Engineering strain for synthesizing serotonin by using 5-hydroxytryptophan as substrate microorganism, construction and application thereof

A technology for hydroxytryptophan and microbial synthesis, applied in the biological field, can solve the problems of long synthesis process, harsh conditions, residual substrate, etc., and achieves the effects of being friendly to the human body and the environment, increasing yield and increasing content.

Pending Publication Date: 2022-03-01
河北维达康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] According to reports, serotonin mainly relies on chemical synthesis, but there are disadvantages such as long synthesis process, many types of raw materials used, harsh conditions, and many wastes; while biosynthesis of serotonin is a green synthesis process, which has the characteristics of less waste and easy disposal. , with broad application prospects
However, the yield of biosynthetic serotonin in the prior art is low, and there is a large amount of surplus substrate, which cannot meet the requirements of industrialized production.

Method used

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  • Engineering strain for synthesizing serotonin by using 5-hydroxytryptophan as substrate microorganism, construction and application thereof
  • Engineering strain for synthesizing serotonin by using 5-hydroxytryptophan as substrate microorganism, construction and application thereof
  • Engineering strain for synthesizing serotonin by using 5-hydroxytryptophan as substrate microorganism, construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of an engineered bacterium expressing 5-hydroxytryptophan decarboxylase

[0054] (1) Using the pRSFDuet-1 vector as a template, PCR amplification was performed using primers P-F and P-R; the pRSFDuet-1 vector was a commercial vector purchased from Novagen; the sequence of the primer P-F was 5'-TGCTTAAGTCGAACAGAAAGTAATCGTATTG-3', The sequence of primer P-R is 5'-GGTATATCCTCCTTATTAAAGTTAAAACAAAATTATTTCTACAGGGG-3'; the linearized vector pRSFDuet-1 is obtained after recovery of the PCR product, and the size of the linearized vector fragment is 3739bp;

[0055] (2) Using artificially synthesized GmDDC, PaDDC, CcDDC, TcDDC and CgDDC genes as templates, the primers were P1 and P2, P3 and P4, P5 and P6, P5 and P6, P7 and P8, P9 and P10 respectively, and carried out PCR amplification, the amplified products are recovered to obtain GmDDC, PaDDC, CcDDC, TcDDC and CgDDC target fragments, the fragment sizes are respectively 1446bp, 1440bp, 1467bp, 1428bp and 143...

Embodiment 2

[0060] Example 2 Cell Fermentation to Produce Serotonin

[0061] (1) engineering bacterium 1, engineering bacterium 2, engineering bacterium 3, engineering bacterium 4 and engineering bacterium 5 are cultivated overnight in the seed culture medium containing 50 μ g / mL kanamycin, obtain seed liquid, described seed culture medium ( mass percent) is 1% tryptone, 1% sodium chloride and 0.5% yeast extract, and the culture condition of the seed solution is 37°C, 225rpm;

[0062] (2) Inoculate the above seed liquid by 2% into a mixture containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH 2 PO 4 and 1.64% K 2 HPO 4 ·3H 2 Fermentation culture is carried out in the fermentation medium (mass percentage) of O;

[0063] (3) After 2 hours of fermentation and cultivation, IPTG with a final concentration of 0.5mM was added to induce expression, the temperature of induction expression was 25°C, and the induction time was 16h; 40g / L substrate 5-hydroxytryptophan was added...

Embodiment 3

[0070] Example 3 The promoting effect of coenzyme pyridoxal phosphate on serotonin production

[0071] (1) Cultivate engineering bacteria 1 overnight in a seed medium containing 50 μg / mL kanamycin to obtain a seed liquid. The seed medium (mass percentage) is 1% tryptone, 1% sodium chloride and 0.5 % yeast extract, the cultivation condition of the seed liquid is 37°C, 225rpm;

[0072] (2) Inoculate the above seed liquid by 2% into a mixture containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH 2 PO 4 and 1.64% K 2 HPO 4 ·3H 2 Fermentation culture is carried out in the fermentation medium (mass percentage) of O;

[0073] (3) After 2 hours of fermentation and cultivation, IPTG with a final concentration of 0.5 mM was added to induce expression, the temperature for induction expression was 25° C., and the induction time was 16 hours;

[0074] (4) Add 40 g / L of substrate 5-hydroxytryptophan to carry out the biotransformation stage, and add different concentra...

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Abstract

The invention discloses an engineering strain for synthesizing serotonin by using 5-hydroxytryptophan as a substrate microorganism as well as construction and application of the engineering strain, and belongs to the technical field of biology. The engineering strain contains a 5-hydroxytryptophan decarboxylase gene, and 5-hydroxytryptophan is finally catalyzed to generate serotonin through expression in a host strain. The method is simple to operate, free of environmental pollution, high in yield, high in purity and low in cost, a basis is provided for industrial production of the serotonin, and the highest yield of the final product serotonin can reach 31.9 g/L.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of an engineering strain for microbially synthesizing serotonin with 5-hydroxytryptophan as a substrate. Background technique [0002] 5-hydroxytryptamine was first discovered in serum, also known as serotonin, also known as 5-HT. It is widely present in mammalian tissues, especially in the cerebral cortex and synapses, and it is also an inhibitory neurotransmitter. In peripheral tissues, serotonin is a strong vasoconstrictor and smooth muscle contraction stimulator. [0003] As a neurotransmitter, 5-HT is mainly distributed in the pineal gland and hypothalamus, and can participate in the regulation of physiological functions such as pain perception, sleep and body temperature. Abnormal 5-HT content and function in the central nervous system may be related to the pathogenesis of various diseases such as mental illness and migraine. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/60C12N15/70C12P17/10C12R1/19
CPCC12N9/88C12N15/70C12N1/20C12P17/10C12Y401/01028C12N2800/22C12N2800/101
Inventor 赵云现冯斌杨志彬崔金旺田昊博胡江林田俊波蒋硕生刘婵
Owner 河北维达康生物科技有限公司
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