ppGpp Extraction from Trichoderma Viride Mycelium
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for obtaining guanosine tetraphosphate (ppGpp) are inefficient and costly, particularly due to difficulties in producing high yields from bacteria and other organisms, and are hindered by ethical restrictions on transgenic organisms, while its high levels can inhibit growth and be toxic, limiting its agricultural applications.
Innovation Solution
A method involving the extraction of ppGpp from Trichoderma viride fungus culture using formic acid and ammonium acetate, followed by purification via weak anion exchange, allowing for cost-effective and high-yield production of this nucleotide.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If ppGpp is produced in bacterial systems, then the level of alarmones can be elevated under stressful conditions, but the production is inefficient and costly with difficulty in producing high yields
Solution Approach 1:
The patent changes the biological system parameter from bacterial to fungal (Trichoderma viride), enabling high-yield production of ppGpp. The fungal system naturally accumulates ppGpp under stress conditions without the growth inhibition problems encountered in bacterial systems, achieving both high quantity and high productivity.
2Quantity of substance
If transgenic organisms are used to increase ppGpp production, then yield can be elevated, but ethical restrictions limit their use
Solution Approach 1:
The patent uses a non-transgenic fungal system that can be easily cultivated and discarded after use, avoiding the ethical and regulatory complexities of transgenic organisms. The Trichoderma viride fungus serves as a temporary, disposable production system that meets agricultural needs without long-term environmental concerns.
3Quantity of substance
If high levels of ppGpp are produced, then it can inhibit growth and be toxic, but this limits its agricultural applications
Solution Approach 1:
The patent extracts ppGpp from the fungal culture and purifies it to obtain high concentrations of the compound without the accompanying toxic cellular components. This separation allows the agricultural application of pure ppGpp at effective concentrations while avoiding the harmful effects associated with whole-cell extracts or crude preparations.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the efficient and cost-effective production of ppGpp from Trichoderma viride, overcoming the challenges of bacterial production limitations and ethical constraints, with Trichoderma viride being easier to cultivate and produce in large quantities, facilitating its use in agriculture for stress protection in crops.
Implementation Method 1
The mycelium is suspended in 1.5-2 N formic acid and incubated on ice preferably for 15 to 30 min
Implementation Method 2
nucleotides are separated in such a manner that the mycelium is suspended in 1.5-2 N formic acid
Implementation Method 3
purification based on weak anions exchange... ammonium acetate NH4OAc... are added and introduced into an already prepared SPE column
Implementation Method 4
then centrifuged, and the supernatant is collected
Implementation Method 5
the thus prepared eluate is lyophilized
Data Source
AI summary
The subject of the invention is the method of obtaining and extraction of secondary fungal metabolites characterized in that the T. Viride mycelium in the amount 75-100 mg is shredded and nucleotides are separated in such a manner that the mycelium is suspended in 1.5-2 N formic acid and incubated on ice preferably for 15 to 30 min, then centrifuged and the supernatant is collected, and then ammonium acetate NH4OAc, the 13C ppGpp internal standard are added and introduced into an already prepared SPE column by rinsing with methanol MeOH and ammonium acetate in the amount from 35 to 60 mM, and then the column with the introduced sample is rinsed with ammonium acetate NH4OAc in the amount from 30 to 70 mM preferably 50 mM at the pH from 4 to 5 preferably pH 4.5 and methanol MeOH and nucleotides are eluted with a MeOH/ H2O/NH4OH solution, and the thus prepared eluate is lyophilized, dissolved in water and filtered in columns for DNA purification. Preferably the mycelium is shredded by grinding in a mortar preferably at the temperature from 20 to 25°C.