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− | + | <h2> | |
+ | GAACF2.0:Multi - level optimization of acrylic cell factory | ||
+ | </h2> | ||
+ | <h3> | ||
+ | On the basis of GAACF1.0, we built a new cell factory of acrylic acid through the four part: CO-PART, SYSTEM, PATHWAY, PRODUCTION! | ||
+ | And the yield has been improved. | ||
+ | </h3> | ||
+ | <br> | ||
+ | <h2> | ||
+ | 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. 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> 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>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. | ||
+ | </h4> | ||
+ | |||
+ | <h2> | ||
+ | 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> Fig1. OD of E.coli MG1655 under acrylic acid of different concentration and time | ||
+ | |||
+ | <br> Fig2. OD of S. cerevisiaeBY4741 under acrylic acid of different concentration and time | ||
+ | <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> Fig3. A comparison of OD of BY4741 and MG1655 under 500mg/L acrylic acid | ||
+ | |||
+ | <br> Fig4. A comparison of OD of BY4741 and MG1655 under 1000mg/L acrylic acid | ||
+ | <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> 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. | ||
+ | </h4> | ||
+ | <h2> | ||
+ | 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 G3P, 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> pET-28a-ceaS2 | ||
+ | <br> [胶图] | ||
+ | <br> pCDFDuet-gld-DAK | ||
+ | <br> [胶图] | ||
+ | <br> pETDuet-NOX-CAT | ||
+ | <br> [胶图] | ||
+ | <br> YCplac33-LEU-ceaS2 | ||
+ | <br> [胶图] | ||
+ | <br> YCplac33-LEU-ceaS2-NOX | ||
+ | <br>[胶图] | ||
+ | <br> YCplac33-URA-gld-DAK | ||
+ | <br> [胶图] | ||
+ | <br> We also used the whole cell catalytic reaction and HPLC determination method to determine the amount | ||
+ | of acrylic acid produced. | ||
+ | <br> 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> | ||
+ | <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>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% | ||
+ | <br>Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the indicator | ||
+ | <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. 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> 【路径图】 | ||
+ | <br> Colonial verification results show that we have successfully knocked out the S. cerevisiae's DLD | ||
+ | genes: | ||
+ | <br> | ||
+ | <br> Fig XX S.C BY4741DLD1gene Agarose gel figure of colonies verification after CRISPR knockout. | ||
+ | <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: | ||
+ | <br> Conditions: reaction time 72h, PH8.0, glycerol concentration 2% | ||
+ | <br> Normalized the results based on the acrylic acid yield of BY4741-ceas2 as the indicator | ||
+ | <br> | ||
+ | <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. | ||
+ | </h4> | ||
+ | <h2> | ||
+ | 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: | ||
+ | </h4> | ||
+ | <h3> | ||
+ | 4.1 The effects of different induction temperatures on the amount of acrylic acid were investigated. The results are as follows: | ||
+ | </h3> | ||
+ | <h4> | ||
+ | Induction time: 14h | ||
+ | <br> | ||
+ | |||
+ | <br>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> | ||
+ | <h3> | ||
+ | |||
+ | |||
+ | 4.2 the results of production of acrylic acid with different carbon sources | ||
+ | </h3> | ||
+ | <h4> | ||
+ | Condition: PH7.4 | ||
+ | <br> Reaction time: 16h | ||
+ | <br> Glucose concentration: 4g/L | ||
+ | <br>Glycerol concentration: 1% | ||
+ | <br> | ||
+ | <br> 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. | ||
+ | </h4> | ||
+ | <h3> | ||
+ | 4.3 The effects of different pH on the amount of acrylic acid were investigated. The results are as follows: | ||
+ | </h3> | ||
+ | <h4> | ||
+ | Reaction conditions: 12h reaction time, 1% concentration of substrate glycerol | ||
+ | <br> | ||
+ | <br> 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. | ||
+ | </h4> | ||
+ | <h3> | ||
+ | 4.4 The effect of different Buffer on the amount of acrylic acid were investigated.The results are as follows: | ||
+ | </h3> | ||
+ | |||
+ | <h4> | ||
+ | It can be seen that the DHa or G3P activity of the two substrates of ceaS2 enzyme was higher under PBS buffer condition. | ||
+ | </h4> | ||
+ | <h3> | ||
+ | 4.5 The effects of different reaction time on the amount of acrylic acid were investigated. The results are shown as follows | ||
+ | </h3> | ||
+ | <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. | ||
+ | </h4> | ||
+ | <h2> | ||
+ | 5. Conclusion | ||
+ | </h2> | ||
+ | <h4> | ||
+ | After the above four levels of independent transformation, we currently obtain the best options, shown as follows: | ||
+ | <br> | ||
+ | <br> 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, we have acquired the highest yield of acrylic acid | ||
+ | in the two cell factories, shown as follows: | ||
+ | <br> | ||
+ | <br> This is currently the highest yield of acrylic acid biosynthesis, where glycerol serves as the carbon | ||
+ | source. | ||
+ | </h4> | ||
+ | |||
</div> | </div> | ||
Revision as of 22:36, 31 October 2017