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Hangtaimycin biosynthetic gene cluster and its application

A biosynthesis, gene cluster technology, applied in the direction of plant genetic improvement, application, genetic engineering, etc., can solve problems that have not been reported yet

Active Publication Date: 2021-12-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biosynthetic gene cluster and biosynthetic mechanism of hangtaimycin and its alcoholysis product hangtaimycinA have not been reported at home and abroad so far

Method used

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  • Hangtaimycin biosynthetic gene cluster and its application
  • Hangtaimycin biosynthetic gene cluster and its application
  • Hangtaimycin biosynthetic gene cluster and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Extraction of total DNA of Hangtaimycin producing bacteria Streptomyces spectabilis CCTCC M2017417:

[0078] Get ABB13 solid medium (soluble starch 0.5%, soybean peptone 0.5%, calcium carbonate 0.3%, 3-(N-morpholine) propanesulfonic acid 0.21%, thiamine hydrochloride 0.001%, ferrous sulfate heptahydrate 0.0012%, agar 2%) grow on the plate for 7 days, the size is about 1cm 2 Streptomyces spectabilis CCTCC M2017417 bacteria blocks were inoculated into 80 mL of TSBY liquid medium (10.5% sucrose, 0.5% yeast extract powder, 0.5% soybean extract) at 28° C., 220 rpm, and cultivated for 48 hours. Centrifuge the mycelium at 6000rmp for 10 minutes, discard the supernatant, and collect the mycelia. Dissolve the bacteria in 15mL of SET buffer (Tris-HCl 0.07%, 0.73% ethylenediaminetetraacetic acid, sodium chloride 4.39%), suspend and mix, repeat the centrifugation and suspension twice, and re-suspend the bacteria Float in 10 mL of buffer solution, add 50 μL of lysozyme solution (1...

Embodiment 2

[0080] Construction of hangtaimycin biosynthetic gene cluster knockout mutant ΔhtmBGC:

[0081] (1) According to the nucleotide sequence of the Hangtaimycin biosynthetic gene cluster, a pair of primers were respectively designed on the upstream of the Hangtaimycin biosynthetic gene cluster htmA4 and downstream of htmK (see Table 2 for the primer sequences). The left and right homologous exchange arms for homologous recombination were obtained by polymerase chain reaction (PCR) amplification, and the lengths were 2061 base pairs and 2060 base pairs respectively. The PCR reaction system and conditions were:

[0082]

[0083] 95°C, 3 minutes

[0084] 95°C, 30 seconds; 58°C, 30 seconds; 72°C, 90 seconds; 30 cycles

[0085] 72°C, 5 minutes

[0086] (2) Digest Escherichia coli and Streptomyces shuttle vector pYH7 with restriction endonucleases NdeI and HindIII, and recover about 9000 base pairs of DNA fragments containing the conjugative transfer initiation replication site and a...

Embodiment 3

[0093] Streptomyces spectabilis CCTCC M2017417 neutralization hangtaimycin biosynthetic gene cluster knockout mutant strain ΔhtmBGC fermentation extraction and detection of hangtaimycin by high performance liquid chromatography (HPLC) and liquid phase high resolution mass spectrometry (LC-ESI-HRMS):

[0094] Grow on SFM solid media plates for 7 days, the size is about 1cm 2 Streptomyces spectabilisCCTCC M2017417 and the mutant strain ΔhtmBGC were inoculated into 40 mL of TSBY liquid medium, cultured at 28°C and 220 rpm for 36 hours, and the mycelium was transferred to 40 mL of SFM liquid fermentation medium (soybean powder 2%, mannitol 2%, macroporous resin 5%), cultivated under the condition of 28 ℃ and 220rmp for 108 hours. After the fermentation broth was centrifuged at 6000rpm for 10 minutes, the precipitate was extracted three times with 40mL ethyl acetate, and the extracted ethyl acetate was combined and evaporated under reduced pressure. HPLC ( Figure 6 ) and LC-ESI-...

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Abstract

The invention discloses a Hangtaimycin biosynthetic gene cluster and application thereof. Its nucleotide sequence is shown in the 1‑91594th position of SEQ ID NO.1, and contains 21 genes. The information on all the genes and proteins of the hangtaimycin biosynthesis cluster provided by the present invention is helpful for people to understand the biosynthesis mechanism of the natural products of the hangtaimycin family, and on this basis to produce a new structure of hangtaimycin that can be used in medicine, industry or agriculture through genetic modification Derivatives provide materials and knowledge, and the gene and protein information provided by the present invention can also be used to find similar compounds, genes or proteins.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a Hangtaimycin biosynthetic gene cluster and its application. Background technique [0002] Hangtaimycin is a novel polyketidepolypeptide hybrid compound with a unique δ-unsaturated six-membered cyclic lactone and a terminal cyclic dipeptide unit ( figure 1 ). Although hangtaimycin was initially isolated from Streptomycesspectabilis CPCC200148 in 2016, only preliminary anti-tumor and anti-Candida albicans activity tests were carried out. We isolated and identified hangtaimycin in the study of active secondary metabolites of Streptomyces spectabilis CCTCC M2017417, and carried out more extensive biological activity investigation and biosynthetic mechanism research on hangtaimycin and its derivatives. [0003] The results of biological activity tests showed that the methanol hydrolyzate of hangtaimycin, hangtaimycinA, had better activity than the first-lin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N15/53C12N15/54C12N15/60C12N15/31C12P17/16
CPCC12N15/52C12N9/1029C12N9/93C12N9/0006C12N9/16C12N9/1025C12N9/104C12N9/88C12N9/90C12N9/1048C12N9/12C07K14/36C12P17/16C12Y603/02C12Y101/01C12Y203/02C12Y402/01C12Y207/11C12Y203/0301
Inventor 孙宇辉罗明和邓子新
Owner WUHAN UNIV
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