Team:Cologne-Duesseldorf/jan

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Secretion

Downstream processing is not only time consuming but also cost and energy intensive. Therefore, we aim to simplify the purification of compounds produced in our artificial compartment. We used a concept based on the peroxicretion described by Sagt and colleagues <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257"> (Sagt et al, 2009) </a>.

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Introduction

Downstream processing is a very important part of industrial biological compound production. For most biotechnological produced compounds, it is the most expensive part of the production <a href=" https://www.ncbi.nlm.nih.gov/pubmed/11602307 "> (Keller et al, 2001) </a> . One step to decrease the costs is to secrete the products into the supernatant <a href=" https://www.ncbi.nlm.nih.gov/pubmed/23385853"> (Berlec et al, 2013) </a>. After secretion, it is possible to remove most cellular compounds from valuable products with one simple centrifugation step. Due to this, secretion is not only a great tool for a compartment toolbox but also has an economic value.
In regards to the whole project, this is an important part for making the compartment more applicable. Through it we go a step further by thinking about the extraction of products after production.
At the end of this sub project it should be possible to secrete every compound produced in the modified compartment to the supernatant. This is not trivial because peroxisomes, which are the basis of our compartment do not possess a known natural secretion mechanism.
We overcome this problem by using the "peroxicretion" concept of Sagt and colleagues <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257"> (Sagt et al, 2009) </a>. They used a v-SNARE (vesicle- synaptosome-associated-Soluble N-ethylmaleimide-sensitive-factor Attachment REceptorprotein) fused to a peroxisomal membrane-protein to secrete the content of peroxisomes. V-SNAREs interact with the t-SNARE (target synaptosome-associated-SNARE) at the cell membrane, which leads to an fusion of the vesicle with the membrane <a href=" https://www.nature.com/nrm/journal/v2/n2/full/nrm0201_098a.html"> (Chen et al, 2001) </a>.

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Secretion

For the peroxisome secretion in S. cerevisiae we designed fusion proteins of the v-SNARE <a href="http://parts.igem.org/Part:BBa_K2271060"> Snc1 </a> with different peroxisomal membrane anchors. We tested the constructs using a GUS Assay. The assays were performed using transformants of the strain BY4742.

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Experimental Design

We will adapt the system of Sagt and colleagues to secrete the content of our modified compartments <a href=" https://www.ncbi.nlm.nih.gov/pubmed/19457257"> (Sagt et al, 2009) </a>.
For the application of this system in S. cerevisiae we use a truncated version of the v-SNARE Snc1 to decorate our compartments(Figure 3.1) <a href=" http://www.jbc.org/content/272/26/16591.short"> (Gerst et al, 1997) </a>.
<figure> <img src="T--Cologne-Duesseldorf--Snc1_gerst.png"> <figcaption> Figure 3.1 A diagram of the general domain structure of Snc1. V is a variable domain which is not important for the binding to the t-SNARE. TM is the transmembrane domain. H1 and H2 are the a-helical segments, forming the SNAREpin with the t-SNARE <a href=" http://www.jbc.org/content/272/26/16591.short"> (Gerst et al, 1997) </a></figcaption> </figure> <p>To decorate the compartments with the SNARE we use a <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#MembraneIntegration"> peroxisomal transmembrane protein </a> . In our case we use the proteins Pex15 or PEX26, which were further investigated in another sub project, and fuse Snc1 to the N-terminus. We expressed these constructs of membrane anchor and Snc1 constitutively under control of the RPL18B promotor. In case of Pex15 we used a truncated version, lacking a large part of the N-terminus, only consisting of the transmembrane domain (315-383) (Figure 3.1). For PEX26 we use the truncated version published in Halbach et al. <a href=" http://jcs.biologists.org/content/119/12/2508"> (Halbach et al, 2006) </a>.

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       <img src="T--Cologne-Duesseldorf--Peroxicretion_concept_Pex26.png">



       <figcaption>Figure 3.2 Concept of secreting peroxisomal contents to the supernatant. For the secretion, the membrane anchor Pex15 or Pex26 is used. This anchor is used to decorate peroxisomes or our modified compartments with the v-SNARE Snc1. For the secretion Snc1 interacts with the t-SNAREs in the cell membrane. Induced from this interaction the vesicle and cell membrane fuse and the content of the compartment is secreted to the supernatant.</figcaption>


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We verified our secretion using Beta-glucuronidase (GUS) as a reporter protein. In 2012 Stock and colleagues described the GUS reporter assay for unconventional secretion <a href=" https://www.ncbi.nlm.nih.gov/pubmed/22446315 "> (Stock et al, 2012) </a> . With it, it is possible to determine whether a protein is secreted conventional and is N-glycosylated or secreted unconventional and not N-glycosylated. GUS is a bacterial protein with an N-glycosylation-site, which is active only if the protein is not N-glycosylated. The GUS-activity can be measured with different reagents in plate or liquid assays. Liquid assays can be applied qualitatively as well as quantitatively to measure differences in activity. If GUS is secreted by the conventional pathway the N-glycosylation leads to inactivation of the enzyme <a href=" https://www.ncbi.nlm.nih.gov/pubmed/22446315 "> (Stock et al, 2012) </a> (Fig 3.2).


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       <img src="T--Cologne-Duesseldorf--Gus_Erkl%C3%A4rung.png">



       <figcaption>Figure 3.3 The GUS Assay.  GUS secreted with an unconventional secreted protein like Cts1 from Ustilago maydis active in the supernatant. GUS secreted with a conventional Signal peptide (Sp) inactive in the supernatant. If GUS is in the cytoplasm there is also no activity (Lysis control) <a href=" https://www.ncbi.nlm.nih.gov/pubmed/23455565">
 
     (Feldbrügge  et al, 2013)

</a>. </figcaption>


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GUS will be imported to the peroxisome with the PTS1 sequence and measured quantitatively in the supernatant. We will use a coexpression of GUS-PTS1 and Snc1-Pex15 or Snc1-PEX26 to identify the secretion of the compounds. Furthermore, we will use GUS-PTS1 expressed in S. cerevisiae without Snc1 fused to a membrane anchor for a control. We will measure the active GUS in the supernatant with a liquid assay based on the turnover of 4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU)<a href=" http://cshprotocols.cshlp.org/content/2007/2/pdb.prot4688.abstract"> (Blázquez et al, 2007) </a>. Here we expect a higher activity of GUS in the supernatant of cultures with Snc1 decorated peroxisomes.
To increase the variability of our constructs we also designed vectors with and without a GS-Linker connecting the Snc1 with the Pex15. Additionally we tested our constructs in strains with a <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> deletion of Pex11 </a>. This deletion leads to formation of <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> larger peroxisomes</a> and may increase the efficiency of our secretion mechanism.


Secretion

<a href="https://static.igem.org/mediawiki/2017/1/14/T--Cologne-Duesseldorf--Secretionlabbook2.pdf">Labook − Team Secretion</a>

Secretion

Using microscopy we were able to show, that our membrane anchors localize in a typical peroxisomal pattern <a href=" http://jcs.biologists.org/content/119/12/2508"> (Halbach et al, 2006) </a>.
The results of the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Secretion"> GUS-assay </a> indicate, that the contents of the peroxisomes were successfully secreted into the supernatant. This is the first time it was shown that this system works in S.Cerevisiae, since to this point it has only been demonstrated in <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Design#Secretion"> Aspergillus niger </a> Even though our secretion is not as efficient as the unconventional secretion in other organisms <a href=" https://www.ncbi.nlm.nih.gov/pubmed/22446315">(Stock et al, 2012)</a>, the possibility to secrete the compounds of our artificial compartment is still a substantial success with exciting implications. One could, for example, not only secrete proteins but also compounds from metabolic pathways.
Here we have only shown our general proof of concept. The next steps would be to develop a more efficient system. An easy way to increase the yield of compounds in the supernatant would be to manipulate the <a href="https://2017.igem.org/Team:Cologne-Duesseldorf/Description#SizeAndNumber"> size and number</a> of the artificial compartments in the cell.
Due to the fact, that peroxisomes are linked to the endoplasmic reticulum (ER) in S. cerevisiae cells, the efficiency of our system could be inhibited. To overcome this problem one possible solution would be to delete INP1 in the background strain. Inp1 works as a molecular link between peroxisomal Pex3 and ER Pex3. Deletion of INP1 leads to mobile peroxisomes which should lead to more fusion events.
After the generation of an optimized background strain, the next goal would be stable genome integration of the secretion system. One of the problems of this integration would be the constitutive expression of the snare constructs. This would lead to a constant secretion and loss of the proteins in our compartment. For an optimal yield it would be better to use inducible promoters to control the secretion of the produced metabolites. Another approach could be to control the fusion of the compartment and the cell membrane by <a href="https://2017.igem.org/Team:Cologne-Duesseldorf"> optogenetics*needs to be change* </a>.