Reformation method for producing coenzyme q10 genetically engineered bacteria and application thereof

Abstract

The application discloses a production coenzyme Q10 gene engineering bacteria modification method and application thereof. The endogenous CDP-diacylglycerol-serine O-phospholipid transferase gene (pssA) and phosphatidylserine decarboxylase gene (psd) are cloned from a Rhodobacter sphaeroides 2.4.1 strain genome, an overexpression vector is constructed, and the pssA and psd are expressed in Rhodobacter sphaeroides 2.4.1 cells, so that the content of phosphatidylethanolamine in the industrial strain cell membrane lipid bilayer can be significantly improved, the capacity of the cell membrane to contain CoQ 10 is strengthened, and the yield of coenzyme CoQ 10 in the Rhodobacter sphaeroides is further improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for modifying genetically engineered bacteria that produce coenzyme Q10 and its application. Background Technology

[0002] Coenzyme Q 10 (Coenzyme Q 10 Or CoQ 10 ), chemically named 2,3-dimethoxy-5-methyl-6-decanoisopentadienylbenzoquinone, is a naturally occurring lipid-soluble quinone compound. Its structure is synthesized from quinone derivatives and multiple isopentenyl monomers. Q 10 CoQ represents a group of quinone compounds with ten isoprene units in their side chains; this is present in both humans and mammals. 10 CoQ is an important electron transporter in the cellular respiratory chain, found on the inner mitochondrial membrane of eukaryotes or the cytoplasmic membrane of prokaryotes, and participates in cellular energy metabolism. 10 CoQ generally exists in two forms: oxidized and reduced. Because its reduced form possesses antioxidant activity, it is present in cells. 10 It exists primarily in a reduced state. CoQ 10 The transition between oxidized and reduced forms activates cell metabolism, provides antioxidant protection, protects lipoproteins, and protects DNA molecules from damage (inhibiting free radical-induced oxidation). Furthermore, CoQ... 10 Synthesized endogenously within cells, CoQ contains few impurities, thus it is receiving increasing attention as an antioxidant in the pharmaceutical, cosmetic, and food industries. Additionally, CoQ... 10 It also plays an important role in anti-tumor and anti-aging effects, as well as in the treatment of gastrointestinal, liver, and heart diseases and the enhancement of immune system function. It also has significant effects in the treatment of scurvy, duodenal ulcers, necrotizing periodontitis, and in promoting pancreatic function and secretion. Its clinical application value is extremely high.

[0003] CoQ 10 CoQ is widely present in plants and animals in nature. 10 The production methods for CoQ include extraction from animal and plant tissues, chemical synthesis, and biosynthesis. Extraction and chemical synthesis methods suffer from limited raw material sources, complex processes, and low yields. In contrast, microbial fermentation offers advantages such as a wide range of raw material sources, high product activity, and high yields, and has become the preferred method for producing CoQ. 10 The main methods of industrial production. (Production of CoQ) 10 There are many types of microorganisms, among which Rhodophyta globulina, a type of photosynthetic bacteria, synthesizes CoQ intracellularly. 10 With a high content and relatively simple extraction steps, it is currently the main method for large-scale industrial production of CoQ. 10Important strains.

[0004] However, the current limitations of microbial fermentation for CoQ production 10 The main factor contributing to the low product yield lies in the genetic characteristics of the strain and the complex fermentation regulation, resulting in poor product yield. CoQ synthesized intracellularly by *Rhodotorula globulus*. 10 The pathways involved are similar to those of most bacteria, mainly including isoprene pyrophosphate synthesis (mevaleric acid pathway), polydecaprene pyrophosphate synthesis (MEP pathway), and quinone ring synthesis and modification (branched acid pathway). 10 It is a lipid-soluble molecule, and after being synthesized within the cell, it is specifically delivered to the lipid bilayer of the cell membrane to exert its function. Therefore, it is found in cells capable of synthesizing large quantities of CoQ. 10 In industrial microbial strains, the cell membrane is often contaminated with excessive CoQ20. 10 The filling and even the state of the cell membrane change, thereby affecting more CoQ cells. 10 Positioning in the lipid bilayer. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art and provide a method for modifying genetically engineered bacteria to produce coenzyme Q10.

[0006] Another object of the present invention is to provide the application of the genetically engineered bacteria for producing coenzyme Q10 obtained by the above-described modification method.

[0007] The technical solution of the present invention is as follows:

[0008] A method for modifying genetically engineered bacteria to produce coenzyme Q10 includes the following steps:

[0009] (1) The CDP-diacylglycerol-serine O-phosphatidyltransferase gene as shown in SEQ ID NO.01 and the phosphatidylserine decarboxylase gene as shown in SEQ ID NO.02 were cloned from the genome of Rhodobacter sphaeroides 2.4.1 (see CN106148263B);

[0010] (2) Insert the above CDP-diacylglycerol-serine O-phosphatidyltransferase gene and phosphatidylserine decarboxylase gene into the overexpression vector to construct a recombinant overexpression vector;

[0011] (3) The above recombinant overexpression vector was transformed into Rhodobacter sphaeroides 2.4.1 for recombinant expression.

[0012] In a preferred embodiment of the present invention, the primers used to clone the CDP-diacylglycerol-serine O-phosphatidyltransferase gene are pssA-F as shown in SEQ ID NO.03 and pssA-R as shown in SEQ ID NO.04.

[0013] In a preferred embodiment of the present invention, the primers used to clone the phosphatidylserine decarboxylase gene are psd-F as shown in SEQ ID NO.05 and psd-R as shown in SEQ ID NO.06.

[0014] In a preferred embodiment of the present invention, the primers used to clone the CDP-diacylglycerol-serine O-phosphatidyltransferase gene are pssA-F as shown in SEQ ID NO.03 and pssA-R as shown in SEQ ID NO.04, and the primers used to clone the phosphatidylserine decarboxylase gene are psd-F as shown in SEQ ID NO.05 and psd-R as shown in SEQ ID NO.06.

[0015] In a preferred embodiment of the present invention, the overexpression vector is plasmid pBBR1MCS-2.

[0016] A genetically engineered bacterium that produces coenzyme Q10 was obtained through the above-mentioned modification method.

[0017] A method for producing coenzyme Q10 by fermentation includes a culture step of culturing the above-mentioned coenzyme Q10-producing genetically engineered bacteria.

[0018] The beneficial effects of this invention are as follows: This invention clones the endogenous CDP-diacylglycerol-serine O-phosphatidyltransferase gene (pssA) and phosphatidylserine decarboxylase gene (psd) from the genome of *Rhodobacter sphaeroides* 2.4.1, constructs an overexpression vector, and performs recombinant expression within *Rhodobacter sphaeroides* 2.4.1 cells. This significantly increases the content of phosphatidylethanolamine in the lipid bilayer of the cell membrane of industrial strains, thereby enhancing the cell membrane's capacity to accommodate CoQ. 10 The ability to further enhance coenzyme CoQ 10 Production within Rhodocytosporum cells. Attached Figure Description

[0019] Figure 1 The spectrum of the pBBR1MCS-2-pssA-psd-his vector constructed in Example 1 of this invention.

[0020] Figure 2It is a result diagram of HPLC detection of CoQ10 produced by the genetically engineered strain for coenzyme Q10 production in Example 1 of the present invention. Detailed Description of Embodiments

[0021] The technical solution of the present invention is further illustrated and described below through specific embodiments in conjunction with the accompanying drawings.

[0022] Example 1

[0023] (1) Construction of pBBR1MCS-2-pssA-psd-his vector:

[0024] a. CDP-diacylglycerol-serine O-phosphotransferase (PssA) gene and phosphatidylserine decarboxylase (PSD) gene are obtained by PCR from the genomic DNA of Rhodobacter sphaeroides 2.4.1: Rhodobacter sphaeroides is inoculated into 50 mL LB medium and cultured overnight at 50 mL, and a genome extraction kit (purchased from TIANGEN) is used to extract the genome of Rhodobacter sphaeroides; with primers pssA-F (SEQ ID NO. 03, Sal I restriction site) and pssA-R (SEQ ID NO. 04, Hind III restriction site), psd-F (SEQ ID NO. 05, Sal I restriction site) and psd-R (SEQ ID NO. 06, Hind III restriction site), and using the genome of Rhodobacter sphaeroides 2.4.1 as a DNA template, pssA gene (SEQ ID NO. 01) and psd gene (SEQ ID NO. 02) are obtained by cloning. The PCR conditions are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 2 min, 30 cycles;

[0025] SEQ ID NO. 01:

[0026]

[0027] SEQ ID NO. 02:

[0028]

[0029] SEQ ID NO. 03: pssA-F: cgtaacaggaggaattaaccatgctgcggcgcgacgaccg

[0030] SEQ ID NO. 04: pssA-R: gtaccagatctaccctcgagtcaggagcgccagcggcgca

[0031] SEQ ID NO.05:psd-F:cgtaacaggaggaattaaccatggcgatt=gacctgctctc

[0032] SEQ ID NO.06:psd-R:gtaccagatctaccctcgagtcagcgcgccgcgcc

[0033] b. Using an agarose gel extraction kit (Omega), the psd and pssA genes obtained by PCR were excised and recovered from the gel. The psd and pssA genes and plasmid pBBR1MCS-2 were double-digested with Sal I and Hind III, respectively. Ligation was then performed at 16℃ for 1 hour. The ligation product was transformed into *E. coli* T1 competent cells using a 42℃ heat shock method. The cells were plated on LB agar plates containing kanamycin and cultured overnight. Positive strains were screened and expanded to extract the positive clone plasmid pBBR1MCS-2 (i.e., pBBR1MCS-2). Figure 1 The pBBR1MCS-2-pssA-psd-his vector shown is ready for use (plasmid extraction kit purchased from TIANGEN).

[0034] (2) Transformation of E. coli S17-1 (donor bacteria) with pBBR1MCS-2-pssA-psd-his vector: Take one tube of competent E. coli S17-1 cells from the ultra-low temperature freezer, place them on ice to thaw, and gently add 0.5 μL of pBBR1MCS-2-pssA-psd-his vector. Incubate together on ice for 10 min, heat shock at 42℃ for 60 s, incubate on ice for 10 min, and finally add 600 μL of antibiotic-free LB liquid medium and mix. Incubate at 37℃ for 45 min, centrifuge for 4 min, take 100 μL and spread it on a plate containing Kans resistance, pick positive strains, extract plasmids and sequence for verification.

[0035] (3) Conjugation transformation of pBBR1MCS-2-pssA-psd-his vector into Rhodobacter phaeroides 2.4.1 (recipient bacterium):

[0036] a. Take 0.1% of the donor bacteria S17-1 (containing plasmid) and culture it overnight in LB liquid medium containing Kan antibiotic. The next day, take 5 mL of the bacterial culture and transfer it to 50 mL of LB liquid medium containing Kan antibiotic. Continue to culture for about 1-2 hours until the donor bacteria S17-1 has fully reached the logarithmic growth phase (OD600: 0.5).

[0037] b. Take 0.1% of the recipient bacterium—Rhodobacter sphaeroides 2.4.1—and culture it in LB liquid medium at 30°C for 24 hours. Transfer 5 mL of the bacterial culture to 50 mL of LB medium containing Kan antibiotic and continue culturing for approximately 20 hours until the recipient bacterium is fully in the logarithmic growth phase (OD). 600 2.5). Take 1.4 mL of bacterial culture, centrifuge, wash twice with fresh LB medium, and dilute the donor and recipient bacteria with 400 μL and 600 μL of LB medium respectively. Mix the donor and recipient bacteria at a ratio of 1:10.

[0038] c. Spread the bacterial culture onto a non-resistant LB solid plate and pre-incubate for 20-24 hours. Wash the bacteria on the plate into a 1.5 mL EP tube with 1 mL of pre-cooled 1×Sistrom's medium (a special medium for conjugation of Rhodopseudomonas aeruginosa).

[0039] d. Centrifuge at 8000 rpm for 2 min at 4℃, discard the supernatant, add 1 mL of 1×Sistrom's medium, mix by pipetting and aspiration, and repeat the step once.

[0040] e. After resuspending the bacteria in 100 μL of 1×Sistrom's solution, spread the solution onto a 1×Sistrom's plate containing a final concentration of 150 mg / L K2TeO3 and 50 mg / mL Kan resistance. Incubate at 32°C for about 5 days until black conjugates grow. Pick out the black conjugates, which are the recombinant Rhodotorula glutinis containing pBBR1MCS-2-pssA-psd (i.e., the genetically engineered bacteria that produce coenzyme Q10).

[0041] (10×Sistrom's medium composition: per 100mL of medium contains 4.0g succinic acid, 0.5g (NH4)2SO4, 0.10g monosodium glutamate, 0.04g aspartic acid, 1.0mg nicotinic acid, 0.50mg vitamin B1, 0.010mg biotin, 0.3g MgSO4·7H2O, 0.0344g CaCl2·H2O, 0.002g FeSO4·7H2O, 0.2mL (NH4)6MO7O) 24 (1% Solution, adjust pH to 4.5-4.9)

[0042] (4) Fermentation culture of genetically engineered bacteria for coenzyme Q10 production and extraction and analysis of coenzyme Q10:

[0043] Plasmids were extracted from the positive clones of the genetically engineered bacteria that produce Coenzyme Q10, and sequenced for verification. 500 μL of the successfully sequenced positive clone was transferred from the preservation tube to 25 mL of LB seed culture medium and incubated at 32°C and 220 rpm for 24 h on a shaker. 9 mL of the seed culture medium was then transferred to 45 mL of Rhodopseudomonas aeruginosa fermentation medium and incubated at 32°C and 220 rpm for 48 h.

[0044] Fermentation medium components of Rhodopseudomonas aeruginosa (per 1L): glucose 7g, peptone 3.2g, monosodium glutamate 1.2g, NH4Cl 3.8g, FeCl3 0.25g / L, MgSO4 6g / L, corn steep liquor 1.2g, Na2HPO4 1.8g, NaH2PO4 2g, ZnCl2 0.0015g, KCl 2.4g, CuSO4 0.06g, Ca(HCO3)2 7.2g, thiamine hydrochloride 20g, riboflavin 3g, folic acid 1g, nicotinamide 25g;

[0045] Take 1 mL of fermentation broth, add 200 μL of 6 mol / L hydrochloric acid, and shake thoroughly to mix; add 2 mL of acetone, and shake thoroughly to mix; add 100 μL of 30% hydrogen peroxide, and shake thoroughly to mix; add anhydrous ethanol to bring the volume to 10 mL, and shake thoroughly to mix; extract by sonication for 45 min in the dark (temperature below 30℃), shake well after sonication, let stand for 10 min, and take the upper organic phase and filter it through a 0.22 μm organic microporous membrane into a liquid chromatography bottle for HPLC quantitative analysis of coenzyme Q. 10 The HPLC detection conditions were as follows: The liquid chromatography instrument was a Thermo DAD3000 high performance liquid chromatograph; the column model was SunFire™, C18 reversed-phase column (4.6×150mm, 1.7μm); the mobile phase was ethanol:methanol = 7:3; the column temperature was 30℃; the flow rate was 1mL / min; the detection wavelength was 275nm; and the injection volume was 10μL.

[0046] HPLC results: Original strain Coenzyme Q 10 The yield was 133.2 ± 3.8 mg / L, while the coenzyme Q produced by the genetically engineered bacteria for coenzyme Q10 production obtained in this example was... 10 The yield was 156.17 + 5.12 mg / L (e.g.) Figure 2 As shown in the figure, the yield increased by 17.2%, indicating that increasing the content of phosphatidylethanolamine in the cell membrane can significantly improve coenzyme Q. 10 Production.

[0047] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for modifying genetically engineered bacteria to produce coenzyme Q10, characterized in that: The steps include the following: (1) From Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides ) 2.4.1 The CDP-diacylglycerol-serine O-phosphatidyltransferase gene, as shown in SEQ ID NO. 01, and the phosphatidylserine decarboxylase gene, as shown in SEQ ID NO. 02, were cloned from the genome. (2) Insert the above CDP-diacylglycerol-serine O-phosphatidyltransferase gene and phosphatidylserine decarboxylase gene into the overexpression vector to construct a recombinant overexpression vector; (3) The above recombinant overexpression vector was transformed into Rhodopseudomonas aeruginosa ( Rhodobacter sphaeroides ) 2.4.1 Recombination expression is carried out in it.

2. The modification method as described in claim 1, characterized in that: The primers used to clone the CDP-diacylglycerol-serine O-phosphatidyltransferase gene are as shown in SEQ ID NO.

03. pssA -F and as shown in SEQ ID NO. 04 pssA -R.

3. The modification method as described in claim 1, characterized in that: The primers used to clone the phosphatidylserine decarboxylase gene are as shown in SEQ ID NO.

05. psd -F and as shown in SEQ ID NO.06 psd -R.

4. The modification method as described in claim 1, characterized in that: The primers used to clone the CDP-diacylglycerol-serine O-phosphatidyltransferase gene are as shown in SEQ ID NO.

03. pssA -F and as shown in SEQ ID NO. 04 pssA -R, the primer used to clone the phosphatidylserine decarboxylase gene is as shown in SEQ ID NO.

05. psd -F and as shown in SEQ ID NO.06 psd -R.

5. The modification method according to any one of claims 1 to 4, characterized in that: The overexpression vector is plasmid pBBR1MCS-2.

6. A genetically engineered bacterium for producing coenzyme Q10, characterized in that: It is obtained by the modification method described in any one of claims 1 to 5.

7. A method for producing coenzyme Q10 by fermentation, characterized in that: It includes the culture step of culturing the coenzyme Q10-producing genetically engineered bacteria as described in claim 6.

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