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Yeast strain promoter with weakened regulation intensity and application thereof in metabolic flux regulation

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

Active Publication Date: 2021-07-23
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 strain promoter with weakened regulation intensity and application thereof in metabolic flux regulation
  • Yeast strain promoter with weakened regulation intensity and application thereof in metabolic flux regulation
  • Yeast strain promoter with weakened regulation intensity and application thereof 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 strain promoter with weakened regulation intensity and application thereof. The invention specifically discloses a DNA molecule, and the DNA molecule is a recombinant promoter with reduced or weakened regulation intensity obtained by integrating a heterologous cis-acting regulatory element with a repression effect and a yeast strain inner source endogenous promoter. According to the invention, the intensity of the yeast strain promoter can be effectively reduced by integrating the marO sequence, the gene expression level is reduced from the transcriptional level, and a new tool is provided for reducing the metabolic flux of a branch way, a decomposition way or a derivative way in yeast metabolic engineering. The metabolic flux from squalene to sterol can be effectively reduced by applying the promoter weakening strategy to weakening of a yeast squalene derivation path. The yeast strain for producing squalene, provided by the invention, can realize efficient accumulation of squalene, reduce the fermentation cost and simplify the fermentation process under the condition of not adding additional enzyme inhibitor terbinafine or other effectors, with a very good industrial application prospect.

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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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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