Recombinant strain of photosynthetic bacteria with high tocols production and its construction method and application

Abstract

This invention relates to a recombinant photosynthetic bacterial strain with high tocopherol production, its construction method, and its applications. The recombinant strain knocks out the photosynthetic gene repressor transcription factor PpsR and integrates the mevalonate MEV pathway gene from *Paragonimococcus zeatans*, the p-hydroxyphenylpyruvate dioxygenase (HPPD) gene from *Pseudomonas putida*, the urokinase chlorophyllase (HPT) gene from *Synostemma pentaphyllum*, the tocopherol cyclase (TC) gene from *Arabidopsis thaliana*, and feedback-resistant 3-deoxy-D-arabinohepenoyl-7-phosphate synthase (AroG*). D146N Genes and prephenyl acid dehydrogenase TyrA* M53I Genes. This recombinant photosynthetic bacterium strain achieves highly efficient heterologous synthesis of tocopherol by knocking out regulatory genes and integrating multiple metabolic modules, with a fermentation yield of up to 3.51 g / L, showing broad prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a recombinant photosynthetic bacterial strain that produces high levels of tocopherol, its construction method, and its applications. Background Technology

[0002] Vitamin E is an essential vitamin for humans and animals that they cannot synthesize on their own. Based on the saturation of its hydrophobic tail and the number and position of methyl groups on its aromatic ring, vitamin E can be classified into eight types: α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol (Tocopherol), and α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol (Tocotrienol). Both tocopherol and tocotrienol can be absorbed in humans and animals, but tocopherol is more active and is preferentially transported and absorbed. α-Tocopherol is the most active type of vitamin E. With the improvement of living standards, vitamin E has been widely used as a nutritional supplement and antioxidant in industries such as feed, pharmaceuticals, food, and cosmetics. Currently, vitamin E is a widely used and highly produced vitamin in the international market, and along with vitamin C and vitamin A, it is considered one of the three pillar vitamins, with a promising market prospect.

[0003] Natural vitamin E is produced only in plants and photosynthetic algae. Currently, the natural vitamin E on the market is mainly extracted and refined from deodorized products and distillates of vitamin E-rich vegetable oils and oilseed crops. Compared with chemically synthesized vitamin E, natural vitamin E has a higher absorption rate and better physiological activity. However, the vitamin E content is only at the microgram level per gram of crop cells, resulting in problems such as higher cost and lower yield.

[0004] Microorganisms have become a research hotspot due to their rapid growth, wide availability of substrates, low land and water requirements, and natural possession of two precursors for vitamin E synthesis: 4-hydroxyphenylpyruvic acid (shikimic acid pathway) and geranylgeranyl pyrophosphate / phytophosphate (GGPP / PDP, terpene pathway). However, while some reports have documented the production of vitamin E by microorganisms, the product configuration is predominantly δ-tocotrienol. This heterologous synthesis exhibits poor substrate compatibility and low yields, indicating that the host cannot efficiently synthesize tocopherols. Further in-depth research is needed to ensure the feasibility of commercial production. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the purpose of this invention is to provide a high-yield tocopherol-producing recombinant photosynthetic bacteria strain, its construction method, and its applications. This recombinant photosynthetic bacteria strain achieves highly efficient heterologous synthesis of tocopherol by knocking out regulatory genes and integrating multiple metabolic pathways, with a fermentation yield of 3.51 g / L, which is the highest level known to date.

[0006] Therefore, in one aspect of the present invention, a recombinant photosynthetic bacterial strain with high tocopherol production is proposed. This recombinant strain has the photosynthetic gene repressor transcription factor PpsR knocked out and integrates the mevalonate MEV pathway gene from *Paragonimococcus zeatans*, the p-hydroxyphenylpyruvate dioxygenase HPPD gene from *Pseudomonas putida*, the urokinase HPT gene from *Synostemma pentaphyllum*, the tocopherol cyclase TC gene from *Arabidopsis thaliana*, and feedback-resistant 3-deoxy-D-arabinohepenoyl-7-phosphate synthase AroG*. D146N Genes and prephenyl acid dehydrogenase TyrA* M53I Gene; the photosynthetic bacteria is spherical red bacterium HY01.

[0007] The recombinant photosynthetic bacterial strain of the present invention, by knocking out the photosynthesis gene repressor transcription factor PpsR, integrates three heterologous expression pathways: the mevalonate (MEV) pathway of *Paragonimococcus zeatans*, the heterologous vitamin E synthesis pathway (*Pseudomonas putida* p-hydroxyphenylpyruvate dioxygenase HPPD, *Synthia spp.* homogentisate phytotransferase HPT, and *Arabidopsis thaliana* tocopherol cyclase TC), and the shikimic acid enhancement pathway of *Escherichia coli* (feedback-tolerant 3-deoxy-D-arabinohepenoyl-7-phosphate synthase AroG*). D146N and prephenyl acid dehydrogenase TyrA* M53I This enables the efficient heterogeneous production of tocopherol.

[0008] In a second aspect, the present invention provides a method for constructing the above-mentioned recombinant photosynthetic bacteria strain with high tocopherol production, comprising the following steps:

[0009] Step 1: Knock out the photosynthesis gene repressor transcription factor PpsR of the photosynthetic bacteria through homologous recombination, and integrate the mevalonate MEV pathway gene of the zeatin-producing paracoccus into the genome of the photosynthetic bacteria to obtain the precursor-enhanced recombinant strain MEV14.

[0010] Step 2: The p-hydroxyphenylpyruvate dioxidase HPPD gene, the urea-methyl chlorophyll transferase HPT gene, the tocopherol cyclase TC gene, and the feedback-tolerant 3-deoxy-D-arabinohepenoyl-7-phosphate synthase AroG* are added. D146N Gene and the prephenylacetic acid dehydrogenase TyrA*M53I The gene was integrated into the genome of the recombinant strain MEV14 to obtain the integrated recombinant strain;

[0011] Step 3: Using the negative correlation effect of pigment accumulation in photosynthetic bacteria, the integrated recombinant strain was selected, and the high-yield photosynthetic bacteria recombinant strain VE30 was obtained through fermentation yield screening.

[0012] According to the construction method of the present invention, by knocking out the photosynthetic gene repressor transcription factor PpsR, and combining it with transposon genome integration, three heterologous expression pathways are identified: the mevalonate (MEV) pathway of *Paragonimococcus zeatans*, the heterologous vitamin E synthesis pathway (*Pseudomonas putida* p-hydroxyphenylpyruvate dioxygenase HPPD, *Synthia spp.* urea-methyl phytotransferase HPT, and *Arabidopsis thaliana* tocopherol cyclase TC), and the shikimic acid enhancement pathway of *Escherichia coli* (feedback-tolerant 3-deoxy-D-arabinohepeptulose-7-phosphate synthase AroG*). D146N and prephenyl acid dehydrogenase TyrA* M53I The study utilizes the pigment color phenotype of photosynthetic bacteria to screen for high-yield strains, thereby achieving heterologous and efficient synthesis of tocopherol.

[0013] In a third aspect, the present invention proposes the application of the recombinant photosynthetic bacteria strain constructed by the above-described construction method in the production of tocopherol.

[0014] According to the application of this invention, the production of natural tocopherol can be achieved. After optimization of fermentation conditions, a tocopherol yield of up to 3.51 g / L was obtained under batch-fed fermentation conditions. This is the first report of de novo synthesis of tocopherol by microorganisms other than plants and oxygen-producing photosynthetic microorganisms, with a yield far exceeding that of previously reported heterologous vitamin E synthesis, and has broad prospects for industrial application.

[0015] 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

[0016] Figure 1 This invention relates to a pathway for the heterologous expression of three enzymes, HPPD, HPT, and TC, in *Rhodops spheroidae* to catalyze the synthesis of vitamin E according to an embodiment of the present invention.

[0017] Figure 2 This is a flowchart and yield diagram of the construction process of a high-tocopherol-producing recombinant strain of *Rhodophyta spheroidae* according to an embodiment of the present invention, wherein a represents a carotenoid pathway engineering strategy based on the supply of the red phenotype-enhancing precursor geraniol pyrophosphate (GGPP) of spheroidin. ppsR Transcription regulators bchP GGPP reductase crtEb is a flowchart of the process of increasing tocopherol production through transposon mutagenesis (GOI: target gene, Tn5: Tn5 transposon recognition site, RM: resistance marker), c is the tocopherol production of recombinant strains screened from mutant library B in culture tubes, d is the representative results of tocopherol titer and distribution of recombinant strains from mutant library B in shake flasks, and e is the LC-MS results of tocopherol produced by recombinant strain VE30 (retention time, range 400-450 m / z).

[0018] Figure 3 This is a secondary mass spectrum of tocopherol synthesis by *Rhodops spheroidae* according to an embodiment of the present invention;

[0019] Figure 4 This is a diagram showing the growth and production of a high-tocopherol-producing recombinant strain of *Rhodotorula spheroides* under the conditions of a batch-fed fermenter according to an embodiment of the present invention, where a is a growth diagram of the recombinant strain of *Rhodotorula spheroides* and b is a distribution diagram of tocopherol production by the recombinant strain of *Rhodotorula spheroides*. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Source of materials used: HY01, a type of spheroidal red bacterium, taxonomically named *Cerebrobacterium cereus* (…). Cereibacter sphaeroidesThe DNA sample was deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC1.16068, on February 15, 2017. Phusion high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from Xiamen Lulong Biotechnology Development Co., Ltd., while kanamycin, ampicillin, and gentamicin were purchased from Shanghai Sangon Biotech Co., Ltd. Plasmid extraction kits, DNA purification kits, gel extraction kits, and genomic DNA extraction kits were purchased from Hangzhou Bori Biotechnology Co., Ltd.

[0024] The composition of LB medium is: 10 g·L -1 Peptone, 5 g·L -1 Yeast powder, 10 g·L -1 NaCl, balance double-distilled water, autoclave at 121℃ for 30 min, add 15 g·L⁻¹ as needed. -1 Agar powder is used to make a solid culture medium.

[0025] The MedA medium consists of: 20 mL / L Solution C, 0.5 g / L (NH4)2SO4, 1 g / L NaCl, 2 g / L sodium succinate, 0.1 g / L glutamic acid, 0.04 g / L aspartic acid, with the remainder being double-distilled water. The pH is adjusted to 7.0, and the medium is autoclaved at 121°C for 30 min. Then, 20 mL / L phosphate buffer and 1 mL / L multivitamin solution are added.

[0026] The compound vitamin solution consists of: 5 mg / L thiamine hydrochloride, 10 mg / L niacin, 0.1 mg / L biotin, with the remainder being double-distilled water.

[0027] The MYG medium consisted of: 5 g / L yeast extract, 20 g / L glucose, 20 mL / L Solution C, 0.5 g / L (NH4)2SO4, 1 g / L NaCl, 2 g / L sodium succinate, 0.1 g / L glutamic acid, 0.04 g / L aspartic acid, with the remainder being double-distilled water. The pH was adjusted to 7.0, and the medium was autoclaved at 121℃ for 30 min. Then, 20 mL / L phosphate buffer and 1 mL / L multivitamin solution were added.

[0028] The Solution C composition in the MedA and MYG media was as follows: 10 g / L nitroglycerin, 29.5 g / L MgCl₂·6H₂O, 3.3 g / L CaCl₂·2H₂O, 0.1 g / L FeSO₄·7H₂O, and 0.1 g / L (NH₄)₆Mo₇O. 24• 4H2O, 50mL / L Trace element, the remainder is double-distilled water, and KOH is used to adjust the pH to 6.8-7.0.

[0029] The trace element composition is: 10.95 g / L ZnSO4·7H2O, 2.5 g / L LEDTA, 5.0 g / L FeSO4·7H2O, 114 mg / L H3BO3, 1.54 g / L MnSO4·H2O, 392 g / L CuSO4·5H2O, 248 g / L Co(NO3)2·6H2O, with the balance being double-distilled water.

[0030] The YCG culture medium consists of: 40 g / L glucose, 4 g / L corn steep liquor powder, 3 g / L sodium glutamate, 2.8 g / L NaCl, 3 g / L (NH4)2SO4, 3 g / L KH2PO4, 6.3 g / L MgSO4, 2 g / L CaCO3, with the remainder being double-distilled water. The pH is adjusted to 6.5, and the medium is autoclaved at 121℃ for 30 min before adding 1 mL / L of compound vitamin solution.

[0031] Tocopherol standards were purchased from Beijing Bailingwei Technology Co., Ltd.

[0032] Extraction and analysis of vitamin E: Collect 1 mL of fermentation broth, centrifuge at 7000 rpm for 3 min, wash the cells twice with distilled water, extract vitamin E with 1 mL of acetone, and transfer to a 2 mL centrifuge tube. Add 100 μL of grinding beads (Ф = 0.5 mm), grind in an automatic sample grinder, and then centrifuge at 13000 rpm for 10 min. Take the organic supernatant, filter it into a liquid chromatography bottle.

[0033] To further determine the composition and yield of the product, α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol standards were dissolved in acetone to prepare standard solutions of a certain concentration. The product extract and standards were then analyzed using a Shimadzu HPLC-MS / MS system with a Shimadzu C18 column (4.6 × 250 mm, 5 μm) and a photodiode array detector (working wavelength 280 nm). The mobile phase was pure water (A) and acetonitrile (B), using a gradient elution program at a flow rate of 0.8 mL / min. -1The column temperature was 40℃, and the injection volume was 10 μL. From 0 to 10 min, the linear gradient was from 30% A / 70% B to 10% A / 90% B; from 10 to 40 min, the linear gradient was from 10% A / 90% B to 0% A / 100% B; from 40 to 70 min, the column was held at 0% A / 100% B; from 70 to 71 min, the linear gradient was from 0% A / 100% B to 30% A / 70% B.

[0034] The plasmids involved in the following examples were constructed in E. coli The gene editing and transposon integration expression were carried out using DH5α λpir (commercially available). After plasmid construction, the plasmid was transformed into Rhodotorula spheroidae HY01 for gene editing and transposon integration expression.

[0035] Table 1 Primers used for PCR amplification

[0036]

[0037] Table 2. DNA sequence of the vector

[0038]

[0039] like Figure 1As shown, the two key precursors in vitamin E biosynthesis are homogentisic acid (HGA) and phytyl diphosphate (PDP). HGA is synthesized via the shikimic acid pathway and is used to synthesize the hydrophilic head of vitamin E, while PDP originates from the 1-deoxy-D-xylitol-5-phosphate (DXP) pathway or the mevalonate (MVA) pathway of terpene synthesis and is used to synthesize the hydrophobic tail of vitamin E. In the shikimic acid pathway, tyrosine is synthesized into 4-hydroxyphenylpyruvate, which is then catalyzed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) to generate HGA. In the DXP pathway, glyceraldehyde-3-phosphate and pyruvate form DXP, which then undergoes multiple catalytic steps to form geraniol-geraniol diphosphate (GGPP); in the MVA pathway, acetyl-CoA undergoes multiple catalytic steps to form GGPP. GGPP is then converted to PDP by geraniol-geraniol reductase (GGR). HGA undergoes condensation with PDP under the catalysis of homogentisate phytotransferase (HPT) to generate 2-methyl-6-phytobenzoquinone (MPBQ). MPBQ is then converted to 2,3-dimethyl-6-phytobenzoquinone (DMPBQ) by MPBQ methyltransferase (MPBQMT). Tocopherol cyclase (TC) can directly act on MPBQ to generate δ-tocopherol, or catalyze the conversion of DMPBQ to γ-tocopherol. δ-tocopherol and γ-tocopherol are then converted to β-tocopherol and α-tocopherol, respectively, by γ-tocopherol methyltransferase (γ-TMT).

[0040] The recombinant photosynthetic bacteria strain constructed in the embodiments of the present invention integrates three heterologous expression pathways by knocking out the photosynthesis gene repressor transcription factor PpsR: the mevalonate (MEV) pathway of *Paragonimus westermani*, the heterologous vitamin E synthesis pathway (*Pseudomonas putida* p-hydroxyphenylpyruvate dioxygenase HPPD, *Synthia spp.* homogentisate phytotransferase HPT, and *Arabidopsis thaliana* tocopherol cyclase TC), and the shikimic acid enhancement pathway of *Escherichia coli* (feedback-tolerant 3-deoxy-D-arabinohepeptulose-7-phosphate synthase AroG*). D146N and prephenyl acid dehydrogenase TyrA* M53I This method enables the efficient heterogeneous production of tocopherol. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) identified the product as containing natural tocopherol, achieving de novo synthesis of tocopherol from photosynthetic bacteria other than aerobic photosynthetic microorganisms and plants. This solves the problem of low levels of natural tocopherol in its natural host and has broad prospects for industrial application.

[0041] 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.

[0042] Example 1 Acquisition ppsR Knockout photosynthetic bacteria recombinant strain Rsp-ΔppsR

[0043] Using the genome of *Rhodopsinia spheroidae* HY01 (NCBI: NZ_CM125964.1) as a template, and primers ppsR-F1 (SEQ ID No. 1) and ppsR-R1 (SEQ ID No. 2) as primers (Table 1), the upstream fragment of the PpsR gene was amplified by PCR. The downstream fragment of the PpsR gene was amplified by PCR using primers ppsR-F2 (SEQ ID No. 3) and ppsR-R2 (SEQ ID No. 4) as primers (Table 1). Using the upstream and downstream fragments of the PpsR gene as templates, and primers ppsR-F1 (SEQ ID No. 1) and ppsR-R2 (SEQ ID No. 4) as primers, overlap extension PCR was performed to amplify the upstream and downstream homologous arms containing the PpsR gene. The target bands were verified by gel electrophoresis, and purified DNA fragments were obtained using a gel extraction and purification kit. PCR amplification conditions: 98 ℃ for 3 min; 98 ℃ for 15 s, 64 ℃ for 1 min, 72 ℃ for 3 min, 30 cycles; 72 ℃ for 5 min, 4 ℃ for maintenance.

[0044] The upstream and downstream homologous arm fragments of the PpsR gene and the nucleotide sequence of the vector pK18mobSacB (Table 2) shown in SEQ ID No. 15 were respectively used... Eco RI and Hin The DNA fragments were obtained after digestion with dIII enzyme and gel electrophoresis. These two fragments (the upstream and downstream homologous arms of the PpsR gene and the DNA fragment of the vector pK18mobSacB) were mixed at a molar ratio of 6:1 and ligated overnight at 16 °C using T4 DNA ligase. The ligated product was transformed into *E. coli* DH5α λpir competent cells, and single colonies were cultured. Using primers ppsR-F1 (SEQ ID No. 1) and ppsR-R2 (SEQ ID No. 4) as primers, positive single colonies were obtained by PCR verification. The recombinant plasmid was extracted, and sequencing yielded the correct recombinant plasmid pK18-ΔppsR.

[0045] The recombinant plasmid pK18-ΔppsR was electroporated into *Rhodotorula spheroides* HY01 and plated onto MedA plates supplemented with kanamycin. The plates were incubated upside down at 35 °C for 2-3 days. One to two single colonies were randomly selected and incubated overnight in 1 mL of MedA. 1 μL, 3 μL, and 5 μL of the culture were then plated onto MedA plates supplemented with 8% sucrose and incubated upside down at 35 °C for 2-3 days. Multiple single colonies were randomly selected, and positive single colonies were obtained by colony PCR verification using primers ppsR-F1 (SEQ ID No. 1) and ppsR-R2 (SEQ ID No. 4). The positive single colonies were cultured on MedA, and the genome was extracted using a bacterial genome extraction kit. PCR verification was performed again using primers ppsR-F1 (SEQ ID No. 1) and ppsR-R2 (SEQ ID No. 4). Sequencing confirmed the correct recombinant strain Rsp-ΔppsR.

[0046] Example 2: Obtaining the precursor-enhanced recombinant photosynthetic bacterial strain MEV14

[0047] The MEV pathway gene fragment of *Paragonimococcus zeatans* synthesized from the gene with nucleotide sequence SEQ ID No. 19 (Table 2) and the gene with nucleotide sequence SEQ ID No. 16 are shown in Table 2. Esp The transposon vector pRL27Ptac-K (Table 2) fragment digested with 3I enzyme was mixed at a molar ratio of 6:1 and ligated overnight at 16 °C using T4 DNA ligase. The ligated product was transformed into E. coli DH5α λpir competent cells, and single colonies were selected for culture. The recombinant plasmid was extracted, and the correct recombinant plasmid pRLK-MEV was obtained after sequencing.

[0048] The recombinant plasmid pRLK-MEV was electroporated into the recombinant strain Rsp-ΔppsR from Example 1, spread on MedA plates supplemented with kanamycin, and incubated upside down at 35 °C for 2-3 days. Darker-colored single colonies were picked and inoculated into shake tubes containing 2 mL of MedA liquid medium, and cultured at 35 °C and 250 rpm for 24 h to obtain the seed culture. The seed culture was then inoculated at a rate of 1% (v / v) into shake flasks containing 20 mL of MYG liquid medium and cultured at 30 °C and 250 rpm for 72 h to obtain the fermentation broth.

[0049] Collect 1 mL of fermentation broth, centrifuge at 7000 rpm for 3 min, wash the cells twice with distilled water, extract the pigment with 1 mL of pre-cooled acetone-methanol (v:v=7:2) mixture, and transfer to a 2 mL centrifuge tube for overnight extraction at 4 °C; then centrifuge at 13000 rpm for 10 min, collect the organic supernatant, and measure the absorbance at 482 nm using a spectrophotometer, combined with 122 mM... -1 cm -1 The extinction coefficient is used to determine the pigment content. For example... Figure 2 a and Figure 2 As shown in b, by comparing pigment content, the optimal photosynthetic bacterial recombinant strain MEV14 with precursor enhancement (high pigment content) was obtained.

[0050] Example 3: Obtaining a recombinant spherical red bacterium strain VE30 with high tocopherol production

[0051] Using the genome of *Pseudomonas putida* KT2440 (NCBI: NC_002947.4) as a template, and using primers (SEQ ID No. 5: HPPD-F and SEQ ID No. 6: HPPD-R) as primers (Table 1), the HPPD gene fragment was amplified by PCR. Using the genome of *Syntropha cytogenes* PCC6803 (NCBI: NC_000911.1) as a template, and using primers (SEQ ID No. 7: HPPD-F and SEQ ID No. 8: HPPD-R) as primers (Table 1), the HPPD gene fragment was amplified by PCR. Using *Arabidopsis thaliana* cDNA (NCBI: CP002687.1) as a template, and using primers (SEQ ID No. 9: TC-F1 and SEQ ID No. 10: TC-R1) as primers (Table 1), the TC gene fragment A was amplified by PCR. Using *Arabidopsis thaliana* cDNA (NCBI: CP002687.1) as a template, and using primers (SEQ ID No. 5: HPPD-F and SEQ ID No. 6: HPPD-R) as primers, the HPPD gene fragment was amplified by PCR. Using TC-F2 (shown in No. 11) and TC-R2 (shown in SEQ ID No. 12) as primers (Table 1), TC gene fragment B was obtained by PCR amplification. Using TC gene fragment A and TC gene fragment B as templates, and TC-F1 (shown in SEQ ID No. 9) and TC-R2 (shown in SEQ ID No. 12) as primers, the complete TC gene fragment was obtained by overlap extension PCR amplification. The target band was verified by gel electrophoresis, and the purified DNA fragment was obtained using a gel extraction and purification kit. PCR amplification conditions: 98 ℃ for 3 min; 98 ℃ for 15 s, 60 ℃ for 45 s, 72 ℃ for 2 min, 30 cycles; 72 ℃ for 3 min, 4 ℃ hold.

[0052] The above HPPD gene fragment, HPT gene fragment, and TC gene fragment were used... Bsa I enzyme digestion, the transposon vector pRL27Ptac-A with the nucleotide sequence shown in SEQ ID No. 17 was used Esp The DNA fragments were obtained after digestion with 3I enzymes and gel electrophoresis. These four fragments (HPPD gene fragment, HPT gene fragment, TC gene fragment, and DNA fragment from the transposon vector pRL27Ptac-A) were mixed at a molar ratio of 2:2:2:1 and ligated overnight at 16 °C using T4 DNA ligase. The ligated products were transformed into *E. coli* DH5α λpir competent cells, and single colonies were cultured. Using primers (HPPD-F as shown in SEQ ID No. 5 and TC-R2 as shown in SEQ ID No. 12), positive single colonies were obtained by PCR verification. The recombinant plasmid was extracted, and sequencing yielded the correct recombinant plasmid pRLA-HPPD-HPT-TC.

[0053] plasmid pCDF-aroG, whose nucleotide sequence is shown in SEQ ID No. 20. fbr -tyrA fbr Using Table 2 as a template, and with AroG-F (nucleotide sequence SEQ ID No. 13) and TyrA-R (nucleotide sequence SEQ ID No. 14) as primers (Table 1), the AroG*-TyrA* fragment was amplified by PCR. The target band was verified by gel electrophoresis, and the purified DNA fragment was obtained using a gel extraction and purification kit. PCR amplification conditions: 98 ℃ for 3 min; 98 ℃ for 15 s, 56 ℃ for 1 min, 72 ℃ for 3 min, 30 cycles; 72 ℃ for 5 min, 4 ℃ hold.

[0054] The above AroG*-TyrA* fragment is used with Bsa I enzyme digestion, the transposon vector pRL27Ptac-G with the nucleotide sequence shown in SEQ ID No. 18 was used Esp The DNA fragments were obtained after digestion with 3I enzyme and gel electrophoresis. These two fragments (AroG*-TyrA* fragment and DNA fragment from the transposon vector pRL27Ptac-G) were mixed at a molar ratio of 6:1 and ligated overnight at 16°C using T4 DNA ligase. The ligated product was transformed into *E. coli* DH5α λpir competent cells, and single colonies were cultured. Using primers (AroG-F as shown in SEQ ID No. 13 and TyrA-R as shown in SEQ ID No. 14), positive single colonies were obtained by PCR verification. The recombinant plasmid was extracted, and sequencing yielded the correct recombinant plasmid pRLG-AroG*-TyrA*.

[0055] The recombinant plasmids pRLA-HPPD-HPT-TC and pRLG-AroG*-TyrA* were electroporated together into the photosynthetic bacterial recombinant strain MEV14 of Example 2. The resulting plating was spread on MedA plates supplemented with kanamycin, ampicillin, and gentamicin, and incubated upside down at 35 °C for 2-3 days. Lighter-colored single colonies were picked and inoculated into shake tubes containing 2 mL of MYG liquid medium, and incubated at 30 °C and 250 rpm for 72 h to obtain the fermentation broth. Figure 2 c and Figure 2 As shown in Figure d, three high-yielding strains (VE30, VE31, and VE45) were initially screened out through tocopherol composition and yield analysis. These strains were then inoculated into shake tubes containing 2 mL of MedA liquid medium and cultured at 35 °C and 250 rpm for 24 h to obtain seed culture. The seed culture was then inoculated at a rate of 1% (v / v) into shake flasks containing 20 mL of MYG liquid medium and cultured at 30 °C and 250 rpm for 72 h to obtain fermentation broth.

[0056] Collect 1 mL of fermentation broth, centrifuge, wash cells twice with distilled water, resuspend in 1 mL acetone to extract vitamin E, transfer to a 2 mL centrifuge tube, add 100 μL of grinding beads (Ф = 0.5 mm), grind in an automated sample grinder, centrifuge, collect the organic supernatant, filter, and sample into a liquid chromatography bottle. Qualitative and quantitative analysis of the product was performed using high-performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLC-MS / MS). The results are as follows: Figure 2 d、 Figure 2 e and Figure 3 As shown, the synthesized vitamin E consists of natural α-tocopherol, γ / β-tocopherol, and δ-tocopherol; by comparing tocopherol yields, the optimal photosynthetic bacterial recombinant strain VE30 for high tocopherol production was obtained.

[0057] Example 4: Increased Tocopherol Yield Through Fed-batch Fermentation

[0058] A single-clone recombinant strain VE30 (obtained in Example 3) was inoculated into 150 mL of MYG culture medium in a shake flask and cultured at 35°C and 250 rpm for 24 h to obtain a seed culture. The seed culture was then inoculated into 1 L of YCG culture medium at a 10% (v / v) inoculation rate and cultured at 30°C in a 5 L fermenter. The dissolved oxygen was maintained at 20% air saturation by adjusting the stirring speed to 300-600 rpm and the constant air input flow rate to 1.0 vvm, and 5 M ammonia was automatically added to maintain the pH at 6.5. After 18 hours of fermentation, a fed-batch culture method was adopted: the glucose concentration in the fermenter was maintained at approximately 15 g / L by adding 600 g / L concentrated glucose solution. After 48 hours, the glucose concentration was adjusted to approximately 5 g / L, and 25 mL of feed medium (glucose-free YCG fermentation medium) was added every 24 hours. In addition, 4 mM methionine should be added to the initial fermentation medium, and then replenished every 60 hours. After 24 hours of fermentation, 2 mM 2-hydroxypropyl-β-cyclodextrin should be manually added to the medium, and then replenished every 48 hours. During fermentation, the fermentation broth should be collected regularly to monitor cell growth, glucose concentration, and tocopherol production. Figure 4 As shown, after 132 h of fermentation, a total of 3.51 g / L of tocopherol was finally obtained, which is the highest reported yield of heterologous vitamin E to date.

[0059] 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.

[0060] 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 recombinant strain of photosynthetic bacteria with high tocols production, characterized in that, The recombinant photosynthetic bacteria strain knocked out the photosynthetic gene repressor transcription factor PpsR and integrated the mevalonate MEV pathway gene from *Paragonimococcus zeatans*, the p-hydroxyphenylpyruvate dioxidase HPPD gene from *Pseudomonas putida* KT2440, the homogentisic acid chlorophyll transferase HPT gene from *Synostemma pentaphyllum* PCC6803, the tocopherol cyclase TC gene from *Arabidopsis thaliana*, and the feedback-resistant 3-deoxy-D-arabinohepeptulose-7-phosphate synthase AroG*. D146N Genes and prephenyl acid dehydrogenase TyrA* M53I Gene; the photosynthetic bacteria mentioned are spherical red bacteria HY01, whose taxonomic name is *Cyperobacter cereus* (…). Cereibacter sphaeroides (), with accession number CGMCC 1.16068; The nucleotide sequence of the mevalonate MEV pathway gene of the zeatin-producing Paracoccus is shown in SEQ ID No. 19; The fragment of the p-hydroxyphenylpyruvate dioxygenase HPPD gene was prepared by using the genome of *Pseudomonas putida* KT2440 as a template, and using the nucleotide sequence HPPD-F shown in SEQ ID No. 5 and the nucleotide sequence HPPD-R shown in SEQ ID No. 6 as primers, and amplified by PCR. The GenBank accession number of the genome of *Pseudomonas putida* KT2440 is NC_002947.

4. The fragment of the homosuccinate chlorophyll transferase HPT gene was prepared by using the genome of Synechocystis PCC6803 as a template, and using HPT-F (nucleotide sequence SEQ ID No. 7) and HPT-R (nucleotide sequence SEQ ID No. 8) as primers for PCR amplification. The GenBank accession number of the genome of Synechocystis PCC6803 is NC_000911.

1. The tocopherol cyclase TC gene fragments were prepared by using TC gene fragment A and TC gene fragment B as templates, and TC-F1 (SEQ ID No. 9) and TC-R2 (SEQ ID No. 12) as primers, followed by overlap extension PCR amplification. TC gene fragment A was prepared by using Arabidopsis cDNA as a template, and TC-F1 (SEQ ID No. 9) and TC-R1 (SEQ ID No. 10) as primers, followed by PCR amplification. TC gene fragment B was prepared by using Arabidopsis cDNA as a template, and TC-F2 (SEQ ID No. 11) and TC-R2 (SEQ ID No. 12) as primers, followed by PCR amplification. The GenBank accession number for the Arabidopsis cDNA is CP002687.

1. said feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase AroG D146N said prephenate dehydrogenase TyrA M53I Preparation of the plasmid pCDF-aroG represented by the nucleotide sequence SEQ ID No. 20 fbr -tyrA fbr AroG-F represented by the nucleotide sequence SEQ ID No. 13 and TyrA-R represented by the nucleotide sequence SEQ ID No. 14 as primers.

2. The application of the recombinant photosynthetic bacteria strain according to claim 1 in the production of tocopherol.

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