Difference between revisions of "Part:BBa K2242005"
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KMADH is an alcohol dehydrogenase from Kluyveromyces marxianus. It can catalyze the ethanol fermentation ongoing in the bacteria with a high efficiency and mild reaction condition, especially temperature.
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KmAdh is an alcohol dehydrogenase from Kluyveromyces marxianus. It can catalyze the ethanol fermentation ongoing in the bacteria with a high efficiency and mild reaction condition, especially temperature.
 
We use T7 promoter and Lac operator to control KMADH’s expression. Both of this two units are induced by IPTG. With this dual switch, we can reduce the leak of the gene expression as much as possible.
 
We use T7 promoter and Lac operator to control KMADH’s expression. Both of this two units are induced by IPTG. With this dual switch, we can reduce the leak of the gene expression as much as possible.
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In our project, Mtr CAB is the most important and fundamental proteins as it plays the role to transfer extracellular electrons into the cytoplasm through the membrane. To examine whether the Mtr protein complex has the function as we expected, we used our engineered strain pMC( strain co-expressed Mtr CAB and Ccm A-H) to construct a bio-cathode. We monitored the current of the bio-cathode to see whether there would be a higher current in the experiment group than the WT strain.
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Here, first we did an bacteria PCR to monitor the maintenance of the recombinant plasmids(pM28 contains the mtr CAB’s gene and the pTBC contains the ccm A-H’s gene). As we can see in figure 11, we could confirm that the strain is fine to use.  
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<span id="first" class="scrollspy label label-pink">1.Transformation and Expression</span>
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    https://static.igem.org/mediawiki/2017/9/94/USTC-result-Mtr-1.png
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                                                  <br>
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    Figure 11. Electrophoresis result of PCR of Mtr and Ccm
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                                                  <p class="indent_word">We transformed a plasmid PET22b containing KmAdh into <span class="italic">E.coli</span> successfully. We use KmAdh’s specific primers to do PCR to verify this achievement. </p>
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    So we started our bio-cathode assay to examine our theory. The protocol of the bio-cathode assay can be found in the notebook part in our wiki, look it up if you want to know more details!! In figure 12, we can easily confirm that the Mtr CAB protein complex was mature, as the pellets were red in pMC group, no matter the strain had been induced or not. When we did it the first time, there was no significant difference of the current of the bio-cathode between WT and our strain pMC(data not shown). We speculated that it was because we did NOT have the starvation step when we first did it, which is to cultivate the bacteria in a minimal salts medium for a certain time, like 4 to 6 hours. Because we did NOT have this starvation step, although we already used PBS to wash the bacteria 2 to 3 times, those nutritions still contained inside of the bacteria, providing another electron source when we were running the bio-cathode. So when the cathode was given a certain voltage, the bacteria still wouldn’t take up the electrons from the electrode.
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                                                  <img src="https://static.igem.org/mediawiki/2017/7/70/USTC-result-harvest-1.jpeg" width="20%" style="margin:0 40%;">
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    https://static.igem.org/mediawiki/2017/6/6f/USTC-result-Mtr-2.jpeg
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                                                  <p style="text-align:center!important">Figure 1. Electrophoresis result of PCR of KmAdh <br>(From left to right: wild type, KmAdh, positive control)</p>
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Figure 12. Bacteria sediments<br>(from left to right, WT, pMC not induced, pMC induced)
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                                                  <p class="indent_word">We can see that the experimental group and the positive control have the same band but WT does not. This shows the transformation is successful. </p>
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                                                  <p class="indent_word">Then we induced the expression of this enzyme. We use 200 mL LB to cultivate our bacteria in 37℃，250 rpm. When its OD<sub>600</sub> reached 0.5-0.8 we added 20μL 1M IPTG(final concentration=0.1mM) in it to induce KmAdh expression. </p>
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===Usage and Biology===
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                                                  <p style="text-align:center!important">Figure 2. SDS-PAGE for KmAdh <br>(From left to right: wild type, KmAdh, KmAdh+IPTG)</p>
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                                                  <p class="indent_word">From the SDS-PAGE result, it can be safely concluded that KmAdh was successfully expressed at a high level. </p>
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                                                  <p class="indent_word">Then we gathered the bacteria to purify the KmAdh for enzyme activity measurement</p>
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                                                  <img src="https://static.igem.org/mediawiki/2017/e/e6/USTC-result-harvest-3.jpeg" width="50%" style="margin:0 20%;">
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                                                  <p style="text-align:center!important">Figure 3. SDS-PAGE for KmAdh <br>(From left to right: wild type, KmAdh, KmAdh+IPTG, raw enzyme, flow throgh, 20mM elution, 300mM elution, Pure enzyme)</p>
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                                                  <p>From figure 3, you can see that we successfully purify the KmAdh from the bacteria lysate.
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                                                  <br><br>
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                                                  <span id="second" class="scrollspy label label-pink">2.Enzyme activity test</span>
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                                                  <p class="indent_word">NADH, as a necessary cofactor of KmAdh, has a significant absorption in 340nm. However, once it's been reduced to NAD<sup>+</sup>, it will have no absorption in 340 nm. So, along the process of the reduction reaction, the consuming of NADH will lead to a decrease of absorption in 340nm which allows us to test the activity of KmAdh by the spectrophotometer. </p>
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                                                  <img src="https://static.igem.org/mediawiki/2017/3/3a/USTC-result-harvest-6.jpeg" width="100%">
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                                                  <p style="text-align:center!important">Figure 4. Reaction mixture</p>
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                                                  <p class="indent_word">Here in figure 4 is how we performed the reaction. After adding every component in to the cuvette, we scan the 340nm UV absorption value over time. Because NADH is easy to be oxidized, we set a blank control to exclude this effect. The system is the same as the above system, with the same amount of PBS to replace KmAdh purified enzyme. </p>
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                                                  <img src="https://static.igem.org/mediawiki/2017/9/96/USTC-result-harvest-4.jpeg" width="100%">
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                                                  <p style="text-align:center!important">Figure 5. The change of OD<sub>340</sub> over time</p>
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                                                  <p class="indent_word">Here in figure 5, there is a rapid increase in the absorption value after adding enzyme, indicating that NADH is drastically consumed. This shows the purified enzyme function is normal and the KmAdh is successfully expressed in E. coli. </p>
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                                                  <br><br>
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                                                  <span id="third" class="scrollspy label label-pink">3.Toxicity test</span>
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                                                  <p class="indent_word">Considering that acetaldehyde and ethanol, the substrate and product of KmAdh, may do harm to the cell, we first made the growth curve of <span class="italic">E.coli</span> at different concentrations of acetaldehyde and ethanol to figure out a proper experimental condition. For acetaldehyde and ethanol, we both set four concentrations: 0%, 0.1%, 0.2% and 0.3%, and the results are shown in the following figures. </p>
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                                                  <img src="https://static.igem.org/mediawiki/2017/0/07/USTC-result-harvest-5%281%29.jpeg" width="100%">
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                                                  <p style="text-align:center!important">Figure 6. The growth curve of wild type and KmAdh at diferent concentration of acetaldehyde and ethanol</p>
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                                                  <div class="col s6">
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                                                  <img src="https://static.igem.org/mediawiki/2017/9/9e/USTC-result-harvest-5%282%29.jpeg" width="100%">
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                                                  <img src="https://static.igem.org/mediawiki/2017/9/99/USTC-result-harvest-5%283%29.jpeg" width="100%">
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                                                  </div>
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                                                  <div class="col s6">
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                                                  <img src="https://static.igem.org/mediawiki/2017/f/fb/USTC-result-harvest-5%284%29.jpeg" width="100%">
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                                                  <img src="https://static.igem.org/mediawiki/2017/e/e2/USTC-result-harvest-5%285%29.jpeg" width="100%">
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                                                  </div>
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                                                  <p style="text-align:center!important">Figure 7. The growth curve of wild type and KmAdh at diferent concentration of acetaldehyde and ethanol</p>
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                                                  <p class="indent_word">As the concentration of ethanol in the system increases, the growth of KMADH and WT is inhibited but KMADH’s growth is clearly better than WT’s at the same concentration. The reason is that KmAdh also has the effect of helping to break down ethanol. For acetaldehyde, the growth of KMADH and WT are both inhibited when the acetaldehyde concentration increases and KMADH’s growth is significantly better than WT’s when the acetaldehyde concentration reaches 0.3% (the highest concentration we set). This result is a rough proof of our KmAdh’s function is normal and the enzyme can be relatively high toxic acetaldehyde into less toxic ethanol to improve cell viability. When the acetaldehyde concentration and ethanol concentration in the system are the same, not only WT’s growth but also KMADH’s growth is inhibited. This indicates that the toxic effects of acetaldehyde on cells are stronger than ethanol. </p>
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                                                  <p class="indent_word">According to the results, we decided to use 0.1% acetaldehyde as the substrate, for <span class="italic">E.coli</span> can live well. </p>
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                                                  <br><br>
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  <span id="fourth" class="scrollspy label label-pink">4.Enzyme Activity Measurement in vivo</span>
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                                                  <p class="indent_word">As you can see above, this reductase KmAdh has strong enzyme activity in vitro. However, in the practical situation, we need this enzyme to function in vivo. So we did a enzyme activity measurement assay in vivo.</p>
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                                                  <p class="indent_word">Here is how we performed this experiment. First, because the condition would be anaerobic when we are running the bio-cathode, so we simulate this anaerobic condition when we are measuring the enzyme activity. Figure 8 here shows how the system was constructed. Same procedure would be taken to create this anaerobic condition as how we did in the conduction system section.Then we put the anaerobic bottles to a incubator at 30˚C and added 0.1% ethanal(final concentration is 251 µmol/L) to initiate the reaction. </p>
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                                                  <p class="indent_word">We took samples from the bottles 2 hour and 26 hour later and used Gas chromatography–mass spectrometry(GC-MS) to analyze the chemical compound in the sample, specifically, the concentration of ethanol and ethanal.</p>
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                                                  <p class="indent_word">Before we use GC-MS to analyze the sample, we need to use standard sample to find out the appearance time for the compound we concerned. Here in figure are the results of standard samples for ethanol, ethanal and acetic acid( used as the internal standard to measure the concentration of ethanol and ethanal).</p>
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                                                  <div class="col s6">
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                                                  <img src="https://static.igem.org/mediawiki/2017/5/51/USTC-result-sepu-yichun.png" width="100%" style="margin:0 0%;">
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                                                  <p style="text-align:center!important">Figure 8. Result of standard sample of ethanol</p>
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                                                  <div class="col s6">
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                                                  <img src="https://static.igem.org/mediawiki/2017/c/c0/USTC-result-sepu-yiquan.png" width="97%" style="margin:0 0%;">
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                                                  <p style="text-align:center!important">Figure 9. Result of standard sample of ethanal</p>
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                                                  </div>
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                                                  <img src="https://static.igem.org/mediawiki/2017/e/eb/USTC-result-sepu-yisuan.png" width="60%" style="margin:0 20%;">
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                                                  <p style="text-align:center!important">Figure 10. Result of standard sample of acetic acid</p>
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                                                  <p class="indent_word">As you can see above, the appearance time for ethanol, ethanal, acetic acid are 3.047, 1.402, 7.109 respectively. The highest peak is the one of acetone, the solvent in our sample.
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                                                  <p class="indent_word">Then we began the analysis of the sample we had. Here is the outcomes of these 4 samples. After calculation, we had an accurate result of the concentration of ethanal in the system. However, the concentration of ethanol in the system is too low to be detected. So we could not measure the concentration of ethanol.</p>
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                                                  <img src="https://static.igem.org/mediawiki/2017/8/85/USTC-result-sepu-jieguo.png" width="100%" style="margin:0 0%;">
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                                                  <p style="text-align:center!important">Figure 10. Result of GC-MS in table</p>
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                                                  <img src="https://static.igem.org/mediawiki/2017/2/2d/USTC-result-sepu-jieguo3.png" width="70%" style="margin:0 15%;">
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                                                  <p style="text-align:center!important">Figure 11. Result of GC-MS in histogram</p>
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                                                  <p class="indent_word">It’s true that the enzyme can catalyze the bio-transformation from ethanal to ethanol. In another word, the decrease of ethanal in the system should lead to the increase of ethanol. But it may be a chance that the ethanol had been used for metabolism. Because in the former procedures before we ran the bio-cathode, there was a step for starvation.The bacteria would be at a state that run out of carbon source, so the ethanol would become the carbon source and be consumed after it was synthesized. So we can not detect ethanol in the system.</p>
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                                                  <p class="indent_word">Although we can’t detect the ethanol’s concentration to confirm whether the reductase has function in vivo, we can still compare the ethanal’s concentration to do the same work. As you can see in figure, the strain that expressed KmAdh utilized more ethanal than WT, which means this enzyme can increase the consumption speed of ethanal. This is a strong evidence to prove that this enzyme can function in vivo!</p>
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                                                  <p class="indent_word">In conclusion, with GC-MS, we can confirm that the KmAdh can function in vivo under anaerobic condition.
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                                                  <p>Reference:</p>
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                                                  [1]Liang, J. J., Zhang, M. L., Ding, M., Mai, Z. M., Wu, S. X., Du, Y., & Feng, J. X. (2014). Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization. BMC biotechnology, 14(1), 45.
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                                                  <br>
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                                                  [2]Deng, M. D., Severson, D. K., Grund, A. D., Wassink, S. L., Burlingame, R. P., Berry, A., ... & Rosson, R. A. (2005). Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metabolic engineering, 7(3), 201-214.
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                                                  <br>
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                                                  [3]He, Y.C., Tao, Z.C., Zhang, X., Yang, Z.X., Xu, J.H., 2014a. Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate and its derivatives by a robust NADH- dependent reductase from E. coli CCZU-K14. Bioresour. Technol. 161, 461–464.
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                                                  <br>
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                                                  [4]Cordell, R. L., Pandya, H., Hubbard, M., Turner, M. A., & Monks, P. S. (2013). GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples—quantifying neonatal exposure. Analytical and bioanalytical chemistry, 405(12), 4139-4147.
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        <p class="indent_word">So we performed this bio-cathode assay for a second time, adding this starvation step into the protocol. In addition, after the starvation step, we used 1 mL of minimal salts medium to resuspend the bacteria and dropped it onto the graphite electrode to form a bio-film, which could help to make a better connection between the bacteria and the electrode, especially when we were using the Mtr pathway to transfer electrons. Here, in figure 13, you can see how we made this biofilm. 2 to 3 hours later, with a sufficient airflow in the laminar flow hood, the graphite electrode would dry up and form a great biofilm. With this biofilm, electrons could be transferred to the Mtr C protein directly from the electrode which can increase the efficiency of electron transferring. Then what we need to do was to construct this bio-cathode, put every part of this “toy” together and get the oxygen out of this container. Here in figure 14 is how we clear the oxygen out of the bio-cathode to create an anaerobic environment. Lastly, we connected the bio-cathode to the electric-chemical station to give a certain voltage to the cathode and monitor the current of the cathode as time went by as how figure 15 shows. </p>
 
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                                                        <p style="text-align:center!important">Figure 13. Preparation for bio-film</p>
 
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                                                        <p style="text-align:center!important">Figure 14. Preparation for reaction system<br>(to exclude oxygen out of the container)</p>
 
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        <img src="https://static.igem.org/mediawiki/2017/thumb/f/f0/USTC-result-Mtr-5.jpeg/800px-USTC-result-Mtr-5.jpeg" width="40%" style="margin:0 30%;">
 
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        <p style="text-align:center!important">Figure 15. Bio-cathode device</p>
 
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        <p class="indent_word">Figure 16 is the result of this experiment. From the figure, we can easily notice that the red line, which is the Mtr-induced group, had a 50% higher current than the other two group after the bio-cathode turned into a stable state . This could strongly prove that the engineered strain pMC can transfer electrons into the cytoplasm, which led to the increasing of the cathode-current. But there would be a chance that this difference between these 3 groups was just the background noise between this three cathode, resulting from the hardware’s varieties. So we added fumarate into the system to see whether there would be a cathode catalyzed current happened in the pMC group. That’s why there was a sharp increasing in the figure. When we added fumarate into the system, the electrons on the electrode finally found a way to leak to—— the fumarate. So there would be a strong electron flow when we added fumarate into the system. But after a short time we introduced this sudden change into the system, the current will become stable again, slowly climbing back to the current it was. However, the time it took to get back to stable state can be a strong evident to prove our assumption——our engineered E.coli can transfer extracellular electrons into the cytoplasm!! The red line’s curve happened after we added fumarate into the system is kind of a typical curve of cathode-catalyze-current!! So, with this result, the cathode’s current to time under a certain voltage, we can confidently say that the Mtr CAB system work!!</p>
 
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        <img src="https://static.igem.org/mediawiki/2017/9/91/USTC-result-Mtr-10.jpeg" width="80%" style="margin:0 10%;">
 
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        <p style="text-align:center!important">Figure 16. The current result of the bio-cathode.</p>
 
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        <p class="indent_word">In conclusion, the Mtr CAB system can really function as an electron pathway to transfer extracellular electrons into the cytoplasm, even though it’s expressed in E.coli, but not it’s origin host Shewanella.!! In another word, our conduction system can function as we expected, transferring those electrons from the electrode into the cytoplasm, which means our E.coli can transform itself like transformer from a normal form to a special form that can “eat” electrons!</p>
 
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        <p class="get_bold1">Reference: </p>
 
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        [1] Thomas, P. E., Ryan, D., & Levin, W. (1976). An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Analytical biochemistry, 75(1), 168-176.
 
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        [2] Jensen, H. M. (2013). Engineering Escherichia coli for molecularly defined electron transfer to metal oxides and electrodes. University of California, Berkeley
 
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Revision as of 13:45, 29 October 2017


T7+lacO+KmAdh

KmAdh is an alcohol dehydrogenase from Kluyveromyces marxianus. It can catalyze the ethanol fermentation ongoing in the bacteria with a high efficiency and mild reaction condition, especially temperature. We use T7 promoter and Lac operator to control KMADH’s expression. Both of this two units are induced by IPTG. With this dual switch, we can reduce the leak of the gene expression as much as possible. 1.Transformation and Expression

We transformed a plasmid PET22b containing KmAdh into E.coli successfully. We use KmAdh’s specific primers to do PCR to verify this achievement.

Figure 1. Electrophoresis result of PCR of KmAdh
(From left to right: wild type, KmAdh, positive control)

We can see that the experimental group and the positive control have the same band but WT does not. This shows the transformation is successful.

Then we induced the expression of this enzyme. We use 200 mL LB to cultivate our bacteria in 37℃，250 rpm. When its OD600 reached 0.5-0.8 we added 20μL 1M IPTG(final concentration=0.1mM) in it to induce KmAdh expression.

Figure 2. SDS-PAGE for KmAdh
(From left to right: wild type, KmAdh, KmAdh+IPTG)

From the SDS-PAGE result, it can be safely concluded that KmAdh was successfully expressed at a high level.

Then we gathered the bacteria to purify the KmAdh for enzyme activity measurement

Figure 3. SDS-PAGE for KmAdh
(From left to right: wild type, KmAdh, KmAdh+IPTG, raw enzyme, flow throgh, 20mM elution, 300mM elution, Pure enzyme)

From figure 3, you can see that we successfully purify the KmAdh from the bacteria lysate.

2.Enzyme activity test

NADH, as a necessary cofactor of KmAdh, has a significant absorption in 340nm. However, once it's been reduced to NAD+, it will have no absorption in 340 nm. So, along the process of the reduction reaction, the consuming of NADH will lead to a decrease of absorption in 340nm which allows us to test the activity of KmAdh by the spectrophotometer.

Figure 4. Reaction mixture

Here in figure 4 is how we performed the reaction. After adding every component in to the cuvette, we scan the 340nm UV absorption value over time. Because NADH is easy to be oxidized, we set a blank control to exclude this effect. The system is the same as the above system, with the same amount of PBS to replace KmAdh purified enzyme.

Figure 5. The change of OD340 over time

Here in figure 5, there is a rapid increase in the absorption value after adding enzyme, indicating that NADH is drastically consumed. This shows the purified enzyme function is normal and the KmAdh is successfully expressed in E. coli.



3.Toxicity test

Considering that acetaldehyde and ethanol, the substrate and product of KmAdh, may do harm to the cell, we first made the growth curve of E.coli at different concentrations of acetaldehyde and ethanol to figure out a proper experimental condition. For acetaldehyde and ethanol, we both set four concentrations: 0%, 0.1%, 0.2% and 0.3%, and the results are shown in the following figures.

Figure 6. The growth curve of wild type and KmAdh at diferent concentration of acetaldehyde and ethanol

Figure 7. The growth curve of wild type and KmAdh at diferent concentration of acetaldehyde and ethanol

As the concentration of ethanol in the system increases, the growth of KMADH and WT is inhibited but KMADH’s growth is clearly better than WT’s at the same concentration. The reason is that KmAdh also has the effect of helping to break down ethanol. For acetaldehyde, the growth of KMADH and WT are both inhibited when the acetaldehyde concentration increases and KMADH’s growth is significantly better than WT’s when the acetaldehyde concentration reaches 0.3% (the highest concentration we set). This result is a rough proof of our KmAdh’s function is normal and the enzyme can be relatively high toxic acetaldehyde into less toxic ethanol to improve cell viability. When the acetaldehyde concentration and ethanol concentration in the system are the same, not only WT’s growth but also KMADH’s growth is inhibited. This indicates that the toxic effects of acetaldehyde on cells are stronger than ethanol.

According to the results, we decided to use 0.1% acetaldehyde as the substrate, for E.coli can live well.



4.Enzyme Activity Measurement in vivo

As you can see above, this reductase KmAdh has strong enzyme activity in vitro. However, in the practical situation, we need this enzyme to function in vivo. So we did a enzyme activity measurement assay in vivo.

Here is how we performed this experiment. First, because the condition would be anaerobic when we are running the bio-cathode, so we simulate this anaerobic condition when we are measuring the enzyme activity. Figure 8 here shows how the system was constructed. Same procedure would be taken to create this anaerobic condition as how we did in the conduction system section.Then we put the anaerobic bottles to a incubator at 30˚C and added 0.1% ethanal(final concentration is 251 µmol/L) to initiate the reaction.

We took samples from the bottles 2 hour and 26 hour later and used Gas chromatography–mass spectrometry(GC-MS) to analyze the chemical compound in the sample, specifically, the concentration of ethanol and ethanal.

Before we use GC-MS to analyze the sample, we need to use standard sample to find out the appearance time for the compound we concerned. Here in figure are the results of standard samples for ethanol, ethanal and acetic acid( used as the internal standard to measure the concentration of ethanol and ethanal).

Figure 8. Result of standard sample of ethanol

Figure 9. Result of standard sample of ethanal

Figure 10. Result of standard sample of acetic acid

As you can see above, the appearance time for ethanol, ethanal, acetic acid are 3.047, 1.402, 7.109 respectively. The highest peak is the one of acetone, the solvent in our sample.

Then we began the analysis of the sample we had. Here is the outcomes of these 4 samples. After calculation, we had an accurate result of the concentration of ethanal in the system. However, the concentration of ethanol in the system is too low to be detected. So we could not measure the concentration of ethanol.

Figure 10. Result of GC-MS in table

Figure 11. Result of GC-MS in histogram

It’s true that the enzyme can catalyze the bio-transformation from ethanal to ethanol. In another word, the decrease of ethanal in the system should lead to the increase of ethanol. But it may be a chance that the ethanol had been used for metabolism. Because in the former procedures before we ran the bio-cathode, there was a step for starvation.The bacteria would be at a state that run out of carbon source, so the ethanol would become the carbon source and be consumed after it was synthesized. So we can not detect ethanol in the system.

Although we can’t detect the ethanol’s concentration to confirm whether the reductase has function in vivo, we can still compare the ethanal’s concentration to do the same work. As you can see in figure, the strain that expressed KmAdh utilized more ethanal than WT, which means this enzyme can increase the consumption speed of ethanal. This is a strong evidence to prove that this enzyme can function in vivo!

In conclusion, with GC-MS, we can confirm that the KmAdh can function in vivo under anaerobic condition.

Reference:

[1]Liang, J. J., Zhang, M. L., Ding, M., Mai, Z. M., Wu, S. X., Du, Y., & Feng, J. X. (2014). Alcohol dehydrogenases from Kluyveromyces marxianus: heterologous expression in Escherichia coli and biochemical characterization. BMC biotechnology, 14(1), 45.
[2]Deng, M. D., Severson, D. K., Grund, A. D., Wassink, S. L., Burlingame, R. P., Berry, A., ... & Rosson, R. A. (2005). Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metabolic engineering, 7(3), 201-214.
[3]He, Y.C., Tao, Z.C., Zhang, X., Yang, Z.X., Xu, J.H., 2014a. Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate and its derivatives by a robust NADH- dependent reductase from E. coli CCZU-K14. Bioresour. Technol. 161, 461–464.
[4]Cordell, R. L., Pandya, H., Hubbard, M., Turner, M. A., & Monks, P. S. (2013). GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples—quantifying neonatal exposure. Analytical and bioanalytical chemistry, 405(12), 4139-4147. Sequence and Features


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