Method for synthesizing spermidine by biological way

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

Active Publication Date: 2020-11-27
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 synthesizing spermidine by biological way
  • Method for synthesizing spermidine by biological way

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 the current literature reports mainly use NADPH (NADP) as a coenzyme, and tend to reduce asparagine to homoserine. Usually there is more NAD in bacteria than NADP, and NAD is cheaper. In this example, homoserine dehydrogenases that can efficiently oxidize homoserine to aspartaldehyde by using NAD as a coenzyme were firstly 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 WP_047622250.1, AIO28186.1, AKV10834.1, KUN83774.1, EEI22323.1, AIL...

Embodiment 2

[0035] Embodiment 2: Screening of carboxyspermidine dehydrogenase

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

[0037]分别从Bacillus coagulans DSM 1、Clostridium sp.DSM 8431、PrevotellabiviaDSM 20514、Ruminococcuscallidus ATCC 27760、Campylobacter jejunisubsp.jejuni ATCC 700819、Porphyromonascatoniae ATCC 51270、Rhodobactersphaeroides ATCC BAA-808、Clostridium symbiosum ATCC 14940克隆得到羧基亚精胺脱氢酶 Genes bccsdh, cccsdh, pbcsdh, rccsdh, cjcsdh, pccsdh, rscsdh, cscsdh. The amino acid sequences of the enzymes encoded by these genes are AJH78404.1, SFU79043.1, EIM32270.1, ERJ96771.1, YP_002343630.1, EWC93538.1, YP_351518.1, ERI79986.1 in N...

Embodiment 3

[0039] Example 3: Selection of carboxyspermidine decarboxylase

[0040] Carboxyspermidine decarboxylase widely exists in a variety of microorganisms. This embodiment obtains carboxyspermidine decarboxylase genes from BacteroidescellulosilyticusDSM 14838, Clostridium symbiosum ATCC 14940, Campylobacter jejunisubsp.jejuni ATCC 700819, RhodobactersphaeroidesATCC BAA-808, respectively. 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 same method as in Example 1 was used to express and purify carboxyspermidine decarboxylase. Carboxyspermidine decarboxylase gene bccsdc, cscsdc, cjcsdc, rscsdc expressed specific activities of enzymes were 78.2, 64.1, 67.4, 76.3 U / mg respectively.

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Abstract

The invention discloses a method for synthesizing spermidine through a biological way, and belongs to the technical field of bioengineering. According to the invention, genetically engineered bacteriaare constructed, and homoserine and putrescine (butanediamine) can be used as substrates to synthesize the spermidine. Specifically, homoserine dehydrogenase is used for dehydrogenating homoserine togenerate aspartate semialdehyde and NADH, carboxyl spermidine dehydrogenase synthesizes the aspartate semialdehyde and the putrescine into carboxyl spermidine with NADH as a coenzyme, coenzyme regeneration is achieved to generate NAD, and finally the carboxyl spermidine decarboxylase decarboxylates the carboxyl spermidine to generate the spermidine. The method for biosynthesizing the spermidine provided by the invention is simple in production process, easily available in raw materials and low in cost, and has a good industrial application prospect.

Description

technical field [0001] The invention relates to a method for biologically synthesizing spermidine, which belongs 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 carboxylation of adenosylmethionine and putrescine, and carboxylation of adenosylmethionine Thionine can be obtained from methionine (Met) through adenylation and decarboxylation catalysis of related enzymes. This pathway is a relatively common traditional...

Claims

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Application Information

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Patent Type & Authority Applications(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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