Unlock instant, AI-driven research and patent intelligence for your innovation.

High-yield strain of Tiancimycin and its fermentation process and application

A fermentation process, the technology of godsendomycin, applied in the directions of fermentation, bacteria, microorganism-based methods, etc., can solve the problems of cumbersome efficiency, low fermentation yield of godsendomycin A, and low yield of enediyne compounds, etc.

Active Publication Date: 2021-11-23
CHANGSHA CHARISM BIOSCIENCES CO LTD +2
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The complex molecular structure of orthomycin A makes the process of obtaining its related derivatives by traditional chemical total synthesis methods cumbersome and inefficient, and the yield of related enediyne compounds is low (J Antibiot.1991,44,1300, Organic Lett.2005,23,5233), Uncialamycin (UCM), and Dynemicin were found to have lower yields (~0.02mg L respectively -1 , ~0.1mg L -1 ), unable to meet the needs of clinical research and the application of industrial production (see figure 1 )
Microbial fermentation can better obtain this compound, however, the fermentation yield of orthomycin A in the original strain CB03234 is extremely low, about ~0.5mg L -1 In order to promote the anti-cancer activity analysis, mechanism of action and other pre-clinical and new drug development research in the later stage, it is necessary to obtain a sufficient amount of orthomycin A

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • High-yield strain of Tiancimycin and its fermentation process and application
  • High-yield strain of Tiancimycin and its fermentation process and application
  • High-yield strain of Tiancimycin and its fermentation process and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Chemical mutagenesis and ribosome engineering of Streptomyces sp. CB03234

[0034] (1) Inoculate Streptomyces CB03234 to G1 solid growth medium (the G1 solid growth medium is: 10g / L soluble starch, 1g / L MgSO 4 ·7H 2O, 1g / L K 2 HPO 4 , 4g / L MgCl 2 , 2g / L (NH 4 ) 2 SO 4 , 2g / LCaCO 3 , 1g / L tryptone, 0.5g / L yeast powder, 0.001g / L MnCl 2 ·7H 2 O, 0.001g / L FeSO 4 ·7H 2 (0, 20g / L agar, pH=7.0) on the flat plate, culture about 7 days under the constant temperature condition of 30 ℃, collect spore with aseptic 20% glycerol. The spores were counted by sparse coating on the plate, and finally made into a uniform concentration of 10 9 per mL of spore suspension.

[0035] (2) take ethanol as solvent, configure the solution of 0.46M diethyl sulfate (DES), then take about 10 9 Mix the spore suspension with it, shake at 200r / min for different times (10-40 minutes) at 30°C, and quickly spread the mixed treatment solution in each time period and pour it on 5 GYM...

Embodiment 2

[0039] Example 2: Overexpression of TNM A-related biosynthetic genes of Streptomyces sp.CB03234-R-16

[0040] Select 200ul of Streptomyces sp.CB03234 and Streptomyces sp.CB03234-R-16 fermented for 48h, 120h, and 160h respectively, extract total RNA, and take the corresponding total RNA for reverse transcription as a template for subsequent real-time quantitative fluorescent PCR. Each sample has three biological replicates. Fluorescent quantitative PCR conditions: 95°C for 10 minutes, a total of 40 cycles, 95°C for 15s, 55°C for 60s. Fluorescent quantitative PCR was used for relative quantification, and the hrdB gene was used as an internal reference gene, and the calculation method was 2 -ΔΔCT Law. Observe the expression of the target gene, specifically including 5 regulatory genes, tnmR1, tnmR3, tnmR2, tnmR4, tnmR7; gene tnmL (cytochrome p450 enzyme), tnmE10 (type 2 thioesterase); ribosome Engineering related gene relA (3,5′ guanosine diphosphate synthase). The results sh...

Embodiment 3

[0041] Embodiment 3: Optimization of the fermentation medium of Streptomyces sp.CB03234-R-16

[0042] (1) Based on the main components of the original medium, try different carbon sources (10g / L) in the laboratory, including soluble starch (original), corn flour, dextrin, Fructose (D-fructose), galactose (D-galactose), maltose (D-maltose), xylose (D-xylose), glycerol (glycerol), glucose (glucose), lactose (α-lactose), mannitol ( mannitol), nonfat milk powder, sucrose. Try different common nitrogen sources (5g / L), including: cottonseed meal (pharmamedia) (raw), beef extract (beef extract), acid hydrolyzed casein (casamino acids), corn steep liquor (corn steep liquor), corn protein powder, fish meal, fish peptone, malt extract, oat meal, peptone, peanut protein powder, Soya peptone, tryptone, yeast extract. Fermentation was carried out without adding resin, and the fermentation supernatant was taken on the 2nd, 4th, and 6th day of fermentation to detect its activity. Finally...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a high-yielding bacterial strain of Tiancimycin as well as its fermentation process and application. The high-yield strain is a strain of Streptomyces sp. CB03234‑R‑16, which was deposited in the China Center for Type Culture Collection on September 30, 2017, with the preservation number CCTCC M 2017556. In the present invention, a high-yield strain CB03234‑R‑16 is finally screened to obtain a high-yielding bacterial strain CB03234‑R‑16 by performing strain evolution based on chemical mutagenesis and ribosome engineering to the original strain CB03234 producing active tiancimycin (tiancimycin). The fermentation yield of (TNM A) was increased by more than 8 times compared with the original yield, reaching (5.8±1.0)mg L ‑1 , after the optimization of the relevant fermentation process, specifically including the fermentation of small-scale shake flasks and large-scale 15L fermenters, the output of shake flasks (250mL) finally reached (22.5±3.1)mg L ‑1 , increased by about 40 times, and the output of the 15L fermenter reached (13±1) mg L ‑1 . The strain has the potential of industrial production of orthomycin A.

Description

technical field [0001] The invention relates to a high-yield strain of Tiancimycin and its fermentation process and application, belonging to the technical field of biopharmaceuticals. Background technique [0002] Facing the R&D dilemma of high investment cost and high failure rate of original new drug R&D, how to effectively speed up the process of new drug R&D has attracted widespread attention. Since 1980, 79% of anticancer drugs are based on natural products (J.Nat.Prod.2012, 75, 311.), and natural products have been proved to be an important source of modern drugs, especially anticancer drugs. Among them, enediyne natural products have the advantages of excellent anti-tumor activity and high drug yield. So far, 11 structures of enediyne compounds have been found, and they are mainly divided into nine-membered rings and ten-membered rings according to their structures. Currently, there are Nine-membered cycloenediyne neocarcinogen, used in the treatment of liver cancer...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P17/18C12R1/465
Inventor 段燕文沈奔黄勇朱湘成刘玲潘建
Owner CHANGSHA CHARISM BIOSCIENCES CO LTD