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Construction and applications of a series of efficient heterologous expression hosts of Streptomyces coelicolor

A technology for Streptomyces coelicolor and natural products, which is applied in the field of synthetic biology and can solve the problems that cannot meet the requirements of new compound identification and combined biosynthesis research, and the heterologous expression yield is low.

Inactive Publication Date: 2018-07-06
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of heterologous expression of these natural products is still very low, sometimes not enough for the identification of new compounds and / or the study of combinatorial biosynthesis

Method used

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  • Construction and applications of a series of efficient heterologous expression hosts of Streptomyces coelicolor
  • Construction and applications of a series of efficient heterologous expression hosts of Streptomyces coelicolor
  • Construction and applications of a series of efficient heterologous expression hosts of Streptomyces coelicolor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Construction of a series of heterologous high-efficiency expression hosts of Streptomyces coelicolor

[0089] Since the four active biosynthetic gene clusters have been knocked out in M1146 and M1152, the present invention directly introduces the attB site (see SEQ ID NO: 54) at the knocked out site, and the introduced sequence is respectively The sites of the deleted yCPK, RED, ACT and CDA synthetic gene clusters ( figure 1 A). The detailed steps of engineering strain construction are as follows: Taking the deleted yCPK synthetic gene cluster site as an example to introduce an attB integration site, first use primers yCPK-up-fw\rev, yCPK-down-fw\rev and yCPK-sgRNA- fw\DM-sgRNA-rev was amplified by PCR to obtain the knockout upstream and downstream homology arms yCPK-UP, yCPK-DOWN and the gRNA expression element yCPK-sgRNA targeting yCPK, and then yCPK-up-fw and sgRNA- Rev is the primer, using the above three PCR products as templates, the yCPK-UP-DOWN-sgRN...

Embodiment 2

[0092] Example 2: Systematic identification of chloramphenicol and YM-216391 synthetic gene cluster insertion efficiency

[0093] In order to explore whether the above-mentioned 8 strains of chassis bacteria can effectively realize the amplification of exogenous secondary metabolite synthesis gene clusters, the present invention selected chloramphenicol and a kind of anti-cancer small molecule YM-216391 synthetic gene clusters for identification (gene clusters respectively in cosmids pAH91 and pST85). Using conjugative transfer, they were introduced into M1152-M1552, respectively. Except when there was no zygote when M1552 was introduced, more than 10 zygotes grew out in all the others. As the copy number increases, the number of zygotes gradually decreases. We randomly selected 10 of them for identification of multi-copy insertion efficiency, and the experiment was repeated twice. The results showed that the two gene clusters could be efficiently integrated into M1252-M145...

Embodiment 3

[0097] Example 3: Test of heterologous expression of chloramphenicol and YM-216391 in M1152-M1452

[0098] By effectively inserting chloramphenicol and YM-216391 synthetic gene clusters into M1152-M1452, we obtained engineering bacteria containing different gene cluster copy numbers. The specific fermentation process is as follows: After obtaining a sufficient amount of fresh bacteria in MS medium, transfer to 2×YT medium at 30°C and 200rpm for 7-10h, then directly transfer the culture solution to GYM medium for 30 Cultivate at 200 rpm, and sample every other day.

[0099] The fermentation results showed that with the increase of the copy number, the yield of the two compounds gradually increased. On the 5th day of fermentation, the chloramphenicol yield of M1452 / pAH91 (containing 4 chloramphenicol synthetic gene clusters) reached the maximum (110.4 mg / L), compared to M1152 / pAH91 (containing 1 chloramphenicol synthetic gene cluster, which The chloramphenicol output is 36.3mg...

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Abstract

The invention relates to construction and applications of a series of efficient heterologous expression hosts of Streptomyces coelicolor, and specifically provides genetically engineered Streptomycescoelicolor, wherein the genome of the genetically engineered Streptomyces coelicolor contains more than two attB sites. The invention further provides a method for improving the efficiency of the transformation of a multi-copy gene cluster into Streptomyces coelicolor, a method for preparing a natural product or improving the yield of a natural product, and corresponding uses thereof. According tothe present invention, a series of Streptomyces coelicolor capable of achieving the multi-copy amplification of the target gene cluster at one time are constructed by introducing a plurality of the attB sites in Streptomyces coelicolor, such that the yield of the target product is increased by increasing the number of the copies of the gene cluster.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology. Specifically, the invention relates to the construction and application of a series of high-efficiency heterologous expression hosts of Streptomyces coelicolor. Background technique [0002] Natural products derived from microorganisms have a wide range of biological activities and play a very important role in the fields of medicine, animal health and plant protection. With large-scale microbial genome and metagenomic sequencing, a large number of secondary metabolite biosynthetic gene clusters (Biosynthetic gene clutsers, BGC) have been revealed. Surprisingly, about 90% of gene clusters are not expressed under normal experimental conditions (called recessive or silent gene clusters, Cryptic orsilent BGCs). These "precious treasures" waiting to be tapped mark that we are entering a second golden age of antibiotic research and development. How to obtain and express these unknown gene ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P13/02C12P17/16C12P19/62C12P21/02C12R1/465
CPCC12N9/1247C12N15/74C12P13/02C12P17/16C12P19/62C12P21/02C12Y207/07006
Inventor 芦银华李雷郑国松杨晟姜卫红
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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