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A method for the production of trehalose by coupled fermentation of a dual-enzyme fusion enzyme with high-efficiency secreted expression

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

Active Publication Date: 2020-12-22
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 the production of trehalose by coupled fermentation of a dual-enzyme fusion enzyme with high-efficiency secreted expression
  • A method for the production of trehalose by coupled fermentation of a dual-enzyme fusion enzyme with high-efficiency secreted expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1: Construction of knockout lysing gene recombinant bacteria

[0077] 1.1 Primer design

[0078] Primer name Primer sequence Restriction sites Xpf-1 CGC GGATCC AGATGTGCAGTGACTATT

BamHI Xpf-2 ACGTATGACAAGCTTCAAATAAAAAACGGACAC Xpf-3 TCCGTTTTTTATTTGAAGCTTGTCATACGTTTG Xpf-4 CCG GAATTC CCGTAGCCGCCGACAAGA

EcoRI SkfA-1 CGC GGATCC GTTGGTGCGTTAGGGGTT

BamHI SkfA-2 CCCTATTCTCAAATGAAGTAAACCTCTCTCAA SkfA-3 AGAGGAGGTTTACTTCATTTGAGAATAGGGAGT SkfA-4 CCG GAATTC ATTTATGACGTGCTTCCC

EcoRI LytC-1 CGC GGATCC ACAGAATTAGTCTTGATG

BamHI LytC-2 TAGATTTGTCTCTTTCGATAAAATAATCAATAACTT LytC-3 AAGTTATTGATTATTTTATCGAAAGAGACAAATCTA LytC-4 CCG GAATTC GTTGAAATACTTGTCCAC

EcoRI SdpC-1 CGC GGATCC TAGTTTAGAAGGTTATAT

BamHI SdpC-2 AAGACACTCAATTATAATATTATTTATACCTCCATTA SdpC-3 TAATGGAGGTATAATAATATTATAATTGAGTGTCTT SdpC-4...

Embodiment 2

[0098] Example 2: pHT01-P 43 - Construction of MTSase / B.subtilis (LM1234)

[0099] 1. PCR amplification of constitutive promoter P 43 and maltooligosaccharide-based trehalose synthase MTSase, the obtained P 43 Ligated with the MTSase gene fragment by overlapping PCR method to obtain P 43 -MTSase gene fragment, the P 43 - MTSase was ligated with the vector pHT01 purified by double digestion with SacI and BamHI, and ligated with T4DNA ligase at 16°C for 12h. The ligation product was transformed and cloned into E.coli (DH5α) coated with LB solid medium (containing 30 μg / mL ampicillin), cultured in a 37°C incubator for 12 hours, picked a single colony, and passed through LB liquid medium (containing 30 μg / mL ampicillin) ), cultured at 37°C for 12 hours, verified by PCR, selected and verified the correct bacterial culture to extract the plasmid, verified by enzyme digestion, and then determined the DNA sequence of the verified recombinant plasmid to obtain the recombinant plasm...

Embodiment 3

[0104] Example 3: pHT01-P 43 - Construction of MTHase / B.subtilis (LM1234)

[0105] 1. PCR amplification of constitutive promoter P 43 and maltooligosaccharide-based trehalose hydrolase MTHase, the resulting P 43 Ligated with the MTHase ​​gene fragment by overlapping PCR method to obtain P 43 -MTHase ​​gene fragment, the P 43 -MTHase ​​was ligated with the vector pHT01 purified by double digestion with SacI and BamHI, and ligated with T4DNA ligase at 16°C for 12h. The ligation product was transformed and cloned into E.coli (DH5α) coated with LB solid medium (containing 30 μg / mL ampicillin), cultured in a 37°C incubator for 12 hours, picked a single colony, and passed through LB liquid medium (containing 30 μg / mL ampicillin) ), cultured at 37°C for 12 hours, verified by PCR, selected and verified the correct bacterial culture to extract the plasmid, verified by enzyme digestion, and then determined the DNA sequence of the verified recombinant plasmid to obtain the recombinan...

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Abstract

The invention relates to a method for producing trehalose by coupling fermentation on double enzyme fusion enzyme expressed by efficient secretion. The method is characterized in that a promoter P43,a signal peptide PhoD, maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohydrolase (MTHase) are inserted into bacillus subtilis for the first time; meanwhile, after lytic genes Xpf, SkfA, LytC and SdpC are knocked out, construction is carried out to obtain recombinant bacteria pHT01-P43-PhoD-MTSase-MTHase(LM1234), and the rigid connection of a special sequence is adoptedto realize the function expression of a target enzyme; after the trehalose is produced by practical fermentation, an amazing discovery shows that the reformed recombinant bacteria exoenzyme activitycan occupy 70% of total enzyme activity, exoenzyme production and conversion are synchronously carried out, so that a process that trehalose production is carried out after enzyme separation is carried out in traditional fermentation liquor is omitted, and the fermentation of the recombinant bacteria and the production of the trehalose are similar to coupling.

Description

technical field [0001] The invention relates to a method for producing trehalose by coupling fermentative production of trehalose by utilizing highly secreted and expressed double-enzyme fusion enzymes, and belongs to the technical fields of genetic engineering and fermentation engineering. Background technique [0002] Trehalose is a stable non-reducing disaccharide composed of two glucopyranose linked by 1,1-glycosidic bonds. [0003] Trehalose widely exists in nature, has special biological functions such as moisture retention, anti-freeze and anti-drying properties, thermal acid stability, etc., and has a non-specific protective effect on biological macromolecules, so it is used in medicine, agriculture, cosmetics, food, etc The industry application potential is huge. Since the 1980s, countries have successively carried out studies on the physiology, biochemistry and molecular biology of trehalose, and the sugar has now become one of the main oligosaccharides recently d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/12C12N15/75C12R1/125
CPCC12N9/1051C12N9/2408C12N15/75C12P19/12C12Y204/01245C12Y302/01141
Inventor 王腾飞刘洪玲王希晖王松
Owner 烟台兆易生物科技有限公司
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