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A high-efficiency and controllable expression system of exogenous genes carried by endogenous

An expression system and gene technology, applied in the field of high-efficiency and controllable expression systems for exogenous genes, can solve the problems of complicated experimental operations, increasing the probability of homologous recombination of genes within transformants, and the length of target DNA fragments, thereby simplifying experimental operation steps, The effect of increasing controllability and stability and improving work efficiency

Active Publication Date: 2021-11-23
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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Problems solved by technology

However, the synthesis of natural products usually involves the expression of multiple genes, which requires separate promoters and transcription terminators for each gene, resulting in complicated experimental operations and long target DNA fragments
Especially in the case of the limited number of promoters currently available, the expression of multiple genes often requires the repeated use of the same promoter and terminator, which greatly increases the probability of homologous recombination of genes within the transformant

Method used

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  • A high-efficiency and controllable expression system of exogenous genes carried by endogenous
  • A high-efficiency and controllable expression system of exogenous genes carried by endogenous
  • A high-efficiency and controllable expression system of exogenous genes carried by endogenous

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the comparison of fungal operon sequence spacer

[0044] 1. Purpose of the experiment

[0045] The auxotrophic Saccharomyces cerevisiae BJ5464-NpgA was used as the chassis cell, and the glycosyltransferase-methyltransferase module encoding genes BbGT and BbMT previously reported by our research team were used as reporter genes (Xie L, Zhang L, Wang C, Wang X , Xu YM, Yu H, Wu P, Li S, Han L, Gunatilaka AAL, Wei X, Lin M, MolnarI, Xu Y. 2018. Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis. Proc Natl Acad Sci USA115:E4980-E4989.), base substitution or deletion was designed for the original sequence of the 26 bp intergenic region, and the sugar methylation efficiency of BbGT and BbMT on the substrate in positive transformants was detected by liquid chromatography, and the intergenic region was elucidated Key elements such as motifs or secondary structures in the sequence that affect downstre...

Embodiment 2

[0058] Example 2. Fungal operon-based exogenous gene fixed-point, quantitative and timed expression system (endogenously equipped exogenous gene efficient and controllable expression system) in desmethyl-lasiodiplodin (DLD) and its methylated product biosynthetic pathway reconstruction in the application.

[0059] 1. Purpose of the experiment

[0060] Using the auxotrophic Saccharomyces cerevisiae BJ5464-NpgA as the chassis cell, using the optimal sequence as the intergenic region, using the fungal operon-based exogenous gene quantitative and timed expression system (endogenously carried exogenous gene efficient and controllable expression system ), reconstructing the biosynthetic pathway of DLD and its methylated products previously discovered by the inventor's research team (Xu Y, Zhou T, Espinosa-Artiles P, Tang Y, Zhan J, Molnar I. 2014. Insights into the biosynthesis of 12-membered resorcylic acid lactones from heterologous production in Saccharomyces cerevisiae ACS Ch...

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Abstract

The invention relates to the field of biotechnology, and discloses an efficient and controllable expression system of an exogenous gene carried by an endogenous source, which includes a sequentially connected sequence: a host target gene stop codon upstream segment containing a stop codon, and a fungal operon gene interval region, the target gene, and the DNA fragment downstream of the stop codon of the host target gene, the target gene is connected using the fungal operon intergenic region sequence, and inserted into the downstream of the stop codon of the host cell expression gene by site-directed gene insertion. The promoter of the target gene completes the transcriptional expression of the target gene. The expression system simplifies the experimental operation steps, improves work efficiency, increases the controllability and stability of target gene expression, and reduces the requirement for known promoter diversity for multi-gene heterologous expression. The excavation of natural products, the improvement of production and the creation of unnatural compounds have high practical application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fixed-point, quantitative and timed expression system for exogenous genes based on fungal operons, that is, an efficient and controllable expression system for exogenous genes carried by endogenous sources. Background technique [0002] Natural products are an important source of modern drug creation. However, it is becoming more and more difficult to obtain new structural natural products with practical value using traditional screening techniques. In addition, some known active natural products are scarce in source, low in natural content, complex in composition and difficult to completely separate. The development of genome sequencing technology and the analysis of natural product synthesis pathways provide the possibility to produce these valuable natural products through heterologous biosynthesis technology. Synthetic biology technology, with its ability to transfer functional ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/81C12N1/19C12R1/865
CPCC12N15/113C12N15/81C12N2830/36
Inventor 徐玉泉岳群张礼文
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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