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CA-3156875-C - HIGHLY EFFICIENT AND CONTROLLABLE EXPRESSION SYSTEM FOR ENDOGENOUS PIGGYBACKING EXOGENEOUS GENES

CA3156875CCA 3156875 CCA3156875 CCA 3156875CCA-3156875-C

Abstract

This patent belongs to the field of biotechnology. Highly efficient and controllable expression system for endogenous piggybacking exogeneous genes includes DNA fragments in the following order: DNA fragment upstream of endogenous target gene stop codon (including stop codon), intergenic gap of operon (I GG). gene of interest (GOI), DNA fragment downstream of endogenous target gene stop codon. It inserts the IGG linked GOI between the endogenous target gene and its terminator to form a bicistron by gene knock-in, leading to GOI expression controlled by the promoter of endogenous target gene. It would greatly simplify the operation, improve work efficiency, increase the controllability and stability of GOI expression, and reduce the requirement of multi-gene heterologous expression for characterized promoters. It would be a great synthetic biology tool for mining of novel natural products, yield improvement of valuable natural products and the development of unnatural compounds.

Inventors

  • Yuquan Xu
  • Qun Yue
  • Liwen Zhang

Assignees

  • BIOTECHNOLOGY RESEARCH INSTITUTE, CHINESE ACADEMY OF AGRICULTURAL SCIENCES

Dates

Publication Date
20260505
Application Date
20200614
Priority Date
20191105

Claims (8)

  1. Claims: 1. A high efficiency controllable system ofheterogenous gene(s) hitched to endogenous gene expression, comprising DNA fragments in the following order: a DNA fragment upstream of endogenous target gene stop codon that includes the stop codon, IGG, GOI, DNA fragment downstream of endogenous target gene stop codon, wherein the IGG includes the following sequences: 5'-CAATCAAAC-3', or 5' -CAATCAAAC-3 'with 1-8 bp at its 5' end and 1-9 bp at its 3' end.
  2. 2. The system of claim 1, wherein the IGG includes the following sequence: 5' - TGTTGAGTCAATCAAACACTCAACAG- 3'.
  3. 3. The system of claim 1 or claim 2, wherein there is a selectable marker gene between the GOI and the DNA fragment downstream of the endogenous target gene stop codon.
  4. 4. The system of any one of claims 1 to 3, wherein the GOI(s) includes multiple genes.
  5. 5. The system of claim 4, wherein the GOI(s) consists of 2, 3 or 4 genes.
  6. 6. The system of any one of claims 1 to 5, wherein a host of the system is a fungus or a plant.
  7. 7. The system of claim 6, wherein the host is a fungus, and the fungus includes one or more of yeast and filamentous fungus.
  8. 8. The system of claim 7, wherein the fungus is Saccharomyces cerevisiae. Date Re9ue/Date Received 2024-03-13

Description

HIGHLY EFFICIENT AND CONTROLLABLE EXPRESSION SYSTEM FOR ENDOGENOUS PIGGYBACKING EXOGENEOUS GENES Technical field This patent belongs to the field of biotechnology. It includes a quantitative and timed expression system for hctcrologous gcnc(s) integrated in the target sitc(s) based on fungal opcron, i.e., a high efficiency controllable system ofhctcrogcnous gcnc(s) hitched to endogenous gene expression. Background technique Natural product is one of important sources of modern medicine. However, it gets harder to obtain new valuable natural products using traditional screening techniques. Furthermore, there arc lots of known valuable natural products from plant biomass or fungal cultures that arc difficult to be obtained because of slow biomass accumulation, low content of the desired product in the native host, and difficulty in purification of desired product from complex crude extract. The development of genome sequencing technology and the reveal of natural product biosynthcsis pathways provide the possibility to produce these valuable natural products through hctcrologous biosynthcsis. Synthetic biology, with its ability to transfer functional clements or modules from different species, have become the main strategy to solve these problems. So far, based on genome information, cell factories of important active natural products, such as artcmisinin precursor artcmisininic acid and opioids, has been constructed using synthetic biology methods. Fungal host cells such as Saccharomyces cerevisiae and Aspergillus nidulans have clear genetic background, and simpler genetic manipulation than that of other eukaryotes. Compared with prokaryotic host cells such as Escherichia coli and Actinomycetes, cukaryotic biosynthctic enzymes arc more likely to be successfully expressed and play their catalytic role in fungal hosts. In fungi, genes arc usually transcribed individually into monocistronic mRNA, which means a promoter-gene-terminator cassette is necessary for gene transcription. Meanwhile, the biosynthcsis of natural product often needs the expression of multiple genes. Thus, individual promoter and transcriptional terminator should be provided for each gene. However, only a limited number of characterized promoters and terminators arc available, leading to the repetitive use of these regulatory clements in the same cell factory. It would not only increase the size of expression cassette, cause inconvenience of construction and decrease of transformation rate, but also brought the danger of internal homologous recombination and metabolic burden, which is an annoying side-effect during cell factory engineering and fermentation production. l CA 0315687 5 2022· 5- 2 In previous study, Yue ct al. (Yue Q, Chen L, Li Y, Bills G, Zhang X, Xiang M, Li S, Che Y, Wang C, Niu X, An Z, Liu X. 2015. Functional opcrons in secondary metabolic gene clusters in Glarea lozoyensis (Fungi, Ascomycota, Lcotiomycctcs). mBio 6:c00703-l 5.) discovered a functional opcron glpks3-glnrps7 containing a 26 bp intcrgcnic gap (JGG 1) in cchinocandin-producing fungus Glarea lozoyensis. The two contiguous genes glpks3 and glnrps7 arc co-transcribed into one bicistronic mRNA under the control of the same promoter, and the mRNA is then translated into two individual proteins. If the opcron organization could be applied to the heterologous expression of multi-genes in fungal hosts, it would greatly reduce the size of expression cassette, simplify the operation, and improve work efficiency. Technical issues The biosynthctic pathway of complex natural product often contains multiple genes with different expression levels. Thus, individual promoter and transcriptional terminator should be provided for each gene. However, only a limited number of characterized promoters and terminators arc available, leading to the repetitive use of these regulatory clements in the same cell factory. It would not only increase the size of expression cassette, cause inconvenience of construction, but also brought the danger of internal homologous recombination and metabolic burden, which is an annoying side-effect during cell factory engineering and fermentation production. If the opcron organization could be applied to the hctcrologous expression of multi-genes in fungal hosts, it would greatly reduce the size of expression cassette, simplify the operation, and improve work efficiency. Invent content A quantitative and timed expression system for hctcrologous gcnc(s) integrated in the target sitc(s) based on fungal opcron uses IGG sequence to link a hctcrologous gene between a host expressed gene and its terminator to form a bicistron. Hctcrogcnous gcnc(s) hitched to endogenous gcnc(s) of a high efficiency controllable system of hctcrogcnous gcnc(s) hitched to endogenous gene expression also means using IGG sequence to link a hctcrologous gene between a host expressed gene (endogenous gene) and its terminator to form a bicistron. Thus, the aim of this invention is t