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+ | <h2 id="section-1" style="padding-top: 100px; margin-top: -50px;">1. Core-part:the activity of rate limiting enzyme ceaS2 has been improved</h2> | ||
+ | <h4> | ||
+ | Acrylic acid is a byproduct of CEAS2 enzyme, the catalytic effect of wild type | ||
+ | ceaS2 enzyme is very weak. | ||
+ | <br/> | ||
+ | We used the AEMD platform to analyze the ceaS2 | ||
+ | enzyme and screened the 38 mutants in the range of 5 Å around the active site | ||
+ | to carry out molecular cloning of point mutation, and then tested the acrylic | ||
+ | acid yield by HPLC after whole cell catalysis. Because there are a large number | ||
+ | of mutants, we divided them into five batches to carry out the reaction, the | ||
+ | results are as follows: | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/8/8b/NPU-image01.png" style="max-width:60%;"><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/d/dc/NPU-image02.png" style="max-width:60%;"><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e6/NPU-image03.png" style="max-width:60%;"><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/a4/NPU-image04.png" style="max-width:60%;"><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/b2/NPU-image05.png" style="max-width:60%;"><br /><br> | ||
+ | In the figure, the horizontal axis stands for each different point mutation. We selected | ||
+ | two reaction times 21h and 42h, the vertical axis is acrylic acid production (mg / L).<br> | ||
+ | Due to the differences in wild type between different batches, we will normalize all | ||
+ | the data in order to facilitate the analysis of the catalytic effect of each mutation point | ||
+ | compared to the respective WT, that is, to compare each mutation point to The batch WT | ||
+ | yield multiple is a new indicator, the result is as follows: | ||
+ | <br /> | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e4/NPU-26.png" style="max-width:110%;"><br /> | ||
+ | The horizontal axis in the figure is the position of each mutational site, and the | ||
+ | vertical axis is the multiple of the acrylic acid yield of each mutational site compared | ||
+ | to each corresponding batch of the wild type. It can be seen that there were 11 | ||
+ | mutational sites, whose yields were higher than the wild type ceaS2, in the 38 mutant | ||
+ | programs, and the F438M mutant had the highest yield of 11 times the wild type. The | ||
+ | effect was significant. | ||
+ | <br /> | ||
+ | </h4> | ||
+ | <h2 id="section-2" style="padding-top: 100px; margin-top: -50px;">2.System:S. cerevisiae is more suitable for chassis cells than E. coli</h2> | ||
+ | <h4> | ||
+ | Acrylic acid has strong chemical reactivity and is very destructive to cell | ||
+ | membrane. Therefore, the chassis cells’ tolerance to acrylic acid is a "roof" factor | ||
+ | that restricts high yield of acrylic acid.<br> | ||
+ | We chose E. coli and S. cerevisiae, the two most convenient model chassis | ||
+ | organisms in prokaryotic and eukaryotic organisms. In order to investigatethe | ||
+ | chassis cells’ tolerance to acrylic acid, we set up a cytotoxicity test where the two | ||
+ | chassis cells grew in different concentrations of acrylic acid medium, and the | ||
+ | bacteria OD changes were monitored.The results are as follows: | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/a8/NPU-image07.png" style="max-width:60%;"><br /> | ||
+ | Fig1. OD of E.coli MG1655 under acrylic acid of different concentration and time | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/8/82/NPU-image08.png" style="max-width:60%;"><br /> | ||
+ | Fig2. OD of S. cerevisiaeBY4741 under acrylic acid of different concentration and time | ||
+ | <br /> | ||
+ | <br> | ||
+ | Two kinds of chassis cells have different tolerance to acrylic acid. Here we selected | ||
+ | 500mg / L and 1000mg / L two kinds of acrylic acid concentration to analyze:<br /> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/1/13/NPU-image09.png" style="max-width:60%;"><br /> | ||
+ | Fig3. A comparison of OD of BY4741 and MG1655 under 500mg/L acrylic acid | ||
+ | <br /> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/4/46/NPU-image10.png" style="max-width:60%;"><br /> | ||
+ | Fig4. A comparison of OD of BY4741 and MG1655 under 1000mg/L acrylic acid | ||
+ | <br /> | ||
+ | <br> | ||
+ | As can be seen from the results, when the concentration of acrylic acid reached | ||
+ | 500mg / L, E. coli bacterial growth was inhibited or even declined while S. | ||
+ | cerevisiae normally grew and entered a stable period. And when the concentration of | ||
+ | acrylic acid reached 1000 mg / L, the growth of S. cerevisiae was then inhibited.<br><br> | ||
+ | Conclusion: S. cerevisiae has a better tolerance to acrylic acid toxicity than E. | ||
+ | coli, and may be more suitable for use as chassis cells, and our results of the | ||
+ | pathway further confirm this conclusion. | ||
+ | <br /> | ||
+ | </h4> | ||
+ | <h2 id="section-3" style="padding-top: 100px; margin-top: -50px;">3.Pathway:Successfully build a new acrylic acid synthesis pathway and increase acrylic acid production</h2> | ||
+ | <h4> | ||
+ | In order to increase the ability of the chassis cells convert ing glycerol to DHAP or | ||
+ | 4G3P, we designed a new GlyDH-DAK glycerol metabolic pathway. To maintain | ||
+ | the supply of the reducing power of GlyDH enzymes, the NOX-CAT reducing | ||
+ | power module was also introduced, which eventually forms the acrylic synthesis | ||
+ | pathway — GDNCC Pathways. | ||
+ | <br> | ||
+ | First, we introduced new pathways into two chassis cells through two or three | ||
+ | plasmid vectors. | ||
+ | <br> | ||
+ | <br>pET-28a-ceaS2; pCDFDuet-gld-DAK; pETDuet-NOX-CAT; YCplac33-LEU-ceaS2; YCplac33-LEU-ceaS2-NOX; YCplac33-URA-gld-DAK | ||
+ | <br> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c7/NPU-image11.png" style="max-width:60%;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/0/00/NPU-image12.png" style="max-width:60%;"> | ||
+ | <br> | ||
+ | Fig5 1:E.gld+DAK;2:S-ceaS2;3,E.NOX-CAT;4.S.NOX-ceaS2;5:DAK;6:NOX;7,ceaS2; | ||
+ | 8:gld;9:s.gld-DAK;10:CAT | ||
+ | <br><br> | ||
+ | We also used the whole cell catalytic reaction and HPLC determination method to determine the amount of acrylic acid produced. | ||
+ | For E. coli, yields of using new and old synthetic pathways of acrylic acid are as follows: | ||
+ | <br> | ||
+ | Conditions: reaction time 42h, PH8.0, glycerol concentration 1% | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/b5/NPU-image13.png" style="max-width:60%;"><br /> | ||
+ | It can be seen that the acrylic acid yield is increased by 3 times after the introduction | ||
+ | of the GlyDH enzyme and the DAK enzyme compared to the introduction of only | ||
+ | the ceaS2 enzyme in old pathway. And the acrylic acid yield is increased by 8 | ||
+ | times compared to the old one after the addition of the reducing power module. The | ||
+ | new pathway does enhance the ability of E. colisynthesizing acrylic acid. | ||
+ | <br> | ||
+ | <br> | ||
+ | As for S. cerevisiae, since S. cerevisiae itself has a higher activity of hydrogen | ||
+ | peroxide reductase, the reducing power module onlyhas NOX enzyme. Theacrylic | ||
+ | acid yields ofapplying new and old synthetic pathways are as follows:<br> | ||
+ | Conditions: reaction time 72h, PH8.0, glycerol concentration 2% | ||
+ | Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the | ||
+ | indicator. | ||
+ | </br> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/5/5e/NPU-image14.png" style="max-width:60%;"><br /> | ||
+ | <br> | ||
+ | It can be seen that, similar to the results of E. coli, the introduction of new | ||
+ | pathways does improve the ability of S. cerevisiae synthesizing acrylic acid. <br> | ||
+ | Compared the old pathway introduced only ceaS2 enzyme, acrylic acid | ||
+ | production was increased by 3 times after introduction of GlyDH enzymes and | ||
+ | DAK enzymes. And the yield of acrylic acid was increased by 5 times compared | ||
+ | to the old pathway after the addition of the reducing power module.<br> | ||
+ | We also used CRISPR-CAS9 to optimize the bypass metabolic pathway of the S. | ||
+ | cerevisiae. | ||
+ | </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/b0/%E9%85%B5%E6%AF%8D%E8%B7%AF%E5%BE%84%E5%9B%BE.png" style="max-width:60%;"><br /> | ||
+ | Colonial verification results show that we have successfully knocked out the S. | ||
+ | cerevisiae's DLD genes: | ||
+ | </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e6/NPU-image16.png" style="max-width:60%;"><br /> | ||
+ | Fig 6 S.C BY4741DLD1gene Agarose gel figure of colonies verification after CRISPR | ||
+ | knockout.<br> | ||
+ | <br> | ||
+ | WT is the corresponding nucleic acid stripe of wild-type S.C BY4741; M is a | ||
+ | GeneRuler 1 kb DNA ladder; lanes 1, 2, 3 are three selected nucleic acid stripes of | ||
+ | monoclonal colonies.<br> | ||
+ | We also tested the acrylic acid synthesis ability of the transformed strain. The results | ||
+ | are as follows: | ||
+ | Conditions: reaction time 72h, PH8.0, glycerol concentration 2% | ||
+ | Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the | ||
+ | indicator.<br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/2e/NPU-image17.png" style="max-width:60%;"><br /> | ||
+ | It can be seen that the optimization of bypass metabolic flux is conducive to the | ||
+ | concentration of metabolic flux and improving the yield of acrylic acid. Of coursewe | ||
+ | also found in the process of the experiment that after knocking out the 9 genes, S. | ||
+ | cerevisiae colony growth became very slow, indicating that a more tender method | ||
+ | should be adopted, such as RNAi, to inhibit the bypass pathway.<br /> | ||
+ | </a> | ||
+ | <br /> | ||
+ | </a> | ||
+ | <h4> </h4> | ||
+ | <h4> </h4> | ||
+ | <h2 id="section-4" style="padding-top: 100px; margin-top: -50px;">4.Product:Multi - Conditional Optimization of Acrylic Cell Factory Catalytic Reaction Process</h2> | ||
+ | <h4> | ||
+ | There are several important conditions for whole cell reaction: enzyme induction | ||
+ | temperature, carbon source, Buffer, PH, reaction time. We set different control | ||
+ | experiments with E.coli BL21 (DE3) as the chassis cells. The results are as follows: | ||
+ | 4.1 The effects of different induction temperatures on the amount of acrylic acid were | ||
+ | investigated. The results are as follows: | ||
+ | Induction time: 14h | ||
+ | </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c3/NPU-image18.png" style="max-width:60%;"><br /> | ||
+ | <h4> | ||
+ | It can be seen that when the induction temperature was 30 ℃, the enzyme expression and activity were the highest, and the yield of acrylic acid was the best.</h4> | ||
+ | </br> | ||
+ | <h4> | ||
+ | 4.2 the results of production of acrylic acid with different carbon sources | ||
+ | Condition: PH7.4 | ||
+ | Reaction time: 16h | ||
+ | Glucose concentration: 4g/L | ||
+ | Glycerol concentration: 1%<br /></h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/8/80/NPU-image19.png" style="max-width:60%;"><br /> | ||
+ | <h4> | ||
+ | It can be seen that the yield of acrylic acid was higher when the glycerol was used | ||
+ | as the carbon source, because the carbon flow rate of the glycerol metabolic | ||
+ | pathway was more concentrated, thus turning more carbon source into acrylic | ||
+ | acid. Plus, the glycerol itself owning a higher reducing powermay also be one of | ||
+ | the reasons. | ||
+ | 4.3 The effects of different pH on the amount of acrylic acid were investigated. | ||
+ | The results are as follows: | ||
+ | Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol </h4><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/d/d7/NPU-image20.png" style="max-width:60%;"><br /> | ||
+ | <h4> | ||
+ | It can be drawn that PH8.0 was most suitable for acrylic acid production; the | ||
+ | reason may be that alkaline environment made E.coli more resistant to acrylic | ||
+ | acid. | ||
+ | 4.4 The effect of different Buffer on the amount of acrylic acid were investigated. | ||
+ | The results are as follows:</h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/8/86/NPU-image21.png" style="max-width:60%;"><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/ef/NPU-image22.png" style="max-width:60%;"><br /> <h4> | ||
+ | It can be seen that the DHa or G3P activity of the two substrates of ceaS2 enzyme was | ||
+ | higher under PBS buffer condition. | ||
+ | 4.5 The effects of different reaction time on the amount of acrylic acid were investigated. The results are shown as follows </h4><br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c2/NPU-image23.png" style="max-width:60%;"><br /> <h4> | ||
+ | It can be drawn that the yield of acrylic acid reached a higher level after the whole | ||
+ | cell catalytic reaction endured for 16h. The sampling point should be set after 16h. | ||
+ | <br /> | ||
+ | <br /> | ||
+ | </h4> | ||
+ | <h2 id="section-5" style="padding-top: 100px; margin-top: -50px;">5.Conclusion </h2> <h4> | ||
+ | Due to the time limit of the experiment, we did not have enough time to replace the | ||
+ | optimal mutation site into the existing cell factory. At present, the highest yield of | ||
+ | acrylic acid that we have acquired is 211.655 mg / L, which is 200 times than that of | ||
+ | GAACF1.0. </h4> | ||
+ | <br /> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/a1/NPU-25.png" style="max-width:60%;"><br /> | ||
+ | <h4> | ||
+ | Fig7. Yeast strain: BY4741-ceaS2-gld-DAK; Condition of whole cell catalysis: PH: | ||
+ | 7.4; Concentration of the substrate glycerol: 2%. | ||
+ | <br> | ||
+ | <br> | ||
+ | The chromatogam of the sample by | ||
+ | HPLC shows the yield is up to 211.655 mg / L according to the standard curve. | ||
+ | 211.655mg/L , currently this is the highest yield of acrylic acid biosynthesis, where | ||
+ | glycerol serves as the carbon source. | ||
+ | As an undergraduate team, in just a few months, we have tried our best to create an | ||
+ | efficient acrylic cell factory. We were surprised by the huge increase in GAACF 2.0 | ||
+ | production, which is only the production of wild-type ceaS2. Because it is a | ||
+ | continuing project, we are planning to screen for more active mutants on the basis of | ||
+ | several productive mutations using HTS for point saturation mutations and | ||
+ | high-throughput screening. And then, we will transform them into the existing chassis | ||
+ | organism. We believe that we will create a new technology for acrylic acid production | ||
+ | which has more industrialization prospect!<br /> | ||
+ | <br /> | ||
+ | |||
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Latest revision as of 02:22, 2 November 2017