Method for producing spermidine by using cheap substrates and engineering bacterium

A spermidine and spermidine decarboxylase technology, applied in the field of bioengineering, can solve the problems of undiscovered microorganisms, low spermidine content and the like

Active Publication Date: 2021-03-05
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the enzymes in these pathways are highly regulated, the content of spermidine in animals and plants is low, and no microorganisms that can produce large amounts of spermidine have been found so far

Method used

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  • Method for producing spermidine by using cheap substrates and engineering bacterium
  • Method for producing spermidine by using cheap substrates and engineering bacterium
  • Method for producing spermidine by using cheap substrates and engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1: Mutation and screening of carboxyspermidine dehydrogenase

[0074] Carboxyspermidine dehydrogenase gene rscsdh was cloned from Rhodobacter sphaeroides ATCC BAA-808. The accession NO of the amino acid sequence on NCBI is YP_351518.1. The expressed product is used to synthesize carboxyspermidine. The cloned genes were respectively connected to the pETDuet-1 vector, transformed into Escherichia coli BL21(DE), and expressed and purified. Induced expression purification method: the recombinant Escherichia coli is transferred to LB fermentation medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) in the amount of 2% by volume ratio, when the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.4mM, induce expression and culture at 20°C for 8h. After induction of expression, the cells were collected by centrifugation at 4°C, 8000 rpm, and 20 minutes. After cell destruction, the enzyme was purified by the histag tag method, and the act...

Embodiment 2

[0076] Example 2: Mutation and screening expression of carboxyspermidine decarboxylase

[0077]The carboxyspermidine decarboxylase gene bccsdc (amino acid sequence is EEF87925.1) was obtained from Bacteroides cellulosilyticus DSM 14838. Induced expression and purification were performed in the same manner as in Example 1. Using isocarboxyspermidine as a substrate, the specific activity of the enzyme expressed by carboxyspermidine decarboxylase gene bccsdc is measured as 0 U / mg. Referring to the aforementioned "5. High-throughput screening of random mutations of enzymes", the genes bccsdc1 and bccsdc2 with the highest enzyme activity were obtained by random mutation screening, and their nucleic acid sequences are shown in SEQ ID NO:3 and SEQ ID NO:4. The specific activities of the expressed enzymes were: 32.1, 56.6U / mg.

Embodiment 3

[0078] Embodiment 3: the construction of the recombinant Escherichia coli expressing 6 kinds of enzymes simultaneously

[0079] Construction of recombinant Escherichia coli: six genes (γ-glutamate kinase ecggk, glutamate-5-semialdehyde dehydrogenase ecgsd, carboxyspermidine dehydrogenase rscsdh, carboxyspermidine decarboxylase bccsdc, glucose dehydrogenase Hydrogenase bsgdh, polyphosphate kinase 2-I smpkk) were connected to pETDuet-1, pACYCDuet-1, pRSFDuet-1 or pCDFduet-1 plasmids respectively, each plasmid expressed 2 kinds of genes, and each gene contained T7 promoter and RBS junction, with T7 terminator behind the gene. The 3 recombinant plasmids were transformed into E. coli Escherichia coli BL21, and the positive transformants were obtained by screening with a mixed antibiotic plate, that is, 6 recombinant E. coli that could enhance the expression of 6 genes as shown in Table 1 were obtained.

[0080] Construct the whole cell transformation production system, the whole c...

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Abstract

The invention discloses a method for producing spermidine by using cheap substrates and engineering bacteria, and belongs to the technical field of bioengineering. The method is characterized in thatrecombinant cells or a combination of recombinant cells expressing gamma-glutamyl kinase, glutamate-5-semialdehyde dehydrogenase, aspartokinase, aspartate-beta-semialdehyde dehydrogenase, amine dehydrogenase, L-2,4-diaminobutanoate decarboxylase, carboxyspermidine dehydrogenase, carboxyspermidine decarboxylase, glucose dehydrogenase, and polyphosphate kinase 2-I are constructed; and aspartic acidand arginine are catalyzed to synthesize the spermidine by utilizing the recombinant cells or the combination of the recombinant cells. According to the method, selected oxidoreductase can efficientlyuse NAD (NADH) as coenzyme, and the product feedback inhibition is avoided in a reaction process; and the method has a good industrial application prospect.

Description

technical field [0001] The invention relates to a method for producing spermidine with a cheap substrate and engineering bacteria, belonging to the technical field of bioengineering. Background technique [0002] Spermidine, the linear molecular formula is NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH 2 , widely present in microorganisms, plants and animals, is an important physiologically active substance. Spermidine has the effect of prolonging the lifespan of animals and counteracting age-related diseases such as cardiovascular disease, neurodegenerative disease and cancer. [0003] Such as figure 1 As shown, there are mainly two ways to synthesize spermidine in organisms: (1) directly synthesize spermidine under the action of spermidine synthetase through carboxylated adenosylmethionine and putrescine, and carboxylated adenosine Methionine can be obtained from common methionine (Met) through adenylation and decarboxylation catalysis of related enzymes. This pathway is a relative...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00C12N1/21C12N15/70C12R1/19
CPCC12P13/001C12N15/70C12N15/52C12N9/1217C12Y207/02004C12N9/0008C12Y102/01011C12N9/0093C12Y117/01C12N9/88C12Y401/01086C12Y207/02011C12Y102/01041C12N9/1229C12Y207/04001C12N9/0006C12Y101/9901C12N9/0028C12Y105/01043C12Y401/01096
Inventor 蔡宇杰梁鑫鑫胡小祥丁彦蕊白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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