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A kind of method for synthesizing spermidine by biological method

A technology of spermidine and spermidine decarboxylase, which is applied in the field of bioengineering, can solve the problems of low spermidine content and no microorganisms, and achieve the advantages of simple production process, easy availability of raw materials and low cost, and good industrial application prospects Effect

Active Publication Date: 2022-08-02
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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  • A kind of method for synthesizing spermidine by biological method
  • A kind of method for synthesizing spermidine by biological method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Screening of homoserine dehydrogenase

[0032] Homoserine dehydrogenase widely exists in a variety of microorganisms, and currently reported in the literature mainly uses NADPH (NADP) as a coenzyme, and tends to reduce aspartic semialdehyde to homoserine. Usually, there is more NAD in the bacteria than NADP, and the price of NAD is cheaper. In this example, a homoserine dehydrogenase capable of efficiently oxidizing homoserine to aspartal semialdehyde using NAD as a coenzyme was screened from multiple strains.

[0033] 分别从Rhizobium leguminosarumATCC 14479、BurkholderiacepaciaATCC25416、Pseudomonas fluorescens NCIMB 11764、Streptomyces griseoruber DSM 40281、Lactobacillus fermentum ATCC 14931、Enterococcus faecalis ATCC 29212、Rhodobactersphaeroides ATCC BAA-808、Clostridium symbiosum ATCC 14940克隆得到高丝氨酸脱氢酶基因rlhsdh、bchsdh、 pfhsdh, sghsdh, lfhsdh, efhsdh, rshsdh, cshsdh. The amino acid sequences of the enzymes encoded by these genes are listed in NCBI Accession NO. W...

Embodiment 2

[0035] Example 2: Screening of carboxyspermidine dehydrogenase

[0036] Carboxyspermidine dehydrogenase widely exists in a variety of microorganisms, usually with NADPH (NADP) as a coenzyme. Usually there is more NAD in the bacteria than NADP, and the price of NAD is cheaper. In this example, a carboxyspermidine dehydrogenase with NADH (NAD) as a coenzyme is obtained by comparing carboxyspermidine dehydrogenases from various sources, which can be used to synthesize carboxyspermidine.

[0037]Carboxylobacter biosimilar symbiosphaeroides ATCC BAA-808, Clostridium dehydrogenase were cloned from Bacillus coagulans DSM 1, Clostridium sp. DSM 8431, Prevotellabivia DSM 20514, Ruminococcuscallidus ATCC 27760, Campylobacter jejunisubsp.jejuni ATCC 700819, Porphyromonascatoniae ATCC 51270, Rhodobactersphaeroides ATCC BAA-808 Genes bccsdh, cccsdh, pbcsdh, rccsdh, cjcsdh, pccsdh, rscsdh, cscsdh. The amino acid sequences of the enzymes encoded by these genes are listed in NCBI Accession ...

Embodiment 3

[0039] Example 3: Selection of carboxyspermidine decarboxylase

[0040] Carboxyspermidine decarboxylase widely exists in a variety of microorganisms. In this example, the carboxyspermidine decarboxylase gene was obtained from Bacteroides cellulosilyticus DSM 14838, Clostridium symbiosum ATCC 14940, Campylobacter jejunisubsp.jejuni ATCC 700819, and Rhodobactersphaeroides ATCC BAA-808. Amine decarboxylase is used to convert carboxyspermidine to spermidine. The amino acid sequences of the enzymes encoded by the genes bccsdc, cscsdc, cjcsdc, and rscsdc are EEF87925.1, and the amino acid sequences are ERI79985.1, YP_002344893.1, and YP_351517.1, respectively. The carboxyspermidine decarboxylase was expressed and purified by the same method as in Example 1. Using carboxyspermidine as the substrate to measure the activity of the enzyme, the specific activities of the enzymes expressed by the carboxyspermidine decarboxylase genes bccsdc, cscsdc, cjcsdc and rscsdc were 78.2, 64.1, 67....

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Abstract

The invention discloses a biological method for synthesizing spermidine, which belongs to the technical field of biological engineering. The invention constructs genetic engineering bacteria, which can synthesize spermidine using homoserine and putrescine (butanediamine) as substrates. Specifically, homoserine dehydrogenase is used to dehydrogenate homoserine to form aspartic semialdehyde and NADH, and carboxyspermidine dehydrogenase uses NADH as a coenzyme to synthesize aspartic semialdehyde and putrescine into carboxyspermidine, and realize Coenzyme regeneration produces NAD, and finally, carboxyspermidine decarboxylase decarboxylates carboxyspermidine to produce spermidine. The method for biosynthesizing spermidine provided by the invention has the advantages of simple production process, readily available raw materials and low cost, and has good industrial application prospect.

Description

technical field [0001] The invention relates to a method for synthesizing spermidine by biological method, and belongs to the technical field of biological engineering. Background technique [0002] Spermidine, the linear molecular formula is NH 2 (CH 2 ) 3 NH(CH 2 ) 4 NH 2 , widely exists in microorganisms, plants and animals, and 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 diseases and cancer. [0003] like figure 1 As shown, there are two main ways to synthesize spermidine in organisms (1) through carboxylated adenosylmethionine and putrescine, and directly synthesize spermidine under the action of spermidine synthase, and carboxylated adenosine methyl Thionine can be obtained from methionine (Met), which is catalyzed by adenylation and decarboxylation of related enzymes. This pathway is a relatively common ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/00C12N1/21C12R1/19
CPCC12P13/001C12N9/0006C12N9/0026C12N9/88C12Y101/01003C12Y105/99006C12Y401/01096
Inventor 蔡宇杰梁鑫鑫邓华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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