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Author: zhiyuanli

[Challenge Topic] MANAGING STRESS FROM ENGINEERED METABOLIC PATHWAYS

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IP  162.105.249.25

Posted at 2012-6-13 00:58:33 | All floors
Edited by bio.lin at 2012-6-13 18:25

Reply hyfang Add Thread


   
I have registered and logged in~ But maybe the papers have not been put on?
The attachments are some papers I found that are related to the aspects on yeast microarray, msn2 deletion and a wine yeast example.

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Posted at 2012-6-13 02:35:08 | All floors
Edited by bio.lin at 2012-6-13 02:36

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I think this is because yeast is favorable for molecular manipulations and cellular studies. Yeast is single-celled organism and so is simple and convenient as a model for eukaryote. Many powerful tools are available for its analysis, for example, the microarray can be employed to have a systematic investigation of gene expression in yeast. In addition, much is known about its genome sequence and genetic regulatory mechanisms, such as the transcription units and regulatory elements. And most metabolic pathways such as that in stress response are evolutionally conserved. So the study of yeast responses can also reveal a lot of information for plant or mammalian cells.

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Posted at 2012-6-13 16:03:47 | All floors
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To achieve the same goal we can transfer genes that encode particular biosynthetic pathways to a heterologous host to form hybrid metabolic networks. Such experiments have been used in some industrial examples to achieve more robust strains or promote productivity.

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Posted at 2012-6-13 17:22:32 | All floors
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To detect gene regulation between normal cell and stressful cell, genomic transcript abundance and global protein synthesis levels have been explored in global studies. For example DNA arrays are used to get whole genomic expression data in yeast cells’ responses to stress. Changes in protein synthesis in the yeast cells can also be measured by two-dimensional
electrophoresis in large scale.

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Posted at 2012-6-13 17:46:38 | All floors
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In my opinion cellular fitness decreased when yeast cannot achieve optimal internal conditions for growth and function. Metabolism engineering approaches usually put yeast strains to unsuitable pH, temperature, osmotic pressure and so forth. Overproduction of foreign proteins or accumulation of toxic intermediates in metabolic pathways may cause changes in enzyme activities and the intracellular environment. Sometimes even low density of unnatural proteins can activate stress responses and influence normal cell functions. All of these affect yeast cells’ viability and fermentation efficiency and result in decrease in cell fitness.
Btw, what are FBA, MOMA, ROOM?

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thx! FBA(flux balance analysis), MOMA(minimization of metabolic adjustment), ROOM(regulatory on off minimization) are static analysis methods for metabolic network.  Posted at 2012-6-20 21:02

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Posted at 2012-6-13 22:10:52 | All floors
I am interested in the idea of “common currency” for stress.
I was considering if we can set up particular reporters for different kinds of stress (temperature, osmotic pressure, nutrition etc). Msn2 /Msn4 were proposed to be “general stress” transcriptional factors in stress response to induce a set of genes. If we characterize the downstream targets that are involved only in condition-specific pathway, it will be convenient to identify and deal with the unwanted characteristics from that stress.

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Posted at 2012-6-21 20:45:22 | All floors
Finally, the exam is over. Maybe we guys should have a meeting.
潘兴杰

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Posted at 2012-6-23 12:52:40 | All floors
Hey Guys,
My name is Ignacio (I am from Argentina!) and I am one of the initators of this topic
Let's think about a very specific case: protein product secretion. Saccharomyces cerevisiae is not  a good protein secretion strain - if we overexpress a protein that misfolds the cells get stressed. On the other side Picchia pastoris is widely used in bioengineering to create all sorts of cool secreted proteins, including enzymes, antibodies and spider silk.
Now, a very interesting question is... how are stress responsive genes differentially regulated in Picchia pastoris when a protein is secreted? How are the genes differentially expressed compared to Saccharomyces cerevisiae?
How many of you have played with genomic microarray datasets?

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Posted at 2012-6-24 23:12:02 | All floors
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o~~~~~
ignacio, guess who am i?

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Posted at 2012-6-25 10:54:12 | All floors
I have no idea about Picchia pastoris and Saccharomyces cerevisiae.
Is these some different between their endoplasmic reticulum?

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Posted at 2012-6-26 00:07:09 | All floors
Well Picchia Pastoris is a yeast that can be used to secrete proteins - and yes somehow their endoplasmic reticulum (ER) works differently. You can make lots of things using Picchia, including spider silk:
http://www.ncbi.nlm.nih.gov/pubmed/9035408

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Posted at 2012-6-26 00:12:32 | All floors
I wanted all of us to read this paper:
Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533675/
and think about what makes the unfolded protein response (UPR), main pathway regulating the ER, different between Picchia pastoris and Saccharomyces cerevisiae.
The question is, what makes a yeast a better biotechnology yeast for production of for example proteins? Can one learn how to make ANY yeast a good protein secretor?

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Posted at 2012-6-26 00:18:43 | All floors
More interesting articles about UPR and comparisons between PP and SC:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144928/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116504/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905327/
http://www.ncbi.nlm.nih.gov/pubmed/17578563
Besides learning about the biology and exploring the transcriptional landscape of this pathway - can we build mathematical models? can we make an FBA model of protein flux in either SC or PP?

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Posted at 2012-6-26 08:06:02 | All floors
OK guys HERE IS THE HOMEWORK !!

0) In what sense is secretion a stress to a cell?
1) Read all papers! Be prepared to discuss your favorite figure from each paper.
2) Retrieve the microarray data from
http://www.ncbi.nlm.nih.gov/pubmed/10847680
and
http://www.ncbi.nlm.nih.gov/pubmed/17578563
http://www.biomedcentral.com/1471-2164/9/390
3) Pick the top 10 targets of UPR in Saccharomyces cerevisiae and contrast their expression changes to their Picchia pastoris ortologs. What do you see? How is PP different from SC? What other aspects of their biology and metabolism are different?
4) Would you rather turn PP into SC or SC into PP if you wanted a super secreter? What would be your strategy?
5) Find and read a tutorial about Flux Balance Analysis (FBA). How could we apply FBA to model the flux of proteins in different stages of processing through the secretory pathway?  Propose a simple FBA model and state how the constraints would be different between SC and PP?
Cheers!

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Posted at 2012-6-26 09:56:07 | All floors
Yes, enhancing the tolerance of yeast to UP is a method.
but can we find a way to go round the stress?

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Posted at 2012-6-28 07:21:48 | All floors
In this case unfolded proteins (UP) are the stress. This is an example in which nature itself has created a species that is naturally resistant to a flux of proteins that for other species would be considered stress. The question is: what is different about the structure of the gene regulation network between the two species besides higher putative pathway activity?

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Posted at 2012-6-30 00:46:09 | All floors
Hi everyone,

I just wanted to introduce myself: I'm Raj and I'll be joining you all in pku soon!  My background is in computational methods and i'm very interested in figuring out a good model for protein secretion.

If you have any questions or thoughts about Ignacio's homework questions feel free to post about it here.  in particular, questions 4 and 5 are really starting points for discussion as there's many possible ways to address them.

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Posted at 2012-7-21 16:48:22 | All floors
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    OK~
王紫薇

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Posted at 2012-7-20 17:46:33 | All floors
OK guys HERE IS THE HOMEWORK !!

0) In what sense is secretion a stress to a cell?
1) Read all  ...
izuleta replied at 2012-6-26 08:06



Sorry for the delayed reply! i had a hectic time.
HOMEWORK 1) & 2) have been done. the PKUers here have discussed 3 of the papers on July 15th and others to be discussed on July 22.
i'm working on 3) now. we are thinking about the questions you raised in HOMEWORK 0), 3), 4), 5) and we'll discuss on July 22nd.
tell us if you have new ideas or other homework for us to do~^0^
looking forward to you guys coming here!

王紫薇

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Posted at 2012-7-20 23:47:01 | All floors
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    When and where will the discussion be in July 22nd? Although I am only a bench warmer now, may I join you?
潘兴杰
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