Ex Nihilo
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How to research biochemical synthesis pathways?
Hello!
What is the general principle and approach to determine the biosynthetic route of the substance?
E.g. I want to figure out the biochemical pathway for the synthesis of the substance of interest - quercetin-3-O-rutinoside from the plant species
Carpobrotus edulis.
What steps should I take to fully explore this entire biosynthetic pathway?
The idea of exploring all these biochemical transformations from the preceding substances to the given to get an answer - what ENZYMES are
involved at what step and which GENES encode these enzymes!Getting answers to these questions is important for the subsequent introduction of these
genes into the microorganisms for the production of described substance on a large scale.
Are there any fundamental literature on these topics that describes in detail the whole process from start to finish (exploring biochemical pathways)?
Thanks a lot!
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Tsjerk
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You should first look at what molecules you think are used to make the product, and see if you can find literature about how these are produced, or
see if they are available in your host species. Next you can try to clone genes you think are needed to produce the metabolites, and to utilize the
metabolites... Check if it works and continue until everything works. Now you know a pathway.
To prove this pathway is used in your original species, you need to knockdown and up regulate all genes in the pathway and see if the metabolites go
down and up as expected.
The above is a really short summary, elucidation and copying a pathway to a host species is many PhDs and post-docs work. Is your species even
transformable?
[Edited on 10-3-2020 by Tsjerk]
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phlogiston
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There are many ways to get to Rome. Typical strategies are:
1) Find mutants that are unable to make the metabolite of interest. Typically, you expose seeds to a treatment that causes random mutations and you
then find a way to select for the mutants you are interested in. Ideally you can find a treatment that your mutants are immune to, but that will kill
wild-types and mutants that are not of interest to you (a 'positive' screen). Then sequence your mutants to find out which genes have been affected.
2) Try to guess the steps in the biosynthetic pathway, and set up assays for the enzymatic reactions involved. Then, purify each enzyme from the plant
using the enzymatic assay to identify the fractions containing your enzyme. When you have a fraction with pure(ish) enzyme, use MALDI-TOF to identify
it.
3) If the genome of your plant is known, and there are other organisms that have a similar biosynthetic pathway: search the genome of your plant for
orthologues of the enzymes in other species.
After identifying an enzyme/gene by any of these or other methods you will then want to express the enzyme you think is responsible for a metabolic
step in a microorganism that can make a lot of the enzyme for you (typically bacteria or yeast), purify this recombinant protein and confirm in-vitro
that it has the enzymatic activity that you think.
If you are interested in a pathway or plant that very little is known about, this will take many years even by experienced people in a well-equipped
lab. Some people have build an entire academic carreer out of resolving only a few steps in a single pathway.
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"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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formallydehyde
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If you aren't already aware of them, KEGG Pathway and BioCyc are often useful places to start.
I find those are usually easier than trying to hunt down literature.
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ethidium
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As mentioned there are many ways to do this, from the reading I have done they all seem tough.
Bioinformatic approaches can be carried out and are likely most accessible. This only works if your organism of interest has been sequenced though.
This works on the premise that eukaryotes have some tendency to cluster genes associated with a pathway (this is not always the case and is by no
means a sure thing). This means that by looking for genes which show differing activity which are adjacent to one another it becomes likely that a
pocket of genes is a cluster which are expressed simultaneously (e.g. a methlytransferase next to an acyl transferase).
Though using this knowledge and your target molecule you could try and work backwards, the molecule you mention quercetin-3-O-rutinoside is a
dissacharide with a ester group linking to another big bit with all those rings (can you tell I'm not a chemist).
You could probably identify all the proteins in your organism which have the ability to form dissacharides based on a search for proteins within the
organism which show a dissacharide forming domain.
There are experimental methods but I think they depend on making knockouts (as mentioned above) and then using techniques like mass spectrometry to
determine how far along the pathway you are getting and then you can figure out which genes were responsible for it.
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