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Insertion of ergotamine gene into yeast DNA
i have started collecting information for a friend that will help me produce the modified yeast with the ergotamine genes inserted.....Does anyone
know of the procedure so i may help him with the required literature....anyway here is an article that cought my eye......solo
Manipulating the Yeast Genome: Deletion, Mutation and Tagging by PCR
Jennifer M. Gardner* and Sue L. Jaspersen*§
Attachment: Manipulating the Yeast Genome- Deletion, Mutation and Tagging by PCR.pdf (984kB)
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solo
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Reference Information
Complete DNA sequence of yeast Chromosone XI
B.Dujon, D. Alexander
Nature
1994, vol. 369 page 371>
-----------------------------------------------------------
Biosynthetic Pathways of Ergot Alkaloids
Nina Gerhards 1, Lisa Neubauer
Toxins
2014, 6, 3281-3295;
doi:10.3390/toxins6123281
Abstract
Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota,
e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups:
clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the
tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific
enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to
recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by
genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the
overproduced enzymes
.
Keywords: ergot alkaloids; biosynthetic pathway; secondary metabolism; natural products; fungi; mycotoxins
Attachment: Complete DNA sequence of yeast Chromosone XI.pdf (1.7MB)
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Attachment: biosynthetic pathways of ergot alkaloids.pdf (732kB)
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solo
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How much are cells effected by their environment , it was once shown that marijuana seeds exposed to Colchicine will produce a diploid parent, and in
Ibogaine the manipulation of ATP production at a cellular level, so what would the effect be to a yeast culture exposed to ergot alkaloids....surely
at some level the changes in the yeast could be measure, but to produce ergot alkaloids ?
See what happens to Ibogaine when a simila experiment was done......
Induction of energy metabolism related enzymes in yeast
Saccharomyces cerevisiae exposed to ibogaine is adaptation to acute decrease in ATP energy pool
Roman Paškulina, Polona Jamnik
European Journal of Pharmacology
627 (2010) 131-135
Abstract
Ibogaine has been extensively studied in the last decades in relation to its anti-addictive properties that have been repeatedly reported as being
addiction interruptive and craving eliminative. In our previous study we have already demonstrated induction of energy related enzymes in rat brains
treated with ibogaine at a dose of 20 mg/kg i.p. 24 and 72 h prior to proteomic analysis. In this study a model organism yeast Saccharomyces
cerevisiae
was cultivated with ibogaine in a concentration of 1 mg/l. Energy metabolism cluster enzymes glyceraldehyde-3-phosphate dehydrogenase,
phosphoglycerate kinase, enolase and alcohol dehydrogenase were induced after 5 h of exposure. This is a compensation of demonstrated ATP pool
decrease after ibogaine.
Yeast in a stationary growth phase is an accepted model for studies of housekeeping metabolism of eukaryotes, including humans. Study showed that
ibogaine's in fluence on metabolism is neither species nor tissue specific.
Effect is not mediated by binding of ibogaine to receptors, as previously described in literature since they are lacking in this mode
Attachment: Induction of energy metabolism related enzymes in yeast Saccharomyces cerevisiae exposed to ibogaine is adaptation to ac (329kB)
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solo
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MetaCyc Pathway: ergotamine biosynthesis
If an enzyme name is shown in bold, there is experimental evidence for this enzymatic activity.
Superclasses: Biosynthesis → Secondary Metabolites Biosynthesis → Nitrogen-Containing Secondary Compounds Biosynthesis → Alkaloids Biosynthesis
→ Indole Alkaloids Biosynthesis
Some taxa known to possess this pathway include ? : Claviceps purpurea , Epichloe festucae
Expected Taxonomic Range: Clavicipitaceae
Summary:
Ergot alkaloids are a complex family of indole derivatives with diverse structures and biological activities. They are toxins produced by some fungi
from the families Clavicipitaceae, Trichocomaceae and Arthrodermataceae. Some of these compounds and their semi-synthetic derivatives are important
drugs in modern medicine, such as ergotamine and dihydroergotamine, which are used for the treatment of migraines. Ergotamine , along with
ergometrine, have also been used for a very long time as uterine contracting agents following childbirth.
Ergotamine is the major alkaloid produced by Claviceps purpurea and related species.
The early part of the pathway is common to all three families and ends with chanoclavine-I aldehyde (see chanoclavine I aldehyde biosynthesis). This
compound is an important branching point, and is converted to different products within the different fungal families. Within the Clavicipitaceae,
such as Claviceps fusiformis, an enzyme encoded by easG converts chanoclavine-I aldehyde to agroclavine [Matuschek11], which is then converted to
assorted amides and peptides of lysergate, known as ergoamides and ergopeptines, respectively. The potent synthetic hallucinogen lysergic acid
diethylamide (LSD) is a diethylamide of this intermediate.
A biosynthetic gene cluster involved in ergot alkaloids biosynthesis has been identified in Claviceps purpurea. While not all steps have been
associated with an enzyme, the cloA gene was shown to encode a cytochrome P-450 enzyme that catalyzes the formation of paspalate from elymoclavine,
while ps1 and ps2 encode two non-ribosomal peptide synthases that form the subunits of a very large complex that catalyzes the addition of a
tripeptidide tail to the lysergic moiety [Liu09].
Superpathways: superpathway of ergotamine biosynthesis
Credits:
Created 16-Apr-2010 by Pujar A , Boyce Thompson Institute
Revised 01-Jun-2012 by Caspi R , SRI International
References
Liu09: Liu X, Wang L, Steffan N, Yin WB, Li SM (2009). "Ergot alkaloid biosynthesis in Aspergillus fumigatus: FgaAT catalyses the acetylation of
fumigaclavine B." Chembiochem 10(14);2325-8. PMID: 19672909
Matuschek11: Matuschek M, Wallwey C, Xie X, Li SM (2011). "New insights into ergot alkaloid biosynthesis in Claviceps purpurea: an agroclavine
synthase EasG catalyses, via a non-enzymatic adduct with reduced glutathione, the conversion of chanoclavine-I aldehyde to agroclavine." Org Biomol
Chem 9(11);4328-35. PMID: 21494745
Other References Related to Enzymes, Genes, Subpathways, and Substrates of this Pathway
Clay02: Clay K, Schardl C (2002). "Evolutionary origins and ecological consequences of endophyte symbiosis with grasses." Am Nat 160 Suppl 4;S99-S127.
PMID: 18707456
Correia03: Correia T, Grammel N, Ortel I, Keller U, Tudzynski P (2003). "Molecular cloning and analysis of the ergopeptine assembly system in the
ergot fungus Claviceps purpurea." Chem Biol 10(12);1281-92. PMID: 14700635
Haarmann06: Haarmann T, Ortel I, Tudzynski P, Keller U (2006). "Identification of the cytochrome P450 monooxygenase that bridges the clavine and
ergoline alkaloid pathways." Chembiochem 7(4);645-52. PMID: 16538694
Jankowski08: Jankowski MD, Henry CS, Broadbelt LJ, Hatzimanikatis V (2008). "Group contribution method for thermodynamic analysis of complex metabolic
networks." Biophys J 95(3);1487-99. PMID: 18645197
Latendresse13: Latendresse Mario (2013). "Computing Gibbs Free Energy of Compounds and Reactions in MetaCyc." Web.
Riederer96: Riederer B, Han M, Keller U (1996). "D-Lysergyl peptide synthetase from the ergot fungus Claviceps purpurea." J Biol Chem
271(44);27524-30. PMID: 8910337
Rigbers08: Rigbers O, Li SM (2008). "Ergot alkaloid biosynthesis in Aspergillus fumigatus. Overproduction and biochemical characterization of a
4-dimethylallyltryptophan N-methyltransferase." J Biol Chem 283(40);26859-68. PMID: 18678866
Tudzynski99: Tudzynski P, Holter K, Correia T, Arntz C, Grammel N, Keller U (1999). "Evidence for an ergot alkaloid gene cluster in Claviceps
purpurea." Mol Gen Genet 261(1);133-41. PMID: 10071219
Wallwey12: Wallwey C, Heddergott C, Xie X, Brakhage AA, Li SM (2012). "Genome mining reveals the presence of a conserved biosynthetic gene cluster for
the biosynthesis of ergot alkaloid precursors in the fungal family Arthrodermataceae." Microbiology. PMID: 22403186
Walzel97: Walzel B, Riederer B, Keller U (1997). "Mechanism of alkaloid cyclopeptide synthesis in the ergot fungus Claviceps purpurea." Chem Biol
4(3);223-30. PMID: 9115414
Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 38 473-D479 2010
Page generated by SRI International Pathway Tools version 17.5 on Sun Dec 6, 2015.
......source,
http://solcyc.solgenomics.net/META/NEW-IMAGE?type=PATHWAY&am...
.....another source ,
http://biocyc.com/META/NEW-IMAGE?type=PATHWAY&object=PWY...
[Edited on 6-12-2015 by solo]
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Are you answering you're own posts ? Geochrichum candidium is a "yeast" type fungus that will produce LA when grown on certain substrates. I can find
source literature if interested. I do not know anything more. Im not into this type of area so please do not ask, if someone does I can't help.
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phlogiston
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It is good practice to explain in your starting post what it is that you are trying to do.
Also, allow people some time to reply before flooding the thread with your own lengthy posts.
In addition, answer these questions:
What yeast are you planning to work with? Give species (and ideally a specific strain as well)
Are you trying to replicate someone else's work or doing something new?
[Edited on 6-12-2015 by phlogiston]
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solo
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I really have limited knowledge on the subject, but a willing to providing information that i can find to try to weave together some information for
willing members with better understanding to provide, not the feeding spoon ,but some input on the direction to go.....as for the starting yeast i
think, "Complete DNA sequence of yeast Chromosone XI" the S. cerviciea may be a good candidate
"Are you answering you're own posts ?".....no i just want to provide as much information as i can find to make it easier for those that want to assist
with some known reference material.....I sometime get carried away and in doing so i almost answer my own questions.....a definite flaw in my part....
"Geochrichum candidium is a "yeast" type fungus that will produce LA when grown on certain substrates".....this is very helpful, thank you....
.....solo
[Edited on 6-12-2015 by solo]
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solo
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Reference Information
Microbiological production of lysergic acid derivatives
US 3219545 A
\
Attachment: Microbiological production of lysergic acid derivatives-US3219545.pdf (434kB)
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Mesa
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Last time I checked, sequencing cost about 50c per base pair. Unless you find the relevant DNA sequence(s) on a free database it'll cost you at least
a few hundred $$$ there. Do you have a protocol for a cloning vector? E. Coli is almost always preferable over yeast here, I'd be surprised if the
required information has been written/published for yeast.
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Nickdul
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http://www.sciencedirect.com/science/article/pii/S0031942205...
The ergot alkaloid synthesis cluster is nearly 70kb!!! Even if some homologous proteins are available in the genetically modified host of your choice,
this is a daunting challenge without access to research level equipment, sequencing and primer synthesis service (and a couple man-years worth of
work).
Perhaps C. purpurea or analogous rye parasite can be cultivated and ergot alkaloids purified. A high throughput in situ screen for lysergic
derivatives can be applied.
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solo
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Reference Information
The ergot alkaloid gene cluster in Claviceps purpurea: Extension of the cluster sequence and intra species evolution
Thomas Haarmanna, Caroline Machadob,
Phytochemistry
Volume 66, Issue 11, June 2005, Pages 1312–1320
Abstract
The genomic region of Claviceps purpurea strain P1 containing the ergot alkaloid gene cluster [Tudzynski, P., Hölter, K., Correia, T., Arntz, C.,
Grammel, N., Keller, U., 1999. Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol. Gen. Genet. 261, 133–141] was explored by
chromosome walking, and additional genes probably involved in the ergot alkaloid biosynthesis have been identified. The putative cluster sequence
(extending over 68.5 kb) contains 4 different nonribosomal peptide synthetase (NRPS) genes and several putative oxidases. Northern analysis showed
that most of the genes were co-regulated (repressed by high phosphate), and identified probable flanking genes by lack of co-regulation. Comparison of
the cluster sequences of strain P1, an ergotamine producer, with that of strain ECC93, an ergocristine producer, showed high conservation of most of
the cluster genes, but significant variation in the NRPS modules, strongly suggesting that evolution of these chemical races of C. purpurea is
determined by evolution of NRPS module specificity.
Attachment: The ergot alkaloid gene cluster in Claviceps purpurea - Extension of the cluster sequence and intra species evolution.pd (389kB)
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confused
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i think your best bet would be to use a yeast artificial chromosome and do all the vector modification in E coli and selection.
Before cloning it into your (yeast? fungus?) strain of choice but nucleotide synthesis on that scale is not practical and could easily cost a few
thousand bucks.
The other way would be to get a strain of Claviceps purpurea that has already been sequenced and PCR to get the genes of interest, ligate them and
clone them into E coli for selection and into your host.
I haven't actually heard of fungus vectors, but you might want to take a look a agrobacteria for plant transformation. To be honest it would be a
daunting task, even with a well funded research lab
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Nickdul
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Check these out:
1. Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome.
Itaya M, Tsuge K, Koizumi M, Fujita K.
Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15971-6. Epub 2005 Oct 18.
PMID:16236728
2. Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis.
Nishizaki T, Tsuge K, Itaya M, Doi N, Yanagawa H.
Appl Environ Microbiol. 2007 Feb;73(4):1355-61. Epub 2006 Dec 28.
PMID:17194842
3. Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector.
Itaya M, Fujita K, Ikeuchi M, Koizumi M, Tsuge K.
J Biochem. 2003 Oct;134(4):513-9.
PMID:14607977
[Edited on 7-12-2015 by Nickdul]
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chemrox
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Quote: Originally posted by solo  | i have started collecting information for a friend that will help me produce the modified yeast with the ergotamine genes inserted.....Does anyone
know of the procedure so i may help him with the required literature....anyway here is an article that cought my eye......solo
Manipulating the Yeast Genome: Deletion, Mutation and Tagging by PCR
Jennifer M. Gardner* and Sue L. Jaspersen*§ |
whoa man this rocks the boat!
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phlogiston
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This will realistically consume thousands of bucks and is risky.
You are focusing on generating the expression vectors now, but this is a relatively simple aspect of the project. Techniques for DNA synthesis,
cloning, restriction, sequencing, etc are well established, and all the tools you need are commercially available. Expression vectors for many species
of yeast are available and often freely shared by researchers if you just ask kindly.
However, you will then face the problem that the proteins you are trying to introduce are likely not all expressed well, sort to the wrong subcellular
compartments or are inactive, for instance because they are incorrectly folded/aggregated, lack cofactors etc. These problems often occur when
introduce genes from one organism into another one, but they are often less severe if both species are evolutionary more closely related.
There are ways to counteract these things but they don’t work in all cases and may take a lot of time to resolve, especially if you need several
enzymes to work together.
Finally, when you resolve all of this, you will likely find that the yield is tiny or zero, because the normal metabolism of the cell diverts
intermediates into other pathways. Further genetic modifications and changing the composition of the growth medium can improve the yield, but this is
not at all easy. It requires a detailed understanding of the metabolism of the species you are working with, expensive equipment (LC-MS and/or GC-MS)
more genetic manipulation and a lot of trial-and-error.
Even for a well-funded research lab, this is a major undertaking. Recently, papers describing succsefull synthesis of precursors of opioids and
artemisine were published in Science and Nature respectively, i.e. high-impact journals. This indicates that these things are possible, but really at
limit of what is currently technically achievable.
https://www.sciencemag.org/content/349/6252/1095.abstract
http://www.nature.com/nature/journal/v496/n7446/full/nature1...
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solo
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"Are you trying to replicate someone else's work or doing something new?"...............phlogiston
Well, we all stand on top of others shoulders, but no i'm gathering the research material that will assist a friend, either to duplicate or advance
the current status of the project.......
Your last post was very informative as i'm sure my friend will be alerted to the difficulties faced ahead, this is not my field and don't pretended
to to know more than what i can read....and thank you those articles summaries are quite insightful i will post them later.....solo/java
Friend= a friend biochemist here in Mexico that currently has no time for the inquiries and research of literature I'm currently doing , for which he
will read and perhaps will assist me in this project.
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phlogiston
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True. What I really meant was whether someone else has achieved and described in scientific literature exactly (or even partially) what your friend is
trying to do. If so, that will enormously simplify things for you.
In principle, you would then only need to copy their method that has been proven to work. A great advantage is that you will have confidence that it
-can- work. The prospect of dumping large amounts of money and time into a project that may never yield any result is not very appealing (but this is
the reality of cutting edge scientific research and one of the reasons it is expensive). You will also save a huge amount of time and money because
they will have resolved many of the potential issues for you and optimised their methods to some extent.
In practice, however, duplicating even a detailed procedure is often frustratingly difficult.
However, even if you never get this to work, you will have gained an invaluable amount of knowledge.
-Especially- the moments when things don't work and you have to troubleshoot are great learning experiences.
So, don't let me talk you out of it.
[Edited on 8-12-2015 by phlogiston]
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chemrox
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| Quote: Originally posted by phlogiston | It is good practice to explain in your starting post what it is that you are trying to do.
Also, allow people some time to reply before flooding the thread with your own lengthy posts.
In addition, answer these questions:
What yeast are you planning to work with? Give species (and ideally a specific strain as well)
Are you trying to replicate someone else's work or doing something new?
[Edited on 6-12-2015 by phlogiston] |
I rather thought he made it perfectly clear. In addition, he posted excellent sources and articles. @Solo: thanks again sir.
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phlogiston
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Yes true actually. He does state what the ultimate goal is. What I meant is that his starting post left me with many questions that prevented me from
formulating a really helpful answer, my apologies.
And, indeed, he does show a lot of effort in finding sources and articles. Excellent, not many people do that these days. However, I am afraid they
demonstrate mostly that the project is till in a very early stage of planning. This is fine of course, one has to start somewhere, but I think we can
produce helpful answer much more quickly if we don't have to read through all the lengthy posts to find out what papers are in them.
[Edited on 8-12-2015 by phlogiston]
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solo
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Requested by phlogiston
Note
Recently, papers describing succsefull synthesis of precursors of opioids and artemisine were published in Science and Nature respectively, i.e.
high-impact journals. This indicates that these things are possible, but really at limit of what is currently technically achievable.
Yeast cell factories on the horizon
Jens Nielsen1
SCIENCE
1050 4 SEPTEMBER 2015 • VOL 349 ISSUE 6252
Modified yeast produce opiates from sugar
Robert F. Service
SCIENCE
14 AUGUST 2015 • VOL 349 ISSUE 6249 677
Complete biosynthesis of opioids in yeast
Stephanie Galanie, Kate Thodey
Sciencexpress
13 August 2015 / Page 1 / 10.1126/science.aac9373
Attachment: Yeast cell factories on the horizon.pdf (923kB)
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Attachment: Modified yeast produce opiates from sugar.pdf (145kB)
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Attachment: complete biosynthesis of opioids in yeast.pdf (973kB)
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[Edited on 9-12-2015 by solo]
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phlogiston
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Well, I just mentioned them to underscore the fact that these things are published in top journals because it is difficult to do. Perhaps the papers
may provide some general inspiration, but I suspect the specific challenges will be different in your case.
However, if you/your friend eventually succeed in this, you should write it up and publish it. A science or nature paper will look very good on your
resume. Even if you are only partially successful, it may well be publishable.
[Edited on 9-12-2015 by phlogiston]
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solo
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Reference Information
D-Lysergyl Peptide Synthetase from the Ergot Fungus Claviceps purpurea
Brigitte Riederer, Mehmet Han, and Ullrich Keller‡
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 271, No. 44, Issue of November 1, pp. 27524–27530, 1996
Abstract
The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid
linked by an amide bond. An enzyme activity capable of non- ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylala- nyl-L-proline lactam, the
non-cyclol precursor of the er- gopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of
radioactively labeled enzyme-substrate com- plexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains
for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthe- tase (LPS 2) copurifying with LPS
1. LPS 1 and LPS 2 contain 4 -phosphopantetheine and bind their sub- strates covalently by thioester linkage. Kinetic analysis of the synthesis
reaction revealed a Km of 1.4 M for both D-lysergic acid and its structural homolog dihydro- lysergic acid, which is one to two orders of magnitude
lower than the Km values for the other amino acids in- volved. The Km values for the amino acids reflect their relative concentrations in the cellular
pool of C. purpu- rea. This may indicate that in in vivo conditions D-lyser- gyl peptide formation is limited by the D-lysergic acid concentration in
the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino ac- ids
supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages
of cultivation and in different media, suggesting that it is produced constitutively.
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It's better to die on your feet, than live on your knees....Emiliano Zapata.
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