Stupid_Noob
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First crack at biosynthing 1-hydroxy-1-phenyl-propan-2-one (l-PAC)
I tossed this together from articles throughout the interwebs. Can anyone give it a look before I take a swing at it? Feedback appreciated.
Strain used: Saccharomyces Cerevisiae
Benzaldehyde toxicity levels for S. Cerevisiae:
0.5g/L = growth reduced
1.0 - 2.0g/L = cell inhibition
3.0g/L = viability reduced
Growth/fermentation media (per L) composition:
Pure cane sugar - 7.5%
Yeast extract - 0.8%
Monopotassium phosphate - 0.1%
Urea - 0.4%
Epsom salt - 0.06%
Procedure (fermentation to be carried out in a 5gal bucket [4.5gal = 17.034L])
1. 5gal (18.927L) of fermentation medium is brought to pH 4.5 using citric acid, then boiled at 110C for 20 mins to sterilize, and cooled to room
temperature (18 - 20C).
2. .5gal (1892ml) is collected and set aside. The remaining 4.5gal is sealed and stored at room temperature until use.
3. 210.22ml of the sterilized medium (pH 4.5) is placed in a 500ml flask. 0.210g (1.0g/L) of benzaldehyde is added, and stirring of the solution is
begun. 1g yeast is added to the solution and stirred for 1 hour. The mixture is filtered and 1g of the solid biomass is set aside.
4. 210.22ml of the benzaldehyde free medium is placed in a flask (pH4.4) and 0.210g benzaldehyde is added. The 1g of biomass from the previous flask
is added to the flask and stirred for 1 hour. The solution is then filtered and 1g of the biomass set aside.
5. Repeat step 4.
6. Repeat step 4, except this time bring pH to 4.3, and add 0.420g benzaldehyde. The goal here is to increase the yeasts resistances to both acidity
and benzaldehyde concentration through successive breeding.
7. Repeat step 6.
8. Repeat step 6.
9. Repeat step 6, except this time bring pH to 4.2 and add 0.630g benzaldehyde.
10. Repeat step 9.
11. Repeat step 9. After filtration, weigh out 3-5g of the solid biomass and set aside.
12. Bring the pH in the remaining 4.5gal of medium to pH 4.2 with citric acid.
13. Add the solid biomass from step 11 to the 4.5gal of medium and begin stirring.
14. Weigh out 51.102g benzaldehyde and add 51.102g acetone. Combine the two in a glass vessel.
15. After 2 hours of stirring, add 25.551g of the acetone\benzaldehyde mixture and stop stirring (pH4.2). Seal the container and wait 1 hour.
16. Add 25.551g of the acetone solution (pH 4.2!). Wait 1 hour.
17. Repeat until all remaining solution is used. Maintain pH 4.2. Wait 2 more hours to ensure completion of the reaction.
18. Extract with 300ml diethyl ether (3x) CHECK pH!
19. Wash extracts with 250ml cold water (2x). CHECK pH!
20. Dry ether extracts by adding 15g MgSO4 and stirring for 10-15 minutes. Filter out MgSO4. CHECK pH!
This extract can be used as is for the reduction to l-ephedrine or distilled to give the product.
Acetone may be substituted for acetaldehyde. Ether, dcm, chloroform, benzene, toluene may be used for xtraction Use lower boiling solvent for
xtraction. Keep extract acidic to avoid racemization of PAC pH should be kept at 4.5-5.0 (if not hardening yeast, yields reduced. 4.2-4.5 for
resistant yeast) using citric or phosphoric acid
Thoughts? Anyone think the attempted 'hardening' of the yeast will work?
[Edited on 11-8-2014 by Stupid_Noob]
[Edited on 11-8-2014 by Stupid_Noob]
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Stupid_Noob
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References
The bulk of this writeup is derived from these references. Bits and pieces are melted together from the billion other articles I've read on this
subject.
The third one is of particular interest, although the yeast strain used is different.
Attachment: fermentation&biochem.guide.pdf (1.6MB)
This file has been downloaded 5176 times
Attachment: nutrienteffects.pdf (1.2MB)
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Attachment: OPTICALLYACTIVECOMPOUNDSBYMICROBIALBIOTRANSFORMATION.pdf (116kB)
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phlogiston
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With respect to your growing medium, are you sure ureum is a good source of nitrogen for <i>S. cerevisiae</i>? I have only seen and used
media containing ammonium sulfate for this organism
The last paper is not a different strain, it is an entirely different organism. <i>H. polymorpha</i> is a methylotrophic yeast, (it does
belong to the same family of Saccharomycetaceae though).
Certain aspects of their metabolism are very different. Are you sure it will still yield the intended product?
In principle, your protocol could allow the yeast to adjust to the conditions, but it does not necesarily lead to more product. For instance, it may
upregulate a multidrug transporter that quickly transports any benzaldehyde precursor out of the cell. Or conjugates it to glutathione, etc. etc.
In this timespan, the yeast may adjust by altering the expession of certain genes. On a longer timespan of exposure to benzaldehyde/acid, you can
perhaps also select and culture strains that are genetically superior for your purpose.
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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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Stupid_Noob
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Quote: Originally posted by phlogiston  | With respect to your growing medium, are you sure ureum is a good source of nitrogen for <i>S. cerevisiae</i>? I have only seen and used
media containing ammonium sulfate for this organism
The last paper is not a different strain, it is an entirely different organism. <i>H. polymorpha</i> is a methylotrophic yeast, (it does
belong to the same family of Saccharomycetaceae though).
Certain aspects of their metabolism are very different. Are you sure it will still yield the intended product?
In principle, your protocol could allow the yeast to adjust to the conditions, but it does not necesarily lead to more product. For instance, it may
upregulate a multidrug transporter that quickly transports any benzaldehyde precursor out of the cell. Or conjugates it to glutathione, etc. etc.
In this timespan, the yeast may adjust by altering the expession of certain genes. On a longer timespan of exposure to benzaldehyde/acid, you can
perhaps also select and culture strains that are genetically superior for your purpose. |
Ammonium sulfate is mentioned frequently in papers of this subject, however I can't find a source locally (not that I have really looked), I just
assumed that urea and NH4SO4 were interchangeable as a N source. The only solid reference said only that the organism cannot use nitrates as sources.
In your experience, is NH4SO4 the nitrogen source of choice?
I also thought that the yeast should be exposed to low pH and the aldehyde for a longer period, but didn't want to end up with an 18 hour process.
From what I understand, S. Cerevisiae can tolerate pH levels from 4.5-5.0. Successive hardening can be used to create organisms tolerant of
pH as low as 4.2. Acidity in this instance serves a dual purpose: Acidic conditions reduce formation of byproducts, as well as prevent the
racemization of the desired product.
I am absolutely certain S. Cerevisiae will produce the desired product.
From Wikipedia:
"L-PAC is widely synthesized by fermentation of benzaldehyde and dextrose. In this process, colonies of yeast (in particular, strains such as Candida
utilis, Torulaspora delbrueckii, or Saccharomyces cerevisiae) are cultivated and added to a broth of water, dextrose, and the enzyme pyruvate
decarboxylase in a vat. The yeast is left to grow for a period of time, after which the benzaldehyde is introduced into the broth. The yeast then
ferments the benzaldehyde into L-PAC.[2]
The majority of L-PAC is generated in pharmaceutical plants in India, as an intermediate precursor in the production of pseudoephedrine.
There are also biochemical reactions where enzymes such as Acetohydroxyacid Synthase I [3] from E. coli condense pyruvate and benzaldehyde into R-PAC.
Such methods have much higher conversion rates in comparison to the conventional yeast fermentation"
Source page: http://en.wikipedia.org/wiki/Phenylacetylcarbinol
Both the benzaldehyde and the desired product are toxic to the biomass... Is there a way to continuously remove the product as it forms?
How long would you recommend each "hardening" cycle last?
Also, I have a list of various nutrient ingredients, but none of the articles really go into the benefits and drawbacks with each feedstock in
relation to the yeast used. I see that generally C. Utilis gives higher yields, but AFAIK it isn't available in my area. I also cannot find
any information on benzaldehyde tolerance by different strains.
Nutrient sources include
Molasses, BBO molasses, corn steep liquor, urea, KH2PO4, MgSO4+7H2O, cane sugar, yeast extract, glucose syrup, whey, MgSQ, thiamine, ammonium sulfate,
ammonia...
Lots of choices, not much info as to why each item was chosen.
Thank you in advance for your insight.
[Edited on 11-8-2014 by Stupid_Noob]
[Edited on 11-8-2014 by Stupid_Noob]
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Chemosynthesis
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| Quote: Originally posted by Stupid_Noob |
Ammonium sulfate is mentioned frequently in papers of this subject, however I can't find a source locally (not that I have really looked), I just
assumed that urea and NH4SO4 were interchangeable as a N source. The only solid reference said only that the organism cannot use nitrates as sources.
In your experience, is NH4SO4 the nitrogen source of choice? |
You picked up on substrate feed pulsing to prolong viability yet can't be bothered to perform a simple acid-base reaction? You don't sound like the
specific "Stupid_Noob" I thought you were, despite appearing to share their interest in meth.
Urea has the additional, unfavorable steps of converting to ammonia via a pathway starting with urea carboxylase prior to intersecting with glutamate
dehydrogenase.
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Stupid_Noob
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Are you referring to NH3 from the sulfate and KOH? Not sure what you're getting at...
And I'm obviously not who you think I am.
[Edited on 11-8-2014 by Stupid_Noob]
...indistinguishable from magic...
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Mesa
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You are going to have a fair bit of inactive/dead yeast cells unless you have some way of removing them pre-filtering.
I'm pretty sure autolysis wont be enough given the short timeframes.
EDIT: Also, while the biosynthesis itself requires anaerobic fermentation, I'm pretty sure the culture growth is done with O2 being fed into the
medium at a constant rate.
[Edited on 19-11-2014 by Mesa]
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karlos³
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Way too complicated. Where´s the sodium pyruvate?
Just use peptone as nutrient source.
Can be done in a day, including condensation of the hydroxyketone as raw extract with your favored amine to the imine.
Reduction takes another few hours, and then if let it sit it for a while overnight(speaking of Al/Hg as reducing agent of course), yield of final
aminoalcohol will be much greater.
Just saw it done few weeks ago, with ethylamine, yield was not rewarding, but it worked fine.
Oh, and dont put sugar in it, that will lower your yield, forming more useless benzyl alcohol.
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