Mutasynthetic biotransformations
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| Number of Parts | 163 | |
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| License | CC Attribution - NoDerivatives 4.0 International: You are free to use, copy, distribute and transmit the work or content in unchanged form for any legal purpose as long as the work is attributed to the author in the manner specified by the author or licensor. | |
| Identifiers | 10.5446/50416 (DOI) | |
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00:00
BiotransformationFunctional groupOrganische ChemieNaturstoffComputer animation
00:15
Functional groupNaturstoffMeeting/Interview
00:25
Derivative (chemistry)NaturstoffSkarnLibrary (computing)Chemical structureMeeting/Interview
00:44
NaturstoffNanoparticleBiosynthesisChemical structureMultiprotein complexOrganische ChemieMeeting/Interview
01:04
HeteroatomRecreational drug useOrganische ChemieTotalsyntheseMedicinal chemistryNaturstoffFluorineLactitolChemistryAtomMeeting/Interview
01:24
ChemistSynthetic oilOrganische ChemieMeeting/Interview
01:40
NaturstoffChemische SyntheseOrganische ChemieChemistFunctional groupMeeting/Interview
02:22
MetabolismBiosynthesisMetabolic pathwayEnzymeLecture/ConferenceMeeting/Interview
02:37
NaturstoffEnzymeMoleculeRecreational drug useLecture/Conference
02:56
Precursor (chemistry)MoleculeAmineChemistOrganische ChemieGeneMetabolic pathwayRing strainSea levelSemioticsEnzymeGenomeMetabolismBiosynthesisMeeting/InterviewLecture/Conference
03:34
MoleculeBenzoic acidPrecursor (chemistry)ChemistDerivative (chemistry)MetabolismToll-like receptorMetabolic pathwayNaturstoffMeeting/Interview
03:54
NaturstoffOrganische ChemieTotalsyntheseMetabolic pathwayDerivative (chemistry)DeathChemistGemstoneLecture/ConferencePanel painting
04:15
Storage tankThermalquelleSolutionPH indicatorLecture/ConferenceMeeting/InterviewChemical experiment
04:37
SolutionStructural steelChemical experiment
04:50
Structural steel
05:01
Chemical experimentMeeting/Interview
05:12
AgarCombustion chamberSample (material)Organische ChemieChemical experiment
05:27
AageSample (material)Organische ChemieMeeting/InterviewChemical experiment
05:37
WursthülleMeeting/InterviewChemical experiment
05:47
WursthülleChemical experiment
05:59
WaterChemical reactorChemical reactionChemical experimentMeeting/Interview
06:13
MixtureWaterGrowth mediumChemical experiment
06:24
WaterGrowth mediumSecondary metaboliteChemical experimentMeeting/Interview
06:47
Substrat <Chemie>SolutionChemical experimentMeeting/Interview
07:05
FiltrationSolutionBlood vesselChemical experimentMeeting/Interview
07:17
SteroidSolutionChemical experimentMeeting/Interview
07:29
Electronic cigaretteSteelChemical compoundChemical experimentMeeting/Interview
07:41
Chemical compoundFeed (film)Growth mediumCultivatorMarch (territory)FlocculationPeriodateChemical experimentMeeting/Interview
07:59
Ion transporterPolytetrafluoroethyleneCultivatorChemical experiment
08:12
PeriodateCultivatorCell divisionChemical compoundCell (biology)Alkoholische GärungMoleculeChemical experiment
08:26
Cell (biology)MacromoleculeAlkoholische GärungProteinEssigsäureethylesterKohlenhydratchemieSystemic therapyChemical compoundMoleculeWater purificationBrothSilicon dioxideHuman subject researchWine tasting descriptorsChemical experiment
09:06
MoleculeDerivative (chemistry)Functional groupProlineCarbon (fiber)Chemical experiment
09:34
AlcoholKetoneChemical compoundAlkaneDiagramProgram flowchart
09:55
Toll-like receptorChemical compoundActivity (UML)Carbon (fiber)Functional groupMetabolismToxicityReducing agentBlue cheeseSaltMetalDiagramProgram flowchartChemical experimentMeeting/Interview
10:33
Cell (biology)ConcentrateChemical compoundController (control theory)Growth mediumFamotidineChemical experimentMeeting/Interview
11:00
Growth mediumChemical experiment
11:13
Growth mediumSolutionChemical experiment
11:28
Arzneimitteldosis3'-Phosphoadenosine-5'-phosphosulfateCell (biology)Beta sheetThermoformingChemical experiment
11:51
Cell (biology)Blue cheeseSaltGene clusterDeterrence (legal)BiosynthesisMetabolismSynthetic oilStiffnessMetabolic pathwayChemische SyntheseSynthetic biologySubstrat <Chemie>Chemical compoundChemical experimentMolecular geometryMeeting/Interview
12:19
BiotransformationChemistryOrganische ChemieISO-Komplex-HeilweiseMolecularityCollisionHydrophobic effectChemische SyntheseSynthetic biologyMeeting/Interview
Transcript: English(auto-generated)
00:15
Welcome to the Institute of Organic Chemistry at the Leibniz University of Hanover.
00:20
My name is Andreas Kirschning and I'm heading a research group that is dedicated to natural products. In this field we are particularly interested in developing new strategies and tactics to create natural product derivatives or even small libraries. The natural products that we usually handle are structurally rather complex
00:40
so they are very difficult to access by synthetic, purely synthetic methods. On the other hand, all of our natural products are biologically very relevant and also pharmacologically highly potent. When we look at natural products, how they are formed by nature, nature pursues a highly effective and specific and precise multi-step biosynthesis to create structural complexity.
01:06
Synthetic organic chemistry however is very flexible. You can introduce easily fluorine into a natural product by total synthesis which is very difficult for nature to achieve. However fluorine is an important heteroatom in medicinal chemistry and the development of drugs.
01:24
We do not base our work specifically on purely chemical knowledge but we rather look in neighbouring disciplines such as engineering and biology, look for established methods and techniques there and try to introduce and implement them into the portfolio of the synthetic organic chemist.
01:44
The new term that is important to mention here is mutosynthesis. It basically uses engineered organisms, the producer of natural products like enzymitosine and combines it with chemical synthesis. Please follow me and take a look at the theoretical aspects of mutosynthesis as well as take a
02:06
look at how this is transformed into a laboratory environment, particularly a laboratory environment that is rather chemical. Hello, my name is Frank Hahn and I am a Junior Research Group Leader at the Institute of Organic Chemistry here in Hannover.
02:22
I am going to introduce you into the concept of mutosynthesis. The present publication deals with enzymitosines and the biosynthesis of enzymitosines is arranged in a stepwise assembly line like fashion. We have several intermediates during the biosynthetic pathway and each intermediate is interconverted into each other by a particular enzyme,
02:47
for example intermediate A reacts with enzyme A to intermediate B, reacts with enzyme B to intermediate C and so on. Finally, we end up with a very complex natural product starting from very simple precursor molecules. One of these precursor molecules is amino hydroxybenzoic acid, AHBA.
03:10
Nowadays it is also possible to manipulate biosynthetic pathways on a genetic level. For example, for mutosynthesis we remove or disrupt genes from the genome. This leads to changes in the biosynthesis.
03:23
If we for example remove the genes for enzyme A, the organism is no longer able to produce intermediate B. We have a so-called mock-out strain or a blocked mutant. What we now do is, we feed a similar precursor molecule for a amino benzoic acid. So we feed it to the blocked mutant strain and see what happens.
03:46
So it happens something which is quite interesting for a chemist because this derivative of the precursor is accepted by the biosynthetic pathway. So it is channeled through it via different intermediates which are not identical but very similar
04:02
to the ones from the native pathway and finally end up in the new natural product derivative. So we end up with death mitoxi, death chloro, chloroansamitosin. So this is a very attractive opportunity for a chemist to hand over the endgame of a total synthesis to an organism.
04:22
I will now hand over to Gerrit who will show you how such an experiment is done in practice. Hello, I am Gerrit Uyens and today I will show you how we actually perform a neutrosynthesis in vacationing group. So first what we need is our microorganism. Actinocinema pretiosum is a spore-relating bacterium and we usually keep it stored as a spore solution at minus 80 degrees.
04:49
And first now I will put those spores onto an agar plate. I use regular toothpicks which are sterilized and then draw narrow lines onto the agar plate.
05:13
After applying them on the agar we incubate them for seven days in an incubation chamber at 28 degrees.
05:28
So over here I have prepared a sample which is roughly four to five days old. So you can see it's not completely grown yet but over here you can see the orange central part is the living organism.
05:41
While the air mycelium on the edges of the colonies consists mainly of the spores. So for the inoculation we only take the air mycelium. So in this case we use the inoculation loop, sterilize it a bit and take out some of the air mycelium.
06:04
And put this air mycelium into a reaction vessel with glass beads and water. And then vortex the whole mixture to suspend the spores in the water.
06:24
And then we can add this water to the preculture medium to inoculate it. This is then incubated for two days at 28 degrees. And afterwards we can use this preculture to inoculate the main cultures.
06:41
Where we use a special production medium which is optimized for the production of secondary metabolites. So after that we have our main cultures which we will incubate for another two days. And after another two days we will add the substrate which we have synthesized before. And we still need to sterile filter it.
07:01
So we take out the solution in a syringe. We then use a sterile filter to filter the solution into another vessel. This sterile solution can then be taken up into a Lulock syringe.
07:24
Which can then be attached to tubings. We attach it to the syringe with our sterile solution. And at the end we can attach a steel cannula.
07:41
This can then be poured through this cotton plug. And we can add the compound continuously over three days to the culture medium. Which is an advantage because usually you can only do this in a bioreactor. Over here you can see a typical feeding experiment.
08:03
Where we supplement the muta-synthem by a syringe in the upper part in the syringe pump. Through a Teflon tubing into our four cultivation flasks. And we add it over a period of three days very slowly. And then afterwards we let it cultivate for another two days. After which we can actually take out the whole cultivation flask and do a workout.
08:30
After fermentation we need to obtain our compound. And we do that by extracting the fermentation broth with ethyl acetate. This obtained crude product is then filtered over a silica pad.
08:40
To get rid of most of the cell fragments. And in order to get rid of macromolecules like proteins or sugars. We use a Sephadex system HL20. Which you can see on the right hand side. And this retains our compound. Especially small molecules. And after that we just need a final purification via HPLC.
09:04
To obtain our final compound. What we would expect is this molecule which I have drawn over here. But we didn't find this molecule. We found other derivatives which are modified. So in fact this is a pro enzymaticin derivative. Which doesn't have these functionalities.
09:26
But the more important thing is that actually it is modified in this carbon moiety. So actually one very important derivative is the one where it doesn't have a carbon moiety at all.
09:41
And we can find a ketone over here. And a complete reduced alcohol to the alkyl group. So this is one important compound. And the other one which we found was where you got a reduced ketone. Which is then carbon moiety later.
10:02
Those two compounds are especially interesting. Because they are both modified in this region. Where we usually have the carbon moiety which is essential for biological activity. And that's why it's also very interesting to evaluate these compounds in biological testing. My name is Francisco Damballe and I'm working on to-do toxicity.
10:23
The MTT assay is an established method to measure the metabolic activity. It is based on the reduction of the yellow MTT to the blue formazan salt. The advantage of this assay is the comparison of the metabolic activity of non-proliferating and proliferating cells.
10:42
So at first the cells are cultivated in a 96 well plate. The test compound in different concentration is added. And the test compound without cells is used as a control. The cells are then incubated for five days at 37 degrees.
11:00
After this time the medium is removed. Now fresh medium with the MTT solution is added.
11:33
And now the cells are incubated again for two hours in the incubator.
11:47
I've prepared one plate for you. So here you can see the results. The number of living cells collates directly to the amount of the blue formazan salt. So now you can quantify the formazan salt with a microplate reader.
12:01
From these results we can calculate a dose-dependent curve. I believe that in the future research has to focus on improved manipulation of biosynthetic pathways in order to broaden the substrate specificity of these biosynthetic gene clusters. In order to get the full scope of flexibility, synthetic chemistry as well as synthetic biology
12:24
has to be combined in order to create a larger structural space to be addressed. I thank you for your attention.