阅读说明：本技术 一种通过强化枯草芽孢杆菌功能膜微域提高七烯甲萘醌产量的方法 (Method for improving yield of heptaene menadione by strengthening functional membrane microdomains of bacillus subtilis ) 是由 刘龙 陈坚 吕雪芹 堵国成 李江华 刘延峰 董雅君 金柯 张智航 王凌锐 于 2021-09-06 设计创作，主要内容包括：本发明公开了一种通过强化枯草芽孢杆菌功能膜微域提高七烯甲萘醌产量的方法,本发明的方法是通过强化表达产七烯甲萘醌的枯草芽孢杆菌功能膜微域中的脚手架蛋白FloA。本发明通过对已高产MK-7的重组枯草芽孢杆菌BS20中的脚手架蛋白使用强启动子P43进行强化,增加FMMs在细胞质膜上的占比后,进一步提高了MK-7的产量,与对照菌株BS20相比,强化FloA的重组枯草芽孢杆菌BSQ1产量最高,发酵6天后,BSQ1菌株产量达到417.08mg/L,较BS20菌株提升了16.57％。(The invention discloses a method for improving the yield of heptamenadione by strengthening a bacillus subtilis functional membrane micro domain. According to the invention, the scaffold protein in the recombinant bacillus subtilis BS20 which has high MK-7 yield is strengthened by using the strong promoter P43, the yield of MK-7 is further improved after the proportion of FMMs on a cytoplasmic membrane is increased, compared with a control strain BS20, the yield of the FloA-strengthened recombinant bacillus subtilis BSQ1 is the highest, after 6 days of fermentation, the yield of BSQ1 strain reaches 417.08mg/L, and is improved by 16.57% compared with BS20 strain.)

1. The method for improving the yield of the heptamenadione by strengthening the functional membrane micro-domain of the bacillus subtilis is characterized in that the method is used for strengthening and expressing the scaffold protein FloA in the functional membrane micro-domain of the bacillus subtilis for producing the heptamenadione.

2. The method of claim 1, wherein said enhancing expression is by using a strong promoter.

3. The method of claim 1, wherein the strong promoter is the P43 promoter.

4. The bacillus subtilis recombinant strain for producing heptaene menadione is characterized in that the bacillus subtilis recombinant strain is obtained by enhancing expression of scaffold protein FloA in a bacillus subtilis host functional membrane micro-domain.

5. The recombinant Bacillus subtilis strain of claim 4, wherein the enhanced expression is achieved by using a strong promoter P43.

6. The recombinant Bacillus subtilis strain of claim 5, wherein the strong promoter P43 has the nucleotide sequence shown in SEQ ID No. 3.

7. The recombinant Bacillus subtilis strain of claim 4, wherein the Bacillus subtilis host is Bacillus subtilis BS 20.

8. The recombinant bacillus subtilis strain of claim 4, wherein the nucleotide sequence of the encoding gene of the scaffold protein FloA is shown as SEQ ID No. 4.

9. The application of the bacillus subtilis recombinant strain of any one of claims 4-8 in fermentation production of heptamenadione.

10. The application of claim 9, wherein the application is specifically that recombinant bacillus subtilis is activated in a seed culture medium, and then the activated seeds are transferred into a fermentation culture medium for fermentation culture to obtain heptamenadione.

Technical Field

The invention relates to a method for improving the yield of heptaene menadione by strengthening a functional membrane micro-domain of bacillus subtilis, belonging to the technical field of metabolism.

Background

Vitamin K is one of important fat-soluble vitamins which are indispensable to human bodies and plays a key role in accelerating blood coagulation, preventing cardiovascular sclerosis, treating osteoporosis and the like. The vitamin K takes 2-methyl-1, 4-naphthoquinone as a framework, and can be divided into different subtypes according to the difference of C3 branch chain structures. Among them, Menaquinone (Menaquinone-7, MK-7) has a long half-life in human body and high affinity, and is currently drawing attention in the fields of functional foods, medicines and the like. The inventor subjects CN110157749B, a Chinese patent application in the earlier stage, has achieved high MK-7 yield in Bacillus subtilis, but it is found in the research that conventional metabolic modification, such as strengthening of synthetic pathway, blocking of byproduct formation and the like, is difficult to further improve MK-7 yield.

Functional Membrane Microdomains (FMMs) are a class of structurally dense Microdomains rich in sterol analogue polyisoprene compounds on bacterial cell lipid membranes and are rich in specific scaffold proteins. Such scaffold proteins, as a membrane binding partner, are localized only on FMMs, can recruit proteins that need to be localized on FMMs and promote their interaction and oligomerization, playing an important role in organizing and forming FMMs. No studies have been made to date to show whether it is possible to enhance the productivity of MK-7 by enhancing FMMs in Bacillus subtilis, increasing the proportion of FMMs in the cytoplasmic membrane, and increasing the storage space of MK-7 in the cytoplasmic membrane.

Disclosure of Invention

In order to solve the technical problem, the invention strengthens FloA and FloT proteins in FMMs, and further improves the MK-7 yield after increasing the occupation ratio of FMMs on cytoplasmic membranes.

The first purpose of the invention is to provide a method for improving the yield of heptamenadione by strengthening the functional membrane micro-domain of bacillus subtilis, and the method is to strengthen the expression of scaffold protein FloA in the functional membrane micro-domain of the bacillus subtilis for producing the heptamenadione.

Further, the enhanced expression is performed by using a strong promoter.

Further, the strong promoter is a P43 promoter.

The second purpose of the invention is to provide a bacillus subtilis recombinant strain for producing heptaene menadione, wherein the bacillus subtilis recombinant strain is obtained by enhancing expression of scaffold protein FloA in a bacillus subtilis host functional membrane micro domain.

Further, the enhanced expression is performed by using a strong promoter P43.

Further, the nucleotide sequence of the strong promoter P43 is shown in SEQ ID NO. 3.

Further, the bacillus subtilis host is bacillus subtilis BS 20. Bacillus subtilis BS20 is disclosed in patent CN 110157749B.

Furthermore, the nucleotide sequence of the encoding gene of the scaffold protein FloA is shown as SEQ ID NO. 4.

The third purpose of the invention is to provide the application of the bacillus subtilis recombinant bacteria in fermentation production of heptamenadione.

Further, the application specifically comprises the steps of activating the recombinant bacillus subtilis in a seed culture medium, transferring the activated seeds into a fermentation culture medium, and performing fermentation culture to obtain the heptaene menadione.

The invention has the beneficial effects that:

according to the invention, the scaffold protein in the recombinant bacillus subtilis BS20 which has high MK-7 yield is strengthened by using the strong promoter P43, the yield of MK-7 is further improved after the proportion of FMMs on a cytoplasmic membrane is increased, compared with a control strain BS20, the yield of the FloA-strengthened recombinant bacillus subtilis BSQ1 is the highest, after 6 days of fermentation, the yield of BSQ1 strain reaches 417.08mg/L, and is improved by 16.57% compared with BS20 strain.

Description of the drawings:

FIG. 1 shows MK-7 production by fermentation of strains BS20, BSQ1, BSQ2 and BSQ12 after 6 days of fermentation.

Detailed Description

The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.

(1) Culture medium

The components of the seed culture medium comprise: 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride.

The components of the fermentation medium comprise: 50g/L glucose, 50g/L glycerol, 50g/L soybean peptone and 0.6g/L potassium dihydrogen phosphate.

(2) MK-7 extractant: mixture of isopropanol and n-hexane (1:2, V/V)

(3) MK-7 production by HPLC: using an Agilent ZORBAX eclipseXDB-C18 separation column (5 μm, 250X 4.6mm), the temperature was measured at 40 ℃ and the mobile phase was purified using methanol: dichloromethane (9:1, v/v), flow rate 1mL/min, detection wavelength 254nm, sample size 10 uL.

(4) Detecting the growth condition of the strain: timed determination of the absorbance OD of the fermentation broth using a UV-Vis Spectrophotometer₆₀₀。

Example 1: construction of recombinant Bacillus subtilis BSQ1, BSQ2

The P43 promoter was integrated into the genome of recombinant Bacillus subtilis BS20 upstream of floA (yuaG) and floT (yqfB) by cre/loxp system to construct recombinant Bacillus subtilis BSQ1 and BSQ2, respectively, as follows.

The following sequences were fragment amplified by overlap extension PCR, the amplification sequences required for the P43-floA integration frame being the floA upstream sequence (length 1000bp, sequence as SEQ ID NO.1), the chloramphenicol resistance gene zeo sequence (1309bp, sequence as SEQ ID NO.2), the P43 promoter (0.3bp, sequence as SEQ ID NO.3) and the floA sequence (996bp, sequence as SEQ ID NO. 4). The amplification sequences required for the P43-floT integration frame were the floT upstream sequence (length 1000bp, sequence as SEQ ID NO.5), the chloramphenicol resistance gene zeo sequence (1309bp), the P43 promoter (0.3bp) and the floT sequence (1000bp, sequence as SEQ ID NO. 6). Primer sequences are shown in Table 1, where floA-P7C6-1R and floA-P43-1F are universal primers for two integration frames. FloA obtained by fusion PCR_up-lox71-zeo-lox66-p43-floA and floT_up-lox71-zeo-lox66-p43-floT fusion expression cassette.

TABLE 1

The obtained fusion expression frame fragment is integrated into the recombinant bacillus subtilis BS20 genome by means of chemical transformation, and the addition amount of the integrated fragment is about 1000 ng. Screening by adding a chloramphenicol-resistant LB solid medium, selecting a single colony for PCR verification and sequencing verification, and confirming the successful integration.

And (3) eliminating chloramphenicol resistance of the successfully integrated recombinant bacillus subtilis, transferring the Cre plasmid into the constructed recombinant bacillus subtilis in a chemical transformation mode, screening the recombinant bacillus subtilis by an LB solid culture medium added with a kanamycin antibiotic, and inducing the expression of the Cre plasmid by IPTG to eliminate resistance genes. Cre plasmid is eliminated by picking single colony and inoculating into LB liquid culture medium, and shake culturing at 50 deg.C for 12 h. Screening by using a point plate non-resistant LB plate, adding chloramphenicol antibiotic and adding kanamycin antibiotic, selecting a single colony which successfully eliminates chloramphenicol resistance and Cre plasmid, and finally obtaining recombinant bacillus subtilis which integrates P43-floA and P43-floT respectively and is named as BSQ1 and BSQ 2.

Example 2: construction of recombinant Bacillus subtilis BSQ12

Recombinant Bacillus subtilis BSQ12 was constructed by integrating the P43 promoter upstream of floT on the BSQ1 genome in a manner similar to that in example 1 on the basis of BSQ1 obtained in example 1.

The constructed floT of example 1_upThe-lox 71-zeo-lox66-P43-floT fusion expression box is transferred into BSQ1 in a chemical conversion mode, and is subjected to chloramphenicol resistance plate screening, colony PCR verification, sequencing, Cre plasmid transfer, Kaner resistance plate screening, IPTG induced expression, Cre plasmid elimination and dot plate verification to finally obtain the recombinant bacillus subtilis BSQ12 integrating the P43-floA and P43-floT expression boxes.

Example 3: MK-7 produced by strain fermentation

(1) Seed liquid preparation

Recombinant Bacillus subtilis BS20, BSQ1, BSQ2 and BSQ12 constructed in examples 1 and 2 were inoculated into 15mL shake tubes containing 2mL liquid seed medium, respectively, and shake-cultured at 37 ℃ and 220rpm for 10 h. BS20 served as a control, with three replicates per strain.

(2) Fermentation culture

Inoculating the seed solution obtained in the step (1) into a 250mL conical flask according to the inoculation amount of 10%, wherein each flask is filled with 20mL of fermentation medium, performing shake culture at 41 ℃ and 220rpm for 3d, sampling every 24h, and performing OD (optical density) extraction₆₀₀Detection and sample preparation. The specific method is as follows.

1.2mL of fermentation broth was taken daily. Taking 20 μ L fermentation broth, diluting with sterile water 50 times, shaking uniformly, and performing OD₆₀₀Detecting; adding 500 μ L fermentation liquid into 4 times of MK-7 extractant, vortex shaking for 10min, filtering to obtain extractive solution, and centrifuging at 8000r/min for 5 min. The supernatant was collected and HPLC was used to determine the amount of MK-7, a whole cell fraction. The results are shown in FIG. 1. After fermentation for 6 days, the yield of the BSQ1 strain reaches 417.08mg/L, which is increased by 16.57% compared with the BS20 strain; BSQ2 the MK-7 content of the strain is slightly increased in the early fermentation period, and the yield is obvious in the late fermentation periodDecrease; BSQ12 the MK-7 content of the strain is lower than that of a control in the fermentation process, and the late decline amplitude is increased. The result shows that the enhanced FMMs scaffold protein synthesis genes of floA and floT have influence on the synthesis of MK-7 yield, wherein the floA has a certain positive effect on the synthesis of MK-7, and the effect of improving the MK-7 yield is achieved.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

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