Method for constructing engineered cyanobacterial strain for improving photosynthetic efficiency based on tyrosine metabolism and application thereof

Abstract

This invention relates to a method for constructing an engineered cyanobacterial strain that enhances photosynthetic efficiency based on tyrosine metabolism. The method includes the following steps: constructing a recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC containing genes encoding 4-hydroxyphenylpyruvate dioxygenase (Hpd), homosuccinate dioxygenase (HmgA), fumarate acetoacetate hydrolase (HmgB), and maleate acetoacetate isomerase (HmgC); integrating the recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC into the neutral site NS1 of the cyanobacterial host genome; and obtaining the engineered cyanobacterial strain through gentamicin resistance screening. This method regulates the intracellular O2 / CO2 ratio by constructing an oxygen scavenging metabolic module, significantly improving RuBisCO carboxylation efficiency without modifying RuBisCO itself, ultimately achieving stable growth in photosynthetic efficiency and biomass.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for constructing an engineered cyanobacterial strain that improves photosynthetic efficiency based on tyrosine metabolism and its application. Background Technology

[0002] Photosynthesis is the core foundation of material cycling and energy flow in Earth's ecosystems. Through the catalysis of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO), carbon dioxide is fixed to produce organic matter, supporting the energy source for almost all higher organisms and the supply of carbon raw materials for industrial biomanufacturing. However, RuBisCO itself possesses dual activities as both a carboxylase and an oxidase: when the intracellular oxygen partial pressure increases, its oxidase activity catalyzes the reaction of ribulose-1,5-bisphosphate with O2 to produce phosphoglycolic acid, triggering photorespiration. This causes the fixed carbon to be released again as CO2, typically resulting in a loss of photosynthetic efficiency and severely limiting biomass accumulation and carbon fixation efficiency.

[0003] Although nature has evolved carboxysomes and CO2 concentration mechanisms (CCM) to improve carboxylation efficiency, intracellular oxygen partial pressure still increases significantly under conditions of global warming, strong light, high oxygen, or intensive culture, severely limiting photosynthetic efficiency.

[0004] Currently, the main approaches to improving photosynthetic efficiency fall into the following three categories: Directly modifying the RuBisCO enzyme structure: improving the carboxylation activity / oxidation activity ratio of RuBisCO through directed evolution. However, the RuBisCO structure is complex and the subunit assembly regulation mechanism is not yet fully clear, making modification difficult and easily leading to a decrease in the overall catalytic efficiency of the enzyme.

[0005] Strengthening the CO2 concentration mechanism: Intracellular CO2 concentration can be increased by overexpressing CCM-related transport proteins and carboxysome structural proteins. However, this pathway has a large regulatory network, and overexpression can easily cause metabolic burden on cells. The actual effect is greatly affected by environmental conditions.

[0006] Introducing the photorespiration bypass pathway: This approach reduces carbon loss through photorespiration by introducing the glycolic acid metabolism pathway from prokaryotes. However, the bypass pathway itself requires additional energy and disrupts intracellular redox homeostasis, making it difficult to achieve a stable increase in photosynthetic efficiency.

[0007] Furthermore, existing oxygen scavenging-related metabolic engineering strategies often suffer from substrate dependence, byproduct accumulation, and carbon loss, failing to sustainably and efficiently regulate intracellular O2 / CO2 homeostasis. Therefore, there is an urgent need to develop a novel strategy that does not directly modify the core photosynthetic enzyme but efficiently inhibits photorespiration by reshaping intracellular gas homeostasis, thereby achieving a stable increase in photosynthetic efficiency. Summary of the Invention

[0008] 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 for constructing an engineered cyanobacterial strain that enhances photosynthetic efficiency based on tyrosine metabolism, and its application. This method regulates the intracellular O2 / CO2 ratio by constructing an oxygen scavenging metabolic module, significantly improving the carboxylation efficiency of RuBisCO without modifying the RuBisCO itself, ultimately achieving stable growth in photosynthetic efficiency and biomass.

[0009] Therefore, in a first aspect, the present invention proposes a method for constructing an engineered cyanobacterial strain that improves photosynthetic efficiency based on tyrosine metabolism, comprising the following steps: A recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC was constructed containing the encoding genes for 4-hydroxyphenylpyruvate dioxygenase Hpd, homosuccinate dioxygenase HmgA, fumaroyl acetoacetate hydrolase HmgB, and maleyl acetoacetate isomerase HmgC. The recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC was integrated into the neutral site NS1 of the cyanobacterial host genome, and the engineered cyanobacterial strain was obtained by screening for gentamicin resistance.

[0010] According to an embodiment of the present invention, a method for constructing an engineered cyanobacterial strain with improved photosynthetic efficiency based on tyrosine metabolism is proposed. This method involves constructing a recombinant expression module containing 4-hydroxyphenylpyruvate dioxygenase (Hpd), homosuccinate dioxygenase (HmgA), fumarate acetoacetate hydrolase (HmgB), and maleate acetoacetate isomerase (HmgC), and integrating it into a neutral site in the cyanobacterial genome to obtain an engineered strain with continuous intracellular oxygen scavenging and CO2 replenishment capabilities. By expressing the Hpd-HmgABC metabolic module, 4HPP reductase (Hpd) oxidizes 4-hydroxyphenylpyruvate to produce carbon dioxide and homosuccinate. Homosuccinate is then further converted into fumarate and acetoacetate by the introduced homosuccinate dioxygenase (HmgA), fumaroyl acetoacetate hydrolase (HmgB), and maleoacetoacetate isomerase (HmgC), thus re-entering the central metabolic pathway of the tricarboxylic acid cycle. The oxygen consumption capacity of the catabolic Hpd-HmgABC module reduces the likelihood of oxygen contact with RuBisCO, thereby increasing the carboxylation of RuBisCO and inhibiting photorespiration. It catalyzes the conversion of 4-hydroxyphenylpyruvate to fumarate and acetoacetate in the L-tyrosine catabolic pathway, consuming oxygen and releasing carbon dioxide during the reaction, thereby regulating the intracellular O2 / CO2 ratio, inhibiting the ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) oxidation reaction, and enhancing carboxylation efficiency. Compared to the wild-type strain, this engineered strain exhibits advantages such as higher biomass accumulation, longer growth cycle, increased photosynthetic pigment content, enhanced thylakoid structure, and increased net photosynthetic rate. This strategy enhances photosynthetic efficiency by regulating intracellular gas homeostasis through metabolic coupling without modifying the RuBisCO structure.

[0011] Optionally, the nucleotide sequence of the gene encoding the 4-hydroxyphenylpyruvate dioxygenase Hpd is shown in SEQ ID NO:1.

[0012] Optionally, the nucleotide sequences of the high-somnol dioxygenase HmgA encoding gene, the fumaroyl acetoacetate hydrolase HmgB encoding gene, and the maleyl acetoacetate isomerase HmgC encoding gene are shown in SEQ ID NO:2. The Hpd gene is derived from *Pseudomonas putida* KT2440, and its encoded Hpd enzyme can specifically catalyze the oxidation of 4-hydroxyphenylpyruvate to high-somnol (HGA). The HmgABC operon, derived from *Pseudomonas putida* KT2440, contains three consecutive coding genes: HmgA encodes homosuccinate dioxygenase, which catalyzes the oxidation and ring-opening of homosuccinate to maleoacetoacetate; HmgC encodes maleoacetoacetate isomerase, which catalyzes the isomerization of maleoacetoacetate to fumarate acetoacetate; and HmgB encodes fumarate acetoacetate hydrolase, which catalyzes the hydrolysis of fumarate acetoacetate to fumaric acid and acetoacetate, which can directly enter the tricarboxylic acid cycle to participate in central carbon metabolism without carbon loss.

[0013] Optionally, the Hpd and HmgABC genes in the recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC are expressed in tandem by the light-inducible promoter pCPCB. Expression is automatically initiated under light conditions without the need for additional inducers, conforming to the growth rhythm of photosynthetic organisms.

[0014] Optionally, the cyanobacterial host is Synechococcus PCC7942.

[0015] In a second aspect, the present invention proposes an engineered cyanobacterial strain with coupled oxygen scavenging and carbon recovery functions, which is constructed by the above-described construction method.

[0016] 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.

[0017] 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

[0018] Figure 1This diagram illustrates the oxygen scavenging cycle mechanism and growth phenotype verification of tyrosine metabolism according to embodiments of the present invention. A shows a schematic diagram of the mechanism by which the Hpd-HmgABC module enhances photosynthesis, demonstrating the complete process of tyrosine metabolism coupled with oxygen scavenging and carbon recovery, as well as the regulatory mechanism on RuBisCO activity and photorespiration. Here, RuBP represents ribulose-1,5-bisphosphate; 2PG represents 2-phosphoglycolic acid; 3PGA represents 3-phosphoglyceric acid; 4HPP represents 4-hydroxyphenylpyruvic acid; and HGA represents homosuccinic acid. B shows the colony growth of wild-type (WT) and engineered strain PCC7942-Hpd-HmgABC on BG11 solid agar plates, with the engineered strain exhibiting larger colonies and more vigorous growth. C compares the growth curves of wild-type (WT) and engineered strain PCC7942-Hpd-HmgABC, showing the OD of the engineered strain after 15 days of culture. 730 The biomass accumulation was significantly higher than that of the wild type; D shows the comparison of cell morphology and size between the wild type (WT) and the engineered strain PCC7942-Hpd-HmgABC. The engineered strain showed normal cell morphology and no obvious growth defects; E shows the comparison of net photosynthetic oxygen evolution rate between the wild type and the engineered strain; F shows the comparison of cell lifespan between the wild type and the engineered strain; the data are expressed as mean ± standard deviation (SD) of three biological replicates. The blue column represents PCC7942-WT, and the red dashed column represents PCC7942-Hpd-HmgABC. Statistical analysis was performed using unpaired two-tailed Student's t test. * indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001. Figure 2 The diagram illustrates the enhanced photosynthetic system of the engineered strains according to embodiments of the present invention. A shows a schematic diagram of the transfer of excitation energy from phycobilisomes on the thylakoid membrane to photosystem II, demonstrating the enhanced effect of increased photosynthetic pigment content on light capture. B shows the whole-cell absorption spectra (550-750 nm) of the WT and PCC7942-Hpd-HmgABC strains, with corresponding phycobilisomes (approximately 630 nm) and chlorophyll labeled in the figure. a Characteristic absorption peak (approximately 680 nm); C represents the quantitative analysis of phycobiliproteins (PC, phycocyanin; APC, allophycocyanin) and photosynthetic pigments in WT and PCC7942-Hpd-HmgABC strains; D shows the ultrastructure of cyanobacterial cells as displayed by transmission electron microscopy: T, thylakoids; C, carboxysomes; N, nucleoid region; CW, cell wall; Data are expressed as mean ± standard deviation (SD) of three biological replicates. Blue bars represent PCC7942-WT; red dashed bars represent PCC7942-Hpd-HmgABC. Statistical analysis was performed using unpaired two-tailed Student's t-test. * indicates p < 0.05, and ns indicates no significant difference. Detailed Implementation

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] The construction principle of the cyanobacterial engineered strains for improving photosynthetic efficiency in this invention embodiment is described in [link to invention]. Figure 1 : Part 1: 4HPP reductase (Hpd) oxidizes 4-hydroxyphenylpyruvate to produce carbon dioxide and homosuccinate. Part 2: Homosuccinate is further converted into fumarate and acetoacetate by introduced homosuccinate dioxygenase (HmgA), fumaroyl acetoacetate hydrolase (HmgB), and maleoacetoacetate isomerase (HmgC), thus re-entering the central metabolic pathway of the tricarboxylic acid cycle. Part 3: The oxygen consumption capacity of the catabolic Hpd-HmgABC module reduces the likelihood of oxygen contact with RuBisCO, thereby reducing its photorespiration-driven carbon fixation.

[0024] Table 1: Primers used for gene and PCR amplification

[0025] 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.

[0026] Example 1: Construction of the oxygen consumption module Hpd-HmgABC The HmaS* gene with nucleotide sequence as shown in SEQ ID NO:1 and the HmgABC gene with nucleotide sequence as shown in SEQ ID NO:2 were inserted into the pUC18-NS17942-GmR-pCPCB vector. Bam HI / Xho Within the I site, the recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC was obtained. The specific procedures are as follows: by P. putidaUsing the KT2440 genome (Genebank: GCA_045571375.1) as a template, the PCR primer combination was Hpd_BsaI_fwd1 (nucleotide sequence shown in SEQ ID NO:3) and Hpd_BsaI_rev1 (nucleotide sequence shown in SEQ ID NO:4); the PCR primer combination was HmgABC_BsaI_fwd2 (nucleotide sequence shown in SEQ ID NO:5) and HmgABC_BsaI_rev2 (nucleotide sequence shown in SEQ ID NO:6). Hpd and HmgABC bands were obtained by PCR amplification. The target bands were 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 was mixed and passed through NEB. Bsa Enzyme I digestion, vector pUC18-NS17942-GmR-pCPCB via NEB Bam HI / Xho After digestion with enzyme I at 37°C for 1 h, the residue was purified and recovered. The ligation reaction was then carried out at room temperature for 1 h, followed by transformation into E. coli Top10 competent cells. The plasmid pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC 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.

[0027] Table 2 Connection System

[0028] Example 2: Verification of photosynthetic efficiency The recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC 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-Hpd-HmgABC was obtained through screening with 5 mg / L gentamicin. Wild-type PCC7942-WT and engineered cyanobacterial strain PCC7942-Hpd-HmgABC 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 Synergy H1). 730The growth of cyanobacteria was assessed regularly. Cell morphology of PCC7942-WT and PCC7942-Hpd-HmgABC was recorded using a microscope (Olympus BX53, Olympus Corporation, Japan). To determine the life cycle of the cyanobacteria, they were cultured continuously for several months until their appearance turned yellow. For solid-phase growth observation, the cyanobacterial culture medium collected on day 5 was diluted to 10⁻⁶. -4 The sample was then spotted onto a BG11 solid agar plate.

[0029] 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). Photosynthetic pigments were quantitatively analyzed using organic solvent extraction. Specifically, cyanobacterial cells collected on day 5 were centrifuged and then lysed in pure methanol using an Omni bead ruptor under light-protected conditions. Subsequently, the cells were centrifuged at 4°C and 8,000 g for 5 min, and the supernatant was collected for chlorophyll analysis. a Determination of carotenoids. OD was measured using a UV-Vis spectrophotometer. 665 OD 649 and OD 470 Chlorophyll a The formula for calculating the content is: Chlorophyll- a =13.7×OD 665 -5.76×OD 649 The formula for calculating carotenoid content is: Carotenoids = (1,000 × OD) 470 -2.05×Chlorophyll- a ) / 245. For phycobiliprotein analysis, 2 mL of cyanobacterial culture medium was centrifuged at 4°C and 8,000 g for 5 min. The cell pellet was resuspended in PBS and disrupted by repeated freeze-thaw cycles in liquid nitrogen. The supernatant was measured at OD200. 620 (Phycocyanin) and OD 650 The absorbance at (allophycocyanin).

[0030] To observe the ultrastructure of cells, PCC7942-WT cells cultured for 5 days and its engineered strain PCC7942-Hpd-HmgABC were collected for TEM analysis. 3 mL of culture medium was centrifuged to collect the cell pellet, washed twice with phosphate-buffered saline (PBS, pH 7.2), and resuspended in 2.5% glutaraldehyde. The pellet was gently shaken and fixed at 4°C for 12–14 h. After overnight fixation, the sample was embedded in 2% low-melting-point agar and post-fixed with 1% osmium tetroxide for 2 h at room temperature, followed by stepwise dehydration with a gradient of ethanol. After permeation, the sample was embedded in epoxy resin (EPON 812). Ultrathin sections of approximately 70 nm were prepared using an ultramicrotome (Leica UC7) and placed on a copper grid, stained with 2% uranium acetate for 5 min. Finally, observation and imaging were performed using a transmission electron microscope (Hitachi HT-7800, 100 kV).

[0031] The photosynthetic oxygen release rate was measured using an algal photosynthesis meter (YZQ-201CJ, Yizongqi Technology). The specific method was as follows: 15 mL of cyanobacterial culture medium from day 5 was added to the photosynthetic reaction chamber, and the measurement was performed 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.

[0032] The results are as follows Figure 1 and Figure 2 As shown, compared with PCC7942-WT, the cyanobacterial engineered strain PCC7942-Hpd-HmgABC of this application exhibits advantages such as higher biomass accumulation, longer growth cycle, increased photosynthetic pigment content, enhanced thylakoid structure, and increased net photosynthetic rate.

[0033] The above results indicate that the engineered strain constructed in this invention has successfully achieved a significant increase in photosynthetic efficiency, biomass, and cell lifespan by coupling the tyrosine catabolism pathway with an oxygen scavenging and carbon recovery system. The technical effect is stable and reliable, and its advantages are even more significant under high-density culture and stress conditions.

[0034] 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.

[0035] 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 an engineered cyanobacterial strain that enhances photosynthetic efficiency based on tyrosine metabolism, characterized in that, Includes the following steps: A recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC was constructed containing the encoding genes for 4-hydroxyphenylpyruvate dioxygenase Hpd, homosuccinate dioxygenase HmgA, fumaroyl acetoacetate hydrolase HmgB, and maleyl acetoacetate isomerase HmgC. The recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC 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 nucleotide sequence of the gene encoding the 4-hydroxyphenylpyruvate dioxygenase Hpd is shown in SEQ ID NO:

1.

3. The construction method according to claim 1, characterized in that, The nucleotide sequences of the genes encoding the homosuccinate dioxygenase HmgA, the fumaroyl acetoacetate hydrolase HmgB, and the maleic acetoacetate isomerase HmgC are shown in SEQ ID NO:

2.

4. The construction method according to claim 1, characterized in that, In the recombinant vector pUC18-NS17942-GmR-pCPCB-Hpd-HmgABC, the Hpd and HmgABC genes are expressed in tandem by the light-inducible promoter pCPCB.

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

6. A cyanobacterial strain with coupled oxygen scavenging and carbon recovery functions, characterized in that, It is constructed by the construction method of any one of claims 1-5.

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

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