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.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com