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A method for preparing trehalose by coupled fermentation

A trehalose and gene technology, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems such as the inability to realize coupled fermentation to produce trehalose and the like

Active Publication Date: 2021-08-31
烟台兆易生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the trehalose synthase in this technology can be secreted outside the cell, it is still impossible to achieve coupled fermentation to produce trehalose

Method used

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  • A method for preparing trehalose by coupled fermentation
  • A method for preparing trehalose by coupled fermentation
  • A method for preparing trehalose by coupled fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 High-efficiency expression of trehalose synthase by various promoters

[0050] 1. Optimization of the promoter

[0051] According to the promoter P reported by NCBI srfA (SEQ ID NO.9), promoter P cry3A (SEQ ID NO.11) and promoter P 43 (SEQ ID NO.13) sequence, carry out codon optimization, P srfA The -35 region (GTGATA) and -10 region (TAAACT) of the promoter were changed to -35 region (TTGACT) and -10 region (TATAAT) respectively, and the optimized promoter P srfA Nucleotide sequence SEQ ID NO.10; P cry3A The -35 region (TTGCAA) and -10 region (TAAGCT) of the promoter were changed to -35 region (TTGACA) and -10 region (TATAAT) respectively, and the optimized promoter P cry3A Nucleotide sequence SEQ ID NO.12; P 43 The promoter sequence was shortened, 13bp less than the original sequence, and the optimized promoter P 43 The nucleotide sequence of SEQ ID NO.1 was artificially synthesized according to the optimized sequence. Using the synthetic promoter se...

Embodiment 2

[0066] Example 2 Secretion and expression of trehalose synthase by 24 kinds of signal peptides

[0067] 1. Construction of recombinant plasmids

[0068] Using the Bacillus subtilis genome as a template, 24 signal peptide sequences were amplified with corresponding primers, and the promoter P 43 P 43 -PhoD-treS-1, P 43 -YwbN-treS-1,P 43 -WprA-treS-1,P 43 -YkuE-treS-1,P 43 -YmaC-treS-1,P 43 -AlbB-treS-1, P 43-AmyX-treS-1,P 43 -AppB-treS-1,P 43 -LipA-treS-1,P 43 -PbpX-treS-1, P 43 -OppB-treS-1, P 43 -YvhJ-treS-1,P 43 -YuiC-treS-1, P 43 -YmzC-treS-1,P 43 -QcrA-treS-1,P 43 -TipA-treS-1,P 43 -WapA-treS-1,P 43 -YesW-treS-1,P 43 -YdeJ-treS-1, P 43 -YesM-treS-1,P 43 -YfkN-treS-1,P 43 -YkpC-treS-1,P 43 -YubF-treS-1, P 43 -SpoIIIJ-treS-1, 24 gene fragments were digested and purified with BamHI and Not I vector pHT01-P 43 -treS, ligated with T4DNA ligase at 4°C for 16h. The ligation product was transformed and cloned into E.coli (DH5α) coated with LB solid medium ...

Embodiment 3

[0111] Example 3 Construction of Knockout Lysis Gene Recombinant Bacteria

[0112] 1. Construction of recombinant plasmids

[0113] Using the Bacillus subtilis genome as a template, the cleavage genes Xpf, SkfA, LytC and Sdpc were respectively amplified by PCR with corresponding primers, and connected to the knockout vector pMAD through restriction sites BamHI and EcoRI to obtain four knockout plasmids pMAD-Xpf , pMAD-SkfA, pMAD-LytC and pMAD-Sdpc.

[0114] 2. Construction of recombinant bacteria

[0115] The knockout plasmids pMAD-Xpf, pMAD-SkfA, pMAD-LytC and pMAD-Sdpc were transferred into the expression host B. subtilis WB800n by electroporation in turn, and LB solid medium (containing 30 μg / mL chloramphenicol) was coated, 37 Cultivate in an incubator at ℃ for 12 hours, perform PCR verification on the single colony on the solid medium, pick and verify the correct single colony, and culture it in LB liquid medium (containing 30 μg / mL chloramphenicol) at 37℃ for 12 hours t...

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PUM

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Abstract

The invention relates to a method for preparing trehalose by coupled fermentation. The present invention inserts the promoter P into Bacillus subtilis by adopting 43 , signal peptide PhoD, and the cleavage genes Xpf, SkfA, LytC, and SdpC and the maltose transporter gene EIICB were simultaneously knocked out, and the recombinant engineered bacteria pHT01‑P was constructed 43 ‑PhoD‑treS (LM12345), after producing trehalose through actual fermentation, it was surprisingly found that the extracellular enzyme activity of the modified recombinant bacteria can account for 70% of the total enzyme activity, realizing the synchronization of extracellular enzyme production and transformation This eliminates the need for enzyme separation in the traditional fermentation broth and then the production of trehalose, and realizes the coupling of the fermentation of recombinant bacteria and the production of trehalose.

Description

technical field [0001] The invention relates to a method for preparing trehalose by coupled fermentation, belonging to the technical fields of genetic engineering and fermentation engineering. Background technique [0002] Bacillus subtlis (Bacillus subttlis) is an important model bacteria in the field of prokaryotic research, people's understanding of its genetic background and physiological characteristics is second only to Escherichia coli (Escherichia coli). Bacillus subtilis is a kind of Gram-positive aerobic bacilli, and it is an ideal host for secreting and expressing foreign proteins in the current prokaryotic expression system. The advantages of this host are: a recognized safe strain, secreted expression, no obvious codon preference; fast growth, easy cultivation, high expression level, clear genetic background and simple molecular operation, and is suitable for engineering strain transformation and other significant advantages . [0003] In genetic engineering, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N15/90C12N15/61C12N15/31C12P19/12C12R1/125
CPCC07K14/32C12N9/90C12N15/75C12N15/902C12N2800/22C12P19/12C12Y504/99016
Inventor 王腾飞王希晖刘洪玲杨少杰
Owner 烟台兆易生物科技有限公司