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Method for increasing thaxtomin fermentation yield

A technology of yield and streptomyces, applied in the field of microbial engineering, can solve the problems of low yield, long fermentation cycle, and failure to effectively improve thaxtomin synthesis pathway thaxtomin yield, etc.

Active Publication Date: 2019-04-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the process of fermentative production of thaxtomin compounds still has defects such as long fermentation period (requiring 7-10 days) and low yield (the fermentation yield of natural thaxtomin-producing strains is generally about 20 μg / mL)
Although the functional genes in the thaxtomin biosynthetic pathway have been predicted in the art (Appl. -10; Francis IM et al., 2015, doi:10.1128 / mBio.02018-14), however, there is no method in this field that can effectively improve the thaxtomin synthesis pathway so as to increase the thaxtomin production

Method used

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  • Method for increasing thaxtomin fermentation yield
  • Method for increasing thaxtomin fermentation yield
  • Method for increasing thaxtomin fermentation yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Preparation of plasmid pSET156-thax with thaxtomin gene cluster

[0100] Such as figure 2 As shown, using the Catch method [6] Cloning of the thaxtomin gene cluster.

[0101] (1) Preparation of sgRNA: Design sgRNA-thaxF and sgRNA-thaxR targeting about 1 kb upstream and downstream of the thaxtomin gene cluster (NCBI gene ID: 132205bp-156019bp of NZ_BCMK01000007.1). The in vitro transcription templates of sgRNA-thaxF and sgRNA-thaxR were prepared by overlapping PCR. Use the primers guide RNA-F, guide RNA-R and thax-gF-P in Table 3 to perform overlapping PCR to obtain the sgRNA-thaxF in vitro transcription template; use the primers in Table 3 guide RNA-F, guide RNA-R and thax -gR-P performs overlapping PCR to obtain sgRNA-thaxR in vitro transcription template. The PCR purification kit (Omega-D6492) was used to purify and recover the sgDNA in vitro transcription template. The NEB commercial kit was used to perform in vitro transcription using T7 RNA polymera...

Embodiment 2

[0111] Expression of embodiment 2 thaxtomin gene cluster in various heterologous Streptomyces hosts

[0112] Transform the pSET156-thax plasmid into the following Streptomyces respectively: Streptomyces albusJ1074 [8] ; Streptomyces lividans TK24 [9] Streptomyces venezuelae ISP5230 (ATCC10712); Streptomyces venezuelae ISP5230; Streptomyces venezuelae M1154 [10] .

[0113] (1) Conjugative transfer of pSET156-thax plasmid [11] :

[0114] First use CaCl 2 Transform the pSET156-thax plasmid into Escherichia coli ET12567 (PUZ8002) strain on the LB plate containing apramycin (50 μg / mL), chloramphenicol (15 μg / mL) and kanamycin (50 μg / mL) to cultivate. After the transformant grows, pick a single colony of the transformant and inoculate it in 2 mL of LB liquid medium containing the above three antibiotics, and culture overnight at 37°C; inoculate 10% of the overnight culture into 4 mL of the same resistant fresh In LB liquid culture medium, shake the culture medium at 37° C. fo...

Embodiment 3

[0119] Embodiment 3 carries out promoter optimization to txtED, txtAB and txtC gene in thaxtomin gene cluster

[0120] Using the method of CN105624146A, the promoters of txtED, txtAB and txtC operons in the thaxtomin gene cluster are transformed into strong constitutive promoters of Streptomyces, and the heterologous expression yield of thaxtomin A is further improved. Such as Figure 4 As shown in a, the wild-type gene cluster fragment Thax of Streptomyces acid scab was transformed into Thax-pAE and Thax-pAEC. Wherein, the fragment Thax-pAE is that the strong promoters SPL42 and SPL43 are inserted into the thaxtomin gene cluster (Thax) gene txtA and txtE upstream of Streptomyces acid scabies respectively; the fragment Thax-pAEC is that the strong promoters SPL42, SPL43 and SPL30 are inserted respectively To the upstream of the thaxtomin gene cluster genes txtA, txtC and txtE of S. acid scab. The above-mentioned promoters are universal promoters for industrial Streptomyces s...

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Abstract

The present invention relates to a method for preparing thaxtomin A, thaxtomin B or thaxtomin D by heterologous expression of a thaxtomin biosynthetic gene cluster. In a preferred embodiment, a synthetic biology strategy is further applied to redesign a thaxtomin biosynthetic approach by using gene cluster editing technology, a constitutive strong promoter element is inserted in upstream of a transcription start site of the txtAB, txtED and / or txtC operons in the gene cluster, so that the yield of thaxtomin is increased.

Description

technical field [0001] The invention relates to the field of microbial engineering. More specifically, the present invention relates to heterologous expression of thaxtomin biosynthetic gene clusters of thaxtomin expression strains by using synthetic biology methods to increase thaxtomin production. Background technique [0002] Thaxtomin is a class of phytotoxins produced by the plant pathogen Streptomyces, which can cause suberized scab-like lesions on plant roots or tubers. It has been found that thaxtomin inhibits the synthesis of cellulose in developing plant cells, so it can be used as a natural herbicide to control the growth of weeds in farmland without toxicity to crops (CN104284583A). In addition, thaxtomin can also be used to control algae pollution (CN101677561A). At present, there are 4 kinds of Streptomyces known to produce thaxtomin, namely Streptomyces scabies, Streptomyces sacidiscabies, Streptomyces ipomoea and Streptomyces turgidiscabies. [0003] The c...

Claims

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Application Information

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IPC IPC(8): C12P17/16C12N1/21C12R1/465C12R1/47C12R1/61
CPCC12N9/0075C12N9/0081C12N9/1085C12N9/93C12N15/52C12P17/165C12Y114/13039C12Y114/15006C12Y205/01034
Inventor 娄春波赵学金
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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