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Yeast promoters with reduced regulatory strength and their application in metabolic flux regulation

A technology of promoters and endogenous promoters, applied in fermentation, fungi, viruses/phages, etc., can solve problems such as lack of regulatory tools

Active Publication Date: 2022-04-26
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, the research on how to effectively reduce the regulatory strength of promoters is still very limited, and there is still a lack of effective regulatory tools for down-regulating the metabolic flux of branch pathways, decomposition pathways or derivative pathways

Method used

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  • Yeast promoters with reduced regulatory strength and their application in metabolic flux regulation
  • Yeast promoters with reduced regulatory strength and their application in metabolic flux regulation
  • Yeast promoters with reduced regulatory strength and their application in metabolic flux regulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1. Integrating heterologous cis-regulatory elements to construct promoters with different regulatory strengths

[0089] 1. Screening of heterologous cis-regulatory elements

[0090] The repressor protein from Escherichia coli was preliminarily determined based on the screening principles of heterogeneity, mechanism of action and DNA binding site, cheap and easy-to-obtain repressor effectors, and non-existence or content below the threshold in Saccharomyces cerevisiae The DNA-binding sequence marO of MarR is a heterologous cis-regulatory element for engineering yeast promoters.

[0091] MarR derived from Escherichia coli belongs to the multidrug resistance pump family repressor protein, and its effector is salicylate. The core binding site of MarR on the target gene promoter is a conserved 12bp pseudo-palindromic repeat sequence marO, the sequence of which is shown in SEQ ID No.1.

[0092] 2. Construction of yeast promoter with heterologous cis-regulatory eleme...

Embodiment 2

[0139] Example 2 Using marO to Transform Yeast ERG1 Promoter to Improve Cell Squalene Content

[0140] 1. Construction of ERG1p series promoters containing marO

[0141] (1) According to the nucleotide sequence of the promoter ERG1p of the Saccharomyces cerevisiae squalene monooxygenase gene ERG1 reported in GenBank (GenBank No.NC_001139.9) and the DNA binding site sequence of the Escherichia coli repressor protein MarR were designed and The following primers were synthesized (the underlined parts are the recognition sites of restriction endonucleases HindIII and BamHI respectively; the lowercase letters are marO sequences):

[0142] ERG1p-F: 5'-CC AAGCTT TGAGCGTGGTTCAGGGCACTCTAC-3'

[0143] ERG1p-R: 5'-CG GGATCC GACCCTTTTCTCGATATGTTTTTCT-3'

[0144] ERG1p(SRE)-1F:5'-GCGATACTGCCGTAGCGGGCCTttgccagggcaaAAAGGCAAGCAGTGTATC-3'ERG1p(SRE)-1R:5'-GATACACTGCTTGCCTTTttgccctggcaaAGGCCCGCTACGGCAGTATCGC-3'ERG1p(SRE)-2F:5'-AGCAGTGTATCGGACAGAttgccagggcaaGCTGATATAACACAATACGC-3'ERG1p(SRE)...

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Abstract

The invention discloses a yeast promoter with weakened regulation strength and application thereof. The invention specifically discloses a DNA molecule. The DNA molecule is a recombinant promoter with a reduced or weakened regulatory strength obtained by integrating a heterologous cis-regulatory element with a repressive effect on a yeast endogenous promoter. The invention can effectively down-regulate the strength of yeast promoter by integrating the marO sequence, reduce the gene expression level from the transcription level, and provide a new tool for down-regulating the metabolic flux of branch pathway, decomposition pathway or derivative pathway in yeast metabolic engineering transformation. Applying this promoter attenuation strategy to the attenuation of the yeast squalene-derived pathway can effectively reduce the metabolic flux from squalene to sterols. The squalene-producing yeast strain provided by the present invention can realize high-efficiency accumulation of squalene without additional addition of enzyme inhibitor terbinafine or other effectors, reduce fermentation cost, simplify fermentation process, and have great advantages. Good prospects for industrial application.

Description

technical field [0001] The invention relates to a yeast promoter with weakened regulatory strength in the fields of genetic engineering and metabolic engineering and its application in metabolic flux regulation. Background technique [0002] Yeast is a very important cell factory in the field of modern biotechnology, and has a very wide range of applications in the fields of bioenergy, bulk chemicals, fine chemicals and biomedicine. Reconstructing, transforming and optimizing metabolic pathways through different strategies using yeast as the chassis cells is an important means to achieve efficient synthesis of target metabolites. [0003] Transcriptional regulation is the key regulatory link to control the expression level of genes and related protein activities. The use of strong promoters to enhance the expression of genes related to the synthesis of target metabolites is the most versatile and effective technical means in metabolic engineering. Therefore, promoters from d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N1/19C12N15/81C12P5/02C12R1/865
CPCC07K14/39C12N15/81C12P5/026C12N2830/001
Inventor 何秀萍周晨瑶李明杰鲁素蕊郭雪娜程艳飞王肇悦
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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