methods for reducing photorespiration and enhancing carbon fixation.

[0005] Therefore, in a first aspect, the present invention proposes a method for constructing cyanobacterial engineered strains with improved photosynthetic efficiency based on the NoxE oxygen scavenging module, which includes the following steps: Constructing a water-generating NADH oxidase NoxE Homologous recombination expression vector for the encoding gene NS17942: GmR-pCPCB-NoxE; The homologous recombination expression vector NS17942:GmR-pCPCB-NoxE was integrated into the neutral site NS1 of the cyanobacterial host genome, and the engineered cyanobacterial strain was obtained by screening for gentamicin resistance.

[0006] This invention proposes a method for constructing cyanobacterial engineered strains with enhanced photosynthetic efficiency based on the NoxE oxygen scavenging module. This method expresses the NoxE module, which continuously catalyzes the redox reaction between NADH and molecular oxygen within the cell, converting NADH and O2 into NAD. + and H2O (NADH + O2 + 2 H) + → 2 NAD + + 2 H2O); the oxygen-consuming capacity of the water-generating NADH oxidase NoxE reduces the probability of O2 competing with RuBisCO for binding, thereby increasing the relative proportion of RuBisCO carboxylation and inhibiting photorespiration. Compared with the wild-type strain, the engineered strain constructed by this method exhibits a faster growth rate; simultaneously, photosynthetic pigment content and absorption spectrum analysis show that the relative content of phycobilisomes and chlorophyll a is increased in the engineered strain, the net photosynthetic rate is increased, and glycogen accumulation is increased. The construction method is simple and provides a new technical means for improving crop yield and addressing climate change in the future.

[0007] Optionally, the NoxE The nucleotide sequence of the encoding gene is shown in SEQ ID NO:1.

[0008] Optionally, in the homologous recombination expression vector NS17942:GmR-pCPCB-NoxE NoxE The gene encoding the gene is expressed by the light-inducible promoter pCPCB.

[0009] Optionally, the cyanobacterial host is Synechococcus PCC7942.

[0010] In a second aspect, the present invention proposes an engineered cyanobacterial strain for improving photosynthetic efficiency, which is constructed by the above-described construction method.

[0011] In a third aspect, the present invention proposes the application of the above-mentioned engineered cyanobacterial strains in bioenergy production, carbon dioxide biofixation, or improving RuBisCO carboxylation efficiency.

[0012] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the O2 scavenging pathway mediated by the water-generating NADH oxidase NoxE and its mechanism for promoting photosynthesis in PCC7942 according to an embodiment of the present invention, wherein RuBP is ribulose-1,5-bisphosphate; 2PG is 2-phosphoglycolic acid; and 3PGA is 3-phosphoglyceric acid. Figure 2 The following figures illustrate the enhanced photosynthetic mechanism of the engineered strain PCC7942-NoxE according to embodiments of the present invention: A shows a comparison of growth rates between WT and PCC7942-NoxE strains; B shows a comparison of net photosynthetic rates between WT and PCC7942-NoxE strains; C shows the whole-cell absorption spectra (550-750 nm) of WT and PCC7942-NoxE strains, with corresponding phycobilisomes (approximately 630 nm) and chlorophyll indicated in the figure. a The characteristic absorption peak is approximately 680 nm; D represents the comparison of glycogen accumulation in WT and PCC7942-NoxE strains; data are expressed as mean ± standard deviation (SD) of three biological replicates, with blue bars representing PCC7942-WT and orange bars representing PCC7942-NoxE. Statistical analysis was performed using unpaired two-tailed Student's t-test, with * indicating p < 0.05 and ns indicating no significant difference. Detailed Implementation

[0014] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0015] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0016] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0017] Materials used: Cyanobacterium PCC7942 (American Center for Microbial Culture Preservation, ATCC 33912), vector pUC18-NS17942-GmR-pCPCB [published in Applied Microbiology and Biotechnology, 2022, 106(24), 8169-8181]. Phusion high-fidelity DNA polymerase and restriction endonucleases were purchased from Xiamen Lulong Biotechnology Development Co., Ltd. Plasmid extraction kit, DNA purification kit, gel extraction kit, and genomic DNA extraction kit were purchased from Hangzhou Bori Biotechnology. BG11 medium used for cyanobacterium was from Qingdao Hopo Bio-Technology, cat. No. HB8793. Gentamicin catalog number: 1405-41-0.

[0018] The construction principle of the cyanobacterial engineered strains for improving photosynthetic efficiency in this invention embodiment is described in [link to invention]. Figure 1 : Water-generating NADH oxidase converts NADH and O2 into NAD. + and H2O (NADH + O2 + 2 H) + → 2 NAD ++2 H2O); Part Two: The oxygen consumption capacity of the NoxE module reduces the probability of O2 competing for binding with RuBisCO, thereby reducing photorespiration and promoting carbon fixation. This reaction process directly consumes intracellular molecular oxygen, thereby reducing the intracellular oxygen partial pressure, decreasing the probability of RuBisCO oxygenation, and increasing the relative proportion of RuBisCO carboxylation. Unlike oxidases that partially produce hydrogen peroxide, the NoxE catalytic process ultimately produces H2O rather than H2O2, thus not adding additional cellular oxidative stress.

[0019] Table 1: Gene Synthesis and Primer Sequences

[0020] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0021] Example 1: Construction of an oxygen consumption module NoxE The nucleotide sequence is as shown in SEQ ID NO:1 NoxE Gene insertion into the pUC18-NS17942-GmR-pCPCB vector Bam HI / Xho Within the I site, the recombinant vector pUC18-NS17942-GmR-pCPCB-NoxE was obtained. The specific procedures are as follows: by Lactococcus lactis (Lopez de Felipe F, Kleerebezem M, de Vos WM, Hugenholtz J. 1998. Cofactor Engineering: a Novel Approach to MetabolicEngineering in Lactococcus lactisUsing the controlled expression of NADHOxidase (https: / / doi.org / 10.1128 / jb.180.15.3804-3808.1998) as a template, and primers NoxE-BamHI-Fwd (SEQ ID NO:2) and NoxE-XhoI-Rev (SEQ ID NO:3) as the PCR primer combination, the NoxE band was obtained by PCR amplification. The target band was recovered using a DNA purification kit to obtain the gel recovery product. PCR amplification conditions: 95°C for 1 min, 95°C for 10 s, 56°C for 15 s, 72°C for 15 min, 30 cycles; 72°C for 2 min. The gel recovery product (NoxE fragment) was purified by NEB. Bam HI and Xho Enzyme I digestion, vector pUC18-NS17942-GmR-pCPCB via NEB Bam HI / Xho Digestion with enzyme I at 37°C for 1 hour, followed by purification and recovery. Ligation reaction was then performed at room temperature for 1 hour, followed by transformation into E. coli Top10 competent cells. Plasmid pUC18-NS17942-GmR-pCPCB-NoxE was extracted and sequenced to ensure that the results were completely consistent with the designed plasmid DNA sequence. The ligation system is shown in Table 2 below.

[0022] Table 2 Connection System

[0023] Example 2: Verification of the Improved Photosynthetic Efficiency in Engineered Cyanobacterial Strains The recombinant vector pUC18-NS17942-GmR-pCPCB-NoxE from Example 1 was integrated into the neutral site NS1 of the cyanobacterial PCC7942 genome. Recombinant engineered strains were obtained through gentamicin resistance plate screening. Starting with this recombinant engineered strain, homozygous engineered strain PCC7942-NoxE was obtained through continuous screening with 5 mg / L gentamicin. Wild-type PCC7942-WT and engineered cyanobacterial strain PCC7942-NoxE were picked from plates. The stock solution was inoculated at a 1% inoculum into 50 mL glass Erlenmeyer flasks containing 20 mL of BG11 medium and cultured on a shaker. The optical density (OD) at 730 nm was measured using a microplate reader (BioTek SynergyH1). 730 Regularly assess the growth of cyanobacteria.

[0024] To determine the absorption spectrum of cyanobacteria, whole-cell absorption spectra in the 550-750 nm range were recorded using a microplate reader (BioTek Synergy H1). Glycogen content was determined using a commercial glycogen assay kit (Bioisco Biotech Co., Ltd., China) according to the manufacturer's instructions. Samples were taken on days 5, 10, 15, and 20 for glycogen content analysis. Photosynthetic oxygen evolution rate was measured using an algal photosynthesis meter (YZQ-201CJ, Yizongqi Technology). Specifically, 15 mL of the cyanobacterial culture medium from day 5 was added to the photosynthetic reaction chamber, and measurements were taken under the following conditions: RGB excitation light intensity 1200 μmol photons / m². - ² s - ¹, Stabilization time 180 s, measurement time 180 s. Net photosynthetic rate is expressed as oxygen release rate.

[0025] The results are as follows Figure 2 As shown, compared to PCC7942-WT, the cyanobacterial engineered strain PCC7942-NoxE of this application exhibits a faster growth rate. Figure 2 A), increased glycogen accumulation effect ( Figure 2 D), photosynthetic pigment content increased ( Figure 2 C) and net photosynthetic efficiency increased ( Figure 2 B).

[0026] The above results indicate that the present invention utilizes Lactococcus lactis (… Lactococcus lactis )of NoxE The gene is integrated into the cyanobacterial genome, enabling it to continuously catalyze the redox reaction of NADH and molecular oxygen within the cell, converting NADH and O2 into NAD. + And H2O. This reaction process directly consumes intracellular molecular oxygen, thereby reducing the intracellular oxygen partial pressure, decreasing the probability of RuBisCO oxygenation, and increasing the relative proportion of RuBisCO carboxylation. Compared with the wild-type strain, the engineered strain of this invention exhibits a faster growth rate; simultaneously, photosynthetic pigment content and absorption spectrum analysis show that the relative content of phycobilisomes and chlorophyll a is increased in the engineered strain, the net photosynthetic rate is increased, and glycogen accumulation is increased. In summary, this invention achieves direct consumption of intracellular oxygen by introducing NoxE, and improves photosynthetic efficiency by regulating the intracellular oxygen microenvironment without changing the structure of RuBisCO itself. The construction method of this invention is simple and provides a new technical means for improving crop yield and addressing climate change in the future.

[0027] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0028] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing cyanobacterial engineered strains with improved photosynthetic efficiency based on the NoxE oxygen scavenging module, characterized in that, Includes the following steps: Constructing a water-generating NADH oxidase NoxE Homologous recombination expression vector for the encoding gene NS17942: GmR-pCPCB-NoxE; The homologous recombination expression vector NS17942:GmR-pCPCB-NoxE was integrated into the neutral site NS1 of the cyanobacterial host genome, and the engineered cyanobacterial strain was obtained by screening for gentamicin resistance.

2. The construction method according to claim 1, characterized in that, The NoxE The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

3. The construction method according to claim 1, characterized in that, The homologous recombination expression vector NS17942:GmR-pCPCB-NoxE contains NoxE The gene encoding the gene is expressed by the light-inducible promoter pCPCB.

4. The construction method according to claim 1, characterized in that, The cyanobacterial host is Synechococcus PCC7942.

5. A cyanobacterial strain designed to improve photosynthetic efficiency, characterized in that, It is constructed by the construction method of any one of claims 1-4.

6. The application of the cyanobacterial engineered strain according to claim 5 in bioenergy production, carbon dioxide bio-fixation, or improving RuBisCO carboxylation efficiency.

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