Difference between revisions of "Team:Tartu TUIT/Design"

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                 <p>In the beginning, we intended to insert the four genes as a cassette to reduce the number of сloning and not to use an excessive amount of markers.We have chosen five different promoters to control the flux and try to maximise the yield of the final products.</p>
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                <h2>Optimization of the yield of the final products by genetic engineering</h2>
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                 <p>We were planning to run the parallel assembly using Vegas, Gibson and CPEC methods. We had some trouble with the PCR: we did not manage to adjust the annealing temperature of the primers.</p>
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                 <p>In the beginning, we intended to insert the four genes as a cassette to reduce the number of cloning steps and to reduce the number of markers used for transformation. We have chosen five different promoters of different strength to use them in various combinations with the genes to maximize the yield of the final products by controlling the flux of intermediates through the pathway.</p>
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                <p>Another reason why we had to reject the initial plan was the inability to predict the activity of a particular gene and the rate of each reaction in the pathway. This restricted us in designing the experiments and forecasting the results.It also complicated the process of modelling.</p>
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                 <p>Initially, it was planned to assemble the genetic parts using Vegas <a href="ref-1">[1]</a>, Gibson <a href="ref-2">[2]</a> and CPEC <a href="ref-3">[3]</a> methods in parallel. However, we have faced some problems making large assemblies. Finally, we have switched to conventional genetic engineering techniques. It was decided to express genes under various promoters from centromeric plasmids carrying different selection markers. Non-identical selection would allow us to insert several genes simultaneously using a double, triple and quadruple selection plates. We would like to create yeast strains with different number and combinations of promoter-gene cassettes. Based on the yield of shinorine and porphyra-334, the optimal combination will be chosen. This work is currently underway.</p>
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                <p>To be able to track each step of the pathway we have decided to insert genes with alternating promoters one by one via centromeric plasmids with different selection markers. Non-identical selection will allow us to insert several genes simultaneously using double, triple and quadruple selection plates.</p>
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                 <h2>The increase of sedoheptulose-7-phosphate (S7P) concentration</h2>
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                <p style="text-align: center">Used plasmids:</p>
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                 <ol class="cols fancy">
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                    <li class="col half">Plasmid 1</li>
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                    <li class="col half">Plasmid 2</li>
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                    <li class="col half">Plasmid</li>
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                    <li class="col half">Plasmid</li>
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                </ol>
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                 <p>Based on our model we are planning to obtain 136 strains as a result of different combinations.</p>
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                 <p>One of the precursors for shinorine and porphyra-334 biosynthesis is S7P. S7P is one of the intermediates of the pentose-phosphate pathway (PPP). It was reported that the concentration of this substance in yeast cells is very low <a href="ref-4">[4]</a>. Therefore, the increase in the level of S7P in the cells potentially may lead to a rise in the shinorine and porphyra-334 yield.</p>
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                <p>We are also planning to run a parallel experiment, following our second model <strong>(link)</strong> and perform a series of gene knockouts to increase the concentration of the Sedoheptulose-7-phosphate - the precursor of our pathway.</p>
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                 <p>The change of carbon source from glucose to xylulose increases the inner cell concentration of S7P <a href="ref-4">[4]</a>. However, due to the fact we plan to scale-up shinorine and porphyra-334 production in the bioreactor, it is inconvenient to switch from glucose to more expensive sugar.</p>
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                <p>We have created primers to switch off TKL1, TAL1, NQM1, PHO13, PGI1.</p>
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                 <p>To increase the S7P concentration in the cells, we decided to delete several genes (TKL1, TAL1, NQM1, PHO13, PGI1) like it was reported in the papers <a href="ref-5">[5]</a><a href="ref-6">[6]</a>.</p>
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                <p>Since it might be complicated to delete several genes at once, we have decided to first delete only TKL1 and TAL1. The deletion of these 2 genes has been proven to have some effect on the S7P concentration. This might help us to estimate the validity of our assumptions and determine whether it is reasonable to continue the knockouts further.</p>
+
                 <p>Eventually, we expect the rise of S7P concentration will enhance the flux through the enzymes of shinorine and porphyra-334 biosynthesis and will lead to the optimal target products’ yield.</p>
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                <p>We are hoping to obtain 2 strains with the increased S7P concentration, that will prove our hypothesis and allow us to continue knock-outs to optimize S7P concentration.</p>
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                 <p> After the creation of the strains we can proceed with measurements.</p>
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                <p>First of all, we will have to estimate the concentration of the precursor using HPLC in the wild-type and in 2 knockout strains.</p>
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                <p> In addition, we will have to approximate the activity of the enzymes in 4 test strains (all genes with pTDH3 promoter) with the help of HPLC.</p>
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                <p> Using the obtained results, we can choose the promoter combinations that are worth checking. Then we perform the same measurements with them and select a few relevant ones to continue the experiments with.</p>
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                 <p>To maximize the yield of the final products we will have to optimize the growth conditions.
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                Initially, we wanted to put our stains under the UV light to prove that they produce UV-absorbing compounds. We also hope that it might help to induce the production of the MAAs.</p>
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                <p>However, S. Churio proposed to also use the visible light.
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                <p>A lot of specialists also noted that it is reasonable to try out different substrates to find an optimal composition. For example, R.Sommaruga and S.Churio have proposed to use nitrogen enriched media.One of the crucial qualities for the sunscreen compounds is photostability. Despite most science groups working on MAAs find them extremely photostable, we think it is necessary to test photostability of these compounds also in yeast extract.</p>
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                 <p>Dr K.Lawrence has provided us with some guidelines on how to check for this property.
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                We expect our idea to have some real applications in the future.In our opinion, our idea of combining MAAs with yeast extract can be fitting for the industry.
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                After consulting with the scientists working with MAA, we have added some details to our long-term goals.
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                The most popular advice was to incorporate more diverse MAAs with distinct absorption maxima to increase the effectiveness of the sunscreen.
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                Unfortunately, the genes for a lot of the MAAs are still not identified.
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                However, there are sources propose possible pathways for palythine and gaudasol.
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                There is a way to produce gaudasol in yeast. We can repeat and modify this method and make a mix out of yeast extracts from 2 strains.
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                Other pieces of advice were about the composition of the future sunscreen.
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                For example, Dr R Garcesa proposed adding ascorbic acid to increase photostability.</p>
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                <h2>Serviceability</h2>
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                <p>Nowadays the demand for accessible, eco-friendly sunscreen became sensible.
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                For example, Hawaii became the first US state that tries to ban certain types of sunscreens.
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                Therefore, the creation of this sunscreen is justified.
+
  
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                Mycosporine-like amino acids are proven to efficiently absorb UV A and B. 
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                 <h2>Optimization of the yield of the final products by cultivation conditions</h2>
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                A number of human studies were also conducted that have shown the superiority of these compounds over the synthetic filters, as it MAAs not only serve as UV absorbents, but also improve skin firmness (anti-wrinkle effect), smoothness and inhibit liquid peroxidation.<a href="https://mibellebiochemistry.com/app/uploads/2015/03/Helioguard-365_Mycosporine-like-amino-acids-from-red-algae-protect-against-premature-skin-aging-EuroCosmetics-09-06.pdf">  https://mibellebiochemistry.com/app/uploads/2015/03/Helioguard-365_Mycosporine-like-amino-acids-from-red-algae-protect-against-premature-skin-aging-EuroCosmetics-09-06.pdf</a>
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                They also have a potential in wound healing, which can be beneficial as the product will not only be able to prevent sunburns but also heal them<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666432/#B8-marinedrugs-15-00326"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666432/#B8-marinedrugs-15-00326</a>). Mycosporine-like amino acids are also considered harmless for human health.<a href="https://www.ewg.org/skindeep/ingredient/705167/PORPHYRA_UMBILICALIS_(RED_ALGAE)_EXTRACT/#.W4zwKJMzb-Y)"> https://www.ewg.org/skindeep/ingredient/705167/PORPHYRA_UMBILICALIS_(RED_ALGAE)_EXTRACT/#.W4zwKJMzb-Y)</a> We also could not find any information about MAAs being allergens.</p>
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                 <h2> Flexibility </h2>
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                <p>Our goal was to make our system adjustable.
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                It affected our choice of gene sequences.
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                A wide variety of organisms produce Mycosporine-like amino acids.
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                Throughout the cyanobacterial phyla, a lot of species synthesize shinorine from the sedoheptulose-7 phosphate.
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                However, there exist 2 evolutionary different paralogous enzymes for the last reaction leading to the final products.
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                <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3194895/"> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3194895/</a>
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                One of them is seryl transferase, and the other belongs to the ATP-grasp family. The latter allows the production of several MAA products, including shinorine and porphyra-334, and we have decided to work with it.
+
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                This combination might be advantageous, since both compounds are useful in the sunscreen composition.
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                The products and their yield is thought to be easily adjusted by the composition of the media.
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                </p>
+
  
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                 <h2>Simplicity</h2>
+
                 <p> Except for genetic engineering, the yield of target MAAs can be increased by optimization of the growth conditions. In order to get an insight into how variations of different parameters may affect the product yield, we have contacted several researchers working in the field.For example, Prof. S. Churio proposed us to use not only UV but also visible light to favor MAAs biosynthesis. </p>
−
                 <p>The KISS principle is very popular with engineers from different backgrounds. It is important to make our system understandable and straightforward while avoiding the loss in functionality.
+
                 <p>Dr. K. P. Lawrence suggested us in the interview to apply some other types of environmental stress to induce shinorine and porphyra-334, such as osmotic stress, thermal stress or enhanced salinity. Also, Prof. R. Sommaruga and Prof. S. Churio proposed to use nitrogen-enriched media.</p>
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                For our team, that mostly consists of first-year bachelor students, it was especially important to account for the lack of the experience and try to design the project in the fashion that together with being applicable and of current interest, it could still be completed following basic protocols without using complicated methods.
+
  
−
                This approach has also some real-life applications. Eco-friendly cosmetics is a big trend in the industry right now, but a lot of this type of products belongs to small, young brands with limited laboratory capabilities and budgets. That is why simplicity and accessibility are so important in our case.
+
                 <h2>MAAs-enriched yeast extract</h2>
−
                </p>
+
                 <p> In nature, shinorine and porphyra-334 are MAAs, which are synthesized by auto- and heterotrophic organisms and protects them from UV radiation <a href="ref-7">[7]</a> <a href="ref-8">[8]</a> <a href="ref-9">[9]</a> <a href="ref-10">[10]</a> and some other types of the stress <a href="ref-11">[11]</a> <a href="ref-12">[12]</a>. However, a limited number of organisms have industrial importance.</p>
−
                 <h2>Robustness</h2>
+
                 <p>Our approach of obtaining shinorine- and porphyra-334-enriched yeast extract would allow us to combine valuable properties of both yeast extracts and MAAs. In this case, there is no need in a purification of the final products, since it can be time- and cost-consuming or even impossible. Also, short life cycle, heterotrophic growth, and well-developed manipulation techniques make yeast extremely attractive as potential producers of MAAs.</p>
−
                 <p> It is important that the created system is able to work properly without frequent additional adjustments. This goal is harder to achieve with prokaryotic organisms, since they are prone to lateral gene transfer and aquire mutations a lot faster.
+
                <p>As a future goal, in order to increase the effectiveness of the sunscreen, the possibility of incorporation of more diverse MAAs with distinct spectral characteristics can be considered. It was also recommended by the researchers in the field (Dr. K. P. Lawrence, Prof. R. Sommaruga, Dr. R. Garcesa). Although, this will require a thorough investigation of the information available since the genes of the enzymes for the biosynthesis of many MAAs are not yet identified.</p>
−
                When integrating foreign genes to prokaryotes, in our case S. cerevisiae, DNA is inserted into the chromosomal genome. Acquired properties are therefore stable for many generations. Moreover, yeast are less susceptible to the changes in the environment and stress factors. <a href="https://www.genwaybio.com/technologies/protein-expression/yeast-expression"> https://www.genwaybio.com/technologies/protein-expression/yeast-expression</a>
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                </p>
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                <h2> 6.Innovativeness</h2>
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                 <p>The novelty of our project is the use MAAs enriched yeast extract.
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                This allows to skip the purification step and therefore get the maximum possible yield.
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                The yeast extract itself is a popular cosmetics ingredient, that is used as a moisturiser and to nourish the skin. It is already used in the after sun care products. <a href="http://www.naturalislife.com/nio-active.html">http://www.naturalislife.com/nio-active.html http://www.naturalislife.com/nio-active.html</a>
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                <div class="hr"></div>
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                <h3>References:</h3>
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+
<ol class="references">
 +
                <li><a name="ref-1"></a>Mitchell, L. A., Chuang, J., Agmon, N., Khunsriraksakul, C., Phillips, N. A., Cai, Y., ... & Blomquist, P. (2015). Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic acids research, 43(13), 6620-6630.</li>
 +
                <li><a name="ref-2"></a>Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison III, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6(5), 343.</li>
 +
                <li><a name="ref-3"></a>Quan, J., & Tian, J. (2011). Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nature protocols, 6(2), 242.</li>
 +
                <li><a name="ref-4"></a>Senac, T., & Hahn-Hägerdal, B. (1990). Intermediary metabolite concentrations in xylulose-and glucose-fermenting Saccharomyces cerevisiae cells. Applied and environmental microbiology, 56(1), 120-126.</li>
 +
                <li><a name="ref-5"></a>Schaaff, I., Hohmann, S., & Zimmermann, F. K. (1990). Molecular analysis of the structural gene for yeast transaldolase. European journal of biochemistry, 188(3), 597-603.</li>
 +
                <li><a name="ref-6"></a>Clasquin, M. F., Melamud, E., Singer, A., Gooding, J. R., Xu, X., Dong, A., ... & Rabinowitz, J. D. (2011). Riboneogenesis in yeast. Cell, 145(6), 969-980.</li>
 +
                <li><a name="ref-7"></a>Miyamoto, K. T., Komatsu, M., & Ikeda, H. (2014). Discovery of gene cluster for mycosporine-like amino acid biosynthesis from Actinomycetales microorganisms and production of a novel mycosporine-like amino acid by heterologous expression. Applied and environmental microbiology, AEM-00727.</li>
 +
                <li><a name="ref-8"></a>Sinha, R. P., Singh, S. P., & Häder, D. P. (2007). Database on mycosporines and mycosporine-like amino acids (MAAs) in fungi, cyanobacteria, macroalgae, phytoplankton and animals. Journal of Photochemistry and Photobiology B: Biology, 89(1), 29-35.</li>
 +
                <li><a name="ref-9"></a>Conde, F. R., Churio, M. S., & Previtali, C. M. (2000). The photoprotector mechanism of mycosporine-like amino acids. Excited-state properties and photostability of porphyra-334 in aqueous solution. Journal of Photochemistry and Photobiology B: Biology, 56(2-3), 139-144.</li>
 +
                <li><a name="ref-10"></a>Singh, S. P., Kumari, S., Rastogi, R. P., Singh, K. L., & Sinha, R. P. (2008). Mycosporine-like amino acids (MAAs): chemical structure, biosynthesis and significance as UV-absorbing/screening compounds.</li>
 +
                <li><a name="ref-11"></a>Lawrence, K. P., Long, P. F., & Young, A. R. (2017). Mycosporine-like amino acids for skin photoprotection. Curr Med Chem, 24, 1-16.</li>
 +
                <li><a name="ref-12"></a>Oren, A., & Gunde-Cimerman, N. (2007). Mycosporines and mycosporine-like amino acids: UV protectants or multipurpose secondary metabolites?. FEMS microbiology letters, 269(1), 1-10.</li>
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Revision as of 21:50, 17 October 2018

Optimization of the yield of the final products by genetic engineering

In the beginning, we intended to insert the four genes as a cassette to reduce the number of cloning steps and to reduce the number of markers used for transformation. We have chosen five different promoters of different strength to use them in various combinations with the genes to maximize the yield of the final products by controlling the flux of intermediates through the pathway.

Initially, it was planned to assemble the genetic parts using Vegas [1], Gibson [2] and CPEC [3] methods in parallel. However, we have faced some problems making large assemblies. Finally, we have switched to conventional genetic engineering techniques. It was decided to express genes under various promoters from centromeric plasmids carrying different selection markers. Non-identical selection would allow us to insert several genes simultaneously using a double, triple and quadruple selection plates. We would like to create yeast strains with different number and combinations of promoter-gene cassettes. Based on the yield of shinorine and porphyra-334, the optimal combination will be chosen. This work is currently underway.

The increase of sedoheptulose-7-phosphate (S7P) concentration

One of the precursors for shinorine and porphyra-334 biosynthesis is S7P. S7P is one of the intermediates of the pentose-phosphate pathway (PPP). It was reported that the concentration of this substance in yeast cells is very low [4]. Therefore, the increase in the level of S7P in the cells potentially may lead to a rise in the shinorine and porphyra-334 yield.

The change of carbon source from glucose to xylulose increases the inner cell concentration of S7P [4]. However, due to the fact we plan to scale-up shinorine and porphyra-334 production in the bioreactor, it is inconvenient to switch from glucose to more expensive sugar.

To increase the S7P concentration in the cells, we decided to delete several genes (TKL1, TAL1, NQM1, PHO13, PGI1) like it was reported in the papers [5][6].

Eventually, we expect the rise of S7P concentration will enhance the flux through the enzymes of shinorine and porphyra-334 biosynthesis and will lead to the optimal target products’ yield.

Optimization of the yield of the final products by cultivation conditions

Except for genetic engineering, the yield of target MAAs can be increased by optimization of the growth conditions. In order to get an insight into how variations of different parameters may affect the product yield, we have contacted several researchers working in the field.For example, Prof. S. Churio proposed us to use not only UV but also visible light to favor MAAs biosynthesis.

Dr. K. P. Lawrence suggested us in the interview to apply some other types of environmental stress to induce shinorine and porphyra-334, such as osmotic stress, thermal stress or enhanced salinity. Also, Prof. R. Sommaruga and Prof. S. Churio proposed to use nitrogen-enriched media.

MAAs-enriched yeast extract

In nature, shinorine and porphyra-334 are MAAs, which are synthesized by auto- and heterotrophic organisms and protects them from UV radiation [7] [8] [9] [10] and some other types of the stress [11] [12]. However, a limited number of organisms have industrial importance.

Our approach of obtaining shinorine- and porphyra-334-enriched yeast extract would allow us to combine valuable properties of both yeast extracts and MAAs. In this case, there is no need in a purification of the final products, since it can be time- and cost-consuming or even impossible. Also, short life cycle, heterotrophic growth, and well-developed manipulation techniques make yeast extremely attractive as potential producers of MAAs.

As a future goal, in order to increase the effectiveness of the sunscreen, the possibility of incorporation of more diverse MAAs with distinct spectral characteristics can be considered. It was also recommended by the researchers in the field (Dr. K. P. Lawrence, Prof. R. Sommaruga, Dr. R. Garcesa). Although, this will require a thorough investigation of the information available since the genes of the enzymes for the biosynthesis of many MAAs are not yet identified.

References:

  1. Mitchell, L. A., Chuang, J., Agmon, N., Khunsriraksakul, C., Phillips, N. A., Cai, Y., ... & Blomquist, P. (2015). Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic acids research, 43(13), 6620-6630.
  2. Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison III, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature methods, 6(5), 343.
  3. Quan, J., & Tian, J. (2011). Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nature protocols, 6(2), 242.
  4. Senac, T., & Hahn-Hägerdal, B. (1990). Intermediary metabolite concentrations in xylulose-and glucose-fermenting Saccharomyces cerevisiae cells. Applied and environmental microbiology, 56(1), 120-126.
  5. Schaaff, I., Hohmann, S., & Zimmermann, F. K. (1990). Molecular analysis of the structural gene for yeast transaldolase. European journal of biochemistry, 188(3), 597-603.
  6. Clasquin, M. F., Melamud, E., Singer, A., Gooding, J. R., Xu, X., Dong, A., ... & Rabinowitz, J. D. (2011). Riboneogenesis in yeast. Cell, 145(6), 969-980.
  7. Miyamoto, K. T., Komatsu, M., & Ikeda, H. (2014). Discovery of gene cluster for mycosporine-like amino acid biosynthesis from Actinomycetales microorganisms and production of a novel mycosporine-like amino acid by heterologous expression. Applied and environmental microbiology, AEM-00727.
  8. Sinha, R. P., Singh, S. P., & Häder, D. P. (2007). Database on mycosporines and mycosporine-like amino acids (MAAs) in fungi, cyanobacteria, macroalgae, phytoplankton and animals. Journal of Photochemistry and Photobiology B: Biology, 89(1), 29-35.
  9. Conde, F. R., Churio, M. S., & Previtali, C. M. (2000). The photoprotector mechanism of mycosporine-like amino acids. Excited-state properties and photostability of porphyra-334 in aqueous solution. Journal of Photochemistry and Photobiology B: Biology, 56(2-3), 139-144.
  10. Singh, S. P., Kumari, S., Rastogi, R. P., Singh, K. L., & Sinha, R. P. (2008). Mycosporine-like amino acids (MAAs): chemical structure, biosynthesis and significance as UV-absorbing/screening compounds.
  11. Lawrence, K. P., Long, P. F., & Young, A. R. (2017). Mycosporine-like amino acids for skin photoprotection. Curr Med Chem, 24, 1-16.
  12. Oren, A., & Gunde-Cimerman, N. (2007). Mycosporines and mycosporine-like amino acids: UV protectants or multipurpose secondary metabolites?. FEMS microbiology letters, 269(1), 1-10.
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