CN112680394B

CN112680394B - Method for improving biosynthesis amount of natural product

Info

Publication number: CN112680394B
Application number: CN202110047419.2A
Authority: CN
Inventors: 王岩; 闫夏漪
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-01-14
Filing date: 2021-01-14
Publication date: 2022-10-04
Anticipated expiration: 2041-01-14
Also published as: CN112680394A

Abstract

The invention provides a method for improving the biosynthesis amount of natural products, which is to add indole into a culture medium so as to stimulate the expression of various secondary metabolites in bacteria. The invention firstly provides a new application of indole, which is an application in stimulating bacteria to metabolize and synthesize natural products; in another aspect, the invention provides a method for enhancing biosynthesis of a natural product in a bacterium by stimulating the bacterium to synthesize the natural product by adding indole to a culture medium during the culturing. According to the invention, the expression of various secondary metabolites can be increased by adding indole in the process of culturing bacteria. The invention has simple and convenient treatment process on bacteria, and can realize the improvement of the expression quantity of various secondary metabolites.

Description

Method for improving biosynthesis amount of natural product

Technical Field

The invention belongs to the field of fermentation preparation of natural products, and particularly relates to a method for increasing the biosynthesis amount of a natural product by adding indole.

Background

Indole of formula C ₈ H ₇ N is a compound of pyrrole and benzene in parallel. Indoles and homologs and derivatives thereof are widely found in nature. Some indole derivatives are closely related to life activities, so indole is an important heterocyclic compound.

The research finds that the lysobacter exhibits two important characteristics, namely sliding movement without flagella, and secretion of various extracellular enzymes and secondary metabolites such as Heat Stable Antifungal Factors (HSAF), WAP-8294A2, lysobactin and the like, has an inhibiting effect on various plant pathogenic bacteria, and is a new source of various active natural products. The HSAF consists of a unique macrolactam system, a tetramine acid structure and a 5,5,6-three-membered ring skeleton, has the characteristics of high thermal stability, broad-spectrum antifungal activity, low toxicity and the like, and is widely applied to agricultural production. For example, it is used to control wheat blight because of its antibacterial activity against fusarium, an antibacterial mechanism that works by interfering with the biosynthesis of a unique group of sphingolipids. WAP-8294As is a cyclic lipid peptide with strong activity against methicillin-resistant Staphylococcus aureus, and is currently in phase I/II clinical trials. At present, the improvement of the HSAF yield by adopting a two-stage temperature control strategy in the culture process is researched, and the amplification reaches 17.95%; in addition, the addition of a suitable amount of glucose to the culture medium also increases the yield of HSAF, but these methods have limited yields.

Disclosure of Invention

The invention aims to provide a method for improving the biosynthesis amount of natural products, which is characterized in that indole is added into a culture medium, so that the expression of various secondary metabolites in a bacterial body is stimulated; thereby making up for the deficiencies of the prior art.

The invention firstly provides a new application of indole, which is an application in stimulating bacteria to metabolize and synthesize natural products;

in another aspect, the invention provides a method for increasing biosynthesis of natural products in bacteria by stimulating the bacteria to synthesize natural products by adding indole to the culture medium during the culturing process;

the natural products comprise HSAF, WAP-8294A2, ianthriptide, lysobactin and the like;

the addition concentration of the indole is not more than 5mM, and preferably 0.05-1mM;

according to the embodiment of the present invention, LB medium, TSB medium, chitin medium or the like is used for culturing lysobacter enzymogenes,

as a concrete record of an embodiment, the LB culture medium comprises the following specific formula: 1% peptone, 0.5% yeast powder, 1% sodium chloride; the TSB culture medium has the following specific formula: 1.5% tryptone, 0.5% soytone, 0.5% sodium chloride; the chitin culture medium is an aqueous solution added with 1% chitin.

According to the invention, the expression of various secondary metabolites can be increased by adding indole in the process of culturing bacteria. The invention has simple and convenient treatment process on bacteria, and can realize the improvement of the expression quantity of various secondary metabolites.

Drawings

FIG. 1 is an analysis diagram of the gene cluster of lysobacter enzymogenes secondary metabolites.

FIG. 2 is a transcriptome results heatmap.

FIG. 3 is a diagram of HSAF gene cluster.

FIG. 4 is a graph showing the results of Real-time PCR.

FIG. 5 is a graph showing HSAF expression in indole conditions.

Detailed Description

According to the invention, indole is added in the process of culturing bacteria, so that the expression quantity of secondary metabolites such as HSAF, WAP-8294A2, ianthriptide, lysobactin and the like can be increased to different degrees.

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1 genomic and transcriptome analysis

Lysobacter enzymogenes among lysobacter bacteria was selected as a subject to be investigated, and the secondary metabolite synthesis gene cluster contained in the lysobacter enzymogenes genome was analyzed. As in fig. 1.

The lysobacter enzymogenes is cultured in 40% of TSB medium, the test group is supplemented with 0.5mM indole, and the test group and the control group are transferred to the transcriptome. Analysis of transcriptome data revealed that in the group of experiments with indole added, the expression levels of secondary metabolites such as HSAF, WAP-8294A2, ianthripeptide and lysobactin were significantly up-regulated in lysobacter enzymogenes, as shown in FIG. 2.

The HSAF biosynthetic gene cluster includes 10 genes, as shown in FIG. 3, which are Major defect family (MSF) transporter (orf 1), alkyl defect enzyme zinc-binding protein (orf 2), FAD-dependent oxide detect-3 (orf 3), FAD-dependent oxide detect-2 (orf 4), FAD-dependent oxide detect-1 (orf 5), PKS/NRPS (KS-AT-DH-KR-ACP-C-A-PCP-TE) (orf 6), FA/primary detect (orf 7), ferroxide DP gene product/FAD/DP-binding protein (orf 8), region (orf 9), and receptor (Torpedo 10), respectively.

Real-time PCR verified the expression of 6 core genes (orf 2-7) in HSAF biosynthetic gene cluster, and the result shows that the expression of the 6 genes is obviously up-regulated after exogenous indole is added (FIG. 4).

Example 2 detection of expression level of HSAF under exogenous indole conditions

The lysobacter enzymogenes strain was inoculated on LB solid medium, cultured at 28 ℃ for 2d, then washed with physiological saline, and then cultured for 24 hours in 50mL 40% TSB medium at 170rpm,28 ℃.

A50mL 40% TSB medium was prepared at a final concentration of 50. Mu.M, 100. Mu.M, 200. Mu.M and 500. Mu.M indole, and the lysobacter enzymogenes strain was inoculated into the above medium at an inoculation amount of 5% and cultured at 170rpm at 28 ℃ for 48 hours.

Centrifuging (6000rpm, 15min), collecting supernatant, reducing pressure by using a rotary evaporator, concentrating, evaporating to dryness, dissolving the fermentation product on the inner wall of the rotary evaporator by using a proper amount of methanol and a small amount of dichloromethane, reducing pressure again, concentrating, evaporating to dryness, dissolving the crude fermentation product on the inner wall of the rotary evaporator by using 500 mu L of methanol and 1mL of DMSO, and transferring to an EP (ethylene propylene glycol) tube. When the mixture is concentrated to dryness under reduced pressure, the temperature is preferably 30-35 ℃.

13000rpm for 10min, 1mL of supernatant was put into a liquid phase vial for HPLC detection, (YMC-Pack Pro C18, 10X 250mm,5 μm,4mL/min, UV 320 nm). The solvent system for analytical HPLC was: solvent A: acetonitrile: formic acid =100:0.1, solvent B: water: formic acid =100:0.1, the system is shown in Table 1.

Table 1: analytical HPLC solvent System Table

The results showed that the production of HSAF increased about 1.3-fold at an indole concentration of 50. Mu.M, about 2-fold at 100. Mu.M, and about 5-fold at 200. Mu.M, whereas the HSAF production increased about 1.5-fold when the indole concentration reached 500. Mu.M. Under low indole concentrations, the amount of expression of HSAF increased in a dose-dependent manner. The results of the detection are shown in FIG. 5.

Example 3 detection of expression level of HSAF under exogenous indole conditions

The lysobacter enzymogenes strain was inoculated on LB solid medium, cultured at 30 ℃ for 2d, then washed with physiological saline, and then cultured in 50mL 40% TSB medium at 200rpm at 30 ℃ for 24 hours.

50mL of chitin medium was prepared at final concentrations of 50. Mu.M, 100. Mu.M, 200. Mu.M and 500. Mu.M indole, and the strain was inoculated to the medium at 5% inoculum size and cultured at 200rpm and 30 ℃ for 72 hours.

The fermentation broth was treated as in example 2, and the results showed that the amount of expression of HSAF increased in a dose-dependent manner after addition of indole despite changing the medium and the fermentation temperature, rotation speed, time.

Claims

1. The application of indole, which is characterized in that the application is the application for stimulating the metabolism of lysobacter enzymogenes to synthesize natural products; the natural product is WAP-8294A2, lanthipeptide or lysobactin; the addition concentration of the indole in the culture medium is 0.05-0.5mM.

2. A method for increasing biosynthesis of natural products in bacteria, comprising adding indole to a culture medium to stimulate lysobacter enzymogenes to synthesize natural products; the natural product is WAP-8294A2, lanthipeptide or lysobactin; the addition concentration of the indole in the culture medium is 0.05-0.5mM.

3. The method of claim 2, wherein the medium is TSB medium, LB medium, or chitin medium.

4. The method as claimed in claim 3, wherein the LB medium is specifically formulated as follows: 1% peptone, 0.5% yeast powder, 1% sodium chloride; the TSB culture medium has the following specific formula: 1.5% tryptone, 0.5% soytone, 0.5% sodium chloride; the chitin culture medium is an aqueous solution added with 1% chitin.