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A sialic acid inducible expression element and its application

A technology for inducing expression and expressing elements, which is applied in the field of genetic engineering, can solve the problems of inability to transport extracellular sialic acid, lack of natural transport pathways for sialic acid, and limited applications, and achieve good application prospects in metabolic engineering. The construction method is simple and easy to use Effect

Active Publication Date: 2021-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, Bacillus subtilis lacks the natural transport pathway of sialic acid and cannot transport extracellular sialic acid into the cell, which also limits the application of sialic acid-inducible expression elements in the field of metabolic engineering of Bacillus subtilis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of Genome Recombination Integration NanT Fragment

[0054] Design primers L-F: 5'-GTATGTCATGATGCGTGAAGGATCAGTCGCCAAAAACACGC-3' and L-R: 5'-GTTATCCGCTCAAAAAGAGGCACTCCCTAAGGGAGTGCCTCTTTTTATTGCAGTG-3', using the Bacillus subtilis 168 genome as a template to amplify the left arm gene sequence of the NanT transporter recombinant fragment shown in SEQ ID NO.9 ; Design primers S-F: 5'-CCTTAGGGAGTGCCTCTTTTTGAGCGGATAACAATTTCACACAGGAAACAG-3' and S-R: 5'-CACCTATCATAACGCCAGGGTTTTTCCCAGTCACGAC-3', using the P7S6 plasmid as a template (disclosed in Zhang, X., Liu, Y., Liu, L., Wang, M. , Li, J., Du, G. et al., 2018. Modular pathway engineering of key carbon-precursor supply-pathways for improved N-acetylneuraminic acid production in Bacillus subtilis. Biotechnol. Bioeng. 115, 2217–31), expanded Spectinomycin resistance left-arm gene sequence SEQ ID NO.10 of the NanT transporter recombinant fragment; designed primers P-F: 5'-GACTGGGAAAACCCTGGCGTTATGATAGGTGGTATGT...

Embodiment 2

[0055] Example 2 Construction of recombinant Bacillus subtilis that can transport sialic acid

[0056] The recombinant integrated NanT gene fragment LSPNR was transformed into Bacillus subtilis (Bacillus subtilis) 168, recombined into the genome, and the recombinant Bacillus subtilis engineering strain was obtained, which was named BSNanT.

Embodiment 3

[0057] Example 3 Construction of sialic acid response element tool plasmid pHT-ydjO-veg plasmid

[0058] Design primers N-1F: 5-AACGACGGCCAGTGAATTCGAGCTCTTATTTCTTTTTGTTGGTGGTCTGACCGAAAG-3' and N-1R: 5-ACGTAAAAACAAAGGAGGTGAAATGTACACATGGGCCTTATGAACGCATTTGATTCGCA-3', using the Bacillus subtilis 168 genome as a template to clone the transcriptional regulatory protein coding gene NanR gene fragment SEQ ID NO.4; design primer N -2F: 5-GTGTACATTTCACCCTCCTTTGTTTTTACGTAATACGATAAATAGGGCCAAAGGT-3' and N-2R: 5-GTATTACGGAGCACTTCCCATATTATCAAAGAAAGCGGGGAATTGTCCTATACC-3', using the Bacillus subtilis 168 genome as a template, cloned P ydjO Promoter gene fragment SEQ ID NO.1, designed primers N-3F: 5-CCCGCTTTCTTGATAATATGGGAAGTGCTCCGTAATACGCTGACAAGAGA-3' and N-3R: 5-CTTTAAACGATATACCTTTTATACCTGTTATACCATTGTACAACACGAGCCCATTTTTGTCAAATAAAATTTAAATT-3', using the Bacillus subtilis 168 genome as a template, cloned P veg Gene fragment SEQ ID NO.6, designed primers N-4F: 5-ATAACAGGTATAAAGGTATATCGTTTAAAGGA...

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Abstract

The invention discloses a sialic acid inducible expression element and its application, belonging to the field of genetic engineering. The present invention uses the pHT plasmid as a carrier, and in Bacillus subtilis, P ydjO The promoter expresses the transcriptional regulatory protein NanR, and optimizes the strong promoter P that integrates the NanR recognition binding sequence veg , realize the leaky expression of Bacillus subtilis (Bacillus subtilis) 168 at a very low level in the environment containing sialic acid, which can reduce the leaky expression rate to about 2%, and has a wide application prospect.

Description

technical field [0001] The invention relates to a sialic acid inducible expression element and its application, belonging to the field of genetic engineering. Background technique [0002] In Bacillus subtilis, the promoter elements that can induce and activate expression are widely used in the field of metabolic engineering. At present, the commonly used inducible promoters in Bacillus subtilis include the xylose-inducible promoter P xyl and IPTG-inducible promoter P grac . However, the xylose-inducible promoter P xyl The inducer xylose can be metabolically consumed by Bacillus subtilis, and IPTG induces the promoter P grac The inducer IPTG has a certain toxicity problem to the cells. In particular, xylose- and IPTG-inducible promoters have higher leakage issues. The above defects severely limit its application in the field of Bacillus subtilis metabolic engineering. Sialic acid is a suitable inducer as a simple carbon source that is not toxic to cells and is not met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/75C12N1/21C12R1/125
CPCC07K14/32C12N15/75
Inventor 刘延峰堵国成刘龙张晓龙王凤玲袁烁栎陈坚
Owner JIANGNAN UNIV