Difference between revisions of "Team:Amsterdam/Produce"

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  and 48.48 mg L
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  produces more fumarate over the course of a natural day. This clearly shows the benefit of having a day/night production system (and it is extremely gratifying to see that all our modeling and experimental efforts were not in vain!).
 
  produces more fumarate over the course of a natural day. This clearly shows the benefit of having a day/night production system (and it is extremely gratifying to see that all our modeling and experimental efforts were not in vain!).
 
</p>
 
</p>
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<figure id="fig29">
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<img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/3/3b/TAmsterdam_amsterdam_production_2.9.png"/>
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  <b>
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  Figure 2.9
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  <i>
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  Qp
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    daily
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  of the four different strains during the fourth 24h period. This experiment has been carried out with similar results 5 times independently for
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    Δ
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    fumC
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  and 6 times for the
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    fumC
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    fumC
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    Δ
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    zwf
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  has a higher Qp
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    daily
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  than the
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    fumC
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    WT and
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    Δ
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    zwf
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    do not produce fumarate during the night.
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</figcaption>
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</figure>
 
<figure id="table21">
 
<figure id="table21">
 
  <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/f/f0/TAmsterdam_amsterdam_production_table21.png"/>
 
  <img class="module-figure-image" src="https://static.igem.org/mediawiki/2017/f/f0/TAmsterdam_amsterdam_production_table21.png"/>
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   </i>
   strain increases the fumarate production during the night. However, by knocking out fumarase, we disrupted the cyclic nature of  the TCA, which may no longer operate as a cycle. This construct yields one fumarate per glycogen catabolized and NNN reducing equivalents. We started wondering if there could be a way to use synthetic biology to improve the nighttime production efficiency in terms of carbon usage. We turned to nature for inspiration…
+
   strain increases the fumarate production during the night. However, by knocking out fumarase, we disrupted the cyclic nature of  the TCA, which may no longer operate as a cycle. This construct yields one fumarate per glycogen catabolized. We started wondering if there could be a way to use synthetic biology to improve the nighttime production efficiency in terms of carbon usage. We turned to nature for inspiration…
 
   <br/>
 
   <br/>
 
   Many microorganisms express a glyoxylate shunt. The glyoxylate shunt consists of two enzymes which are not natively present in
 
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Revision as of 03:36, 2 November 2017

Production


A major requisite of cyano-cell factories, according to expert's opinion, is that they must be able to produce in a stable fashion under industrial conditions. A recent quantitative analysis of the various ways to convert the energy of photons to chemical bonds has revealed that the direct utilization of sunlight is the most efficient [1]. This however means that cells will be exposed to diurnal regimes in which they will inevitably be exposed to periods of darkness. Our goal here is to achieve the first photoautotrophic cell factories that are able to stably produce fumarate around the clock.


Produce

Overview

Synechocystis does not naturally produce fumarate. However, model guided engineering found that removing a single gene within Synechocystis leads to a stable cell factory that produces fumarate as it grows during the day. Nevertheless, at night our cells do not produce fumarate, since at night, they don't grow. To overcome this challenge, we have taken a systems biology approach which interweaves theory, modeling, and experimentation to implement stable nighttime production of fumarate. We theorized that we can redirect the nighttime flux towards fumarate production by removing a competing pathway via knockout of the zwf gene. Additionally, we also took inspiration from nature and speculated that the incorporation of the glyoxylate shunt would further increase our nighttime production of fumarate. Our models corroborate these predictions, however, they also suggest that the stability of the glyoxylate shunt is sensitive to the timing of when the shunt is turned on (i.e. expressed). We therefore took a robust approach to incorporate the glyoxylate shunt enzymes under ideal expression conditions.

Highlights

  • Engineered a Δ fumC Δ zwf Synechocystis strain, that uses different fumarate production strategies during day and night.
  • Developed a method to make fully segregated libraries in polyploid organisms
  • Created the first fully segregated library representing the entire genome (99.9% confidence) of Synechocystis upstream of the glyoxylate shunt genes. This library is now ready to be tested to further increase nighttime fumarate production.
  • Stable production of fumarate directly from CO 2 around the clock (Nighttime fumarate production rate of 2.96 mM grDW -1 hour -1 Daytime fumarate production rate of 9.24 l mM grDW -1 hour -1 Titer of 48.48 mg L -1 ) [Disclaimer: our experimental design was aimed mostly at proof-of-principle. Much higher titers (>230 mg/L) are possible if economically more favorable due to downstream costs].

Overall production in conditions mimicking industrial settings

The productivity of our strains in an industrial setting is the combined production of day and night. We calculated the Qp daily over the course of a 24h period (figure 2.9). We find that the Δ fumC Δ zwf has a Qp daily of 32.83 µM grDW -1 hour -1 , while the Δ fumC has a Qp daily of 23.00 µM grDW -1 hour -1 . The titers of the strains are 27.39 mg L -1 and 48.48 mf L -1 after four 24h periods. We can thus conclude that the Δ fumC Δ zwf produces more fumarate over the course of a natural day. This clearly shows the benefit of having a day/night production system (and it is extremely gratifying to see that all our modeling and experimental efforts were not in vain!).

<figure id="fig29">

<img class="module-figure-image" src="TAmsterdam_amsterdam_production_2.9.png"/>
<figcaption class="module-figure-text">
 
  Figure 2.9
 
 
  Qp
  
   daily
  
  of the four different strains during the fourth 24h period. This experiment has been carried out with similar results 5 times independently for
  <i>
   Δ
  
  
   fumC
  
  
   Δ
  
  
   zwf
  
  and 6 times for the
  
   Δ
  
  
   fumC
  
  .
  
   Δ
  
  
   fumC
  
  
   Δ
  
  
   zwf
  
  has a higher Qp
  
   daily
  
  than the
  
   Δ
  
  
   fumC
  
  .
  
   WT and
   <i>
    Δ
   
   
    zwf
   
   do not produce fumarate during the night.
  </i>
 </i>
</figcaption>

</figure> <figure id="table21">

<img class="module-figure-image" src="TAmsterdam_amsterdam_production_table21.png"/>
<figcaption class="module-figure-text">
 
  Table 2.1
 
 
  Fumarate production parameters for the four different strains.
  <i>
   Qp values are in mM grDW
   
    -1
   
   hour
   
    -1
   
   measured after the fourth  day/night cycle ,
  
 </i>
</figcaption>

</figure>

Conclusion

We showed that the Δ fumC and the Δ fumC Δ zwf are both able to produce fumarate during the daytime using the growth coupled strategy. During the night, the Δ fumC Δ zwf produces more fumarate than the Δ fumC , which confirms the extensive <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Model#ppp" target="_blank"> modeling </a> we did for this part of the project. We can confirm that at night the Δ fumC Δ zwf is forced to direct carbon from glycogen catabolism towards the TCA cycle to form fumarate. We thus engineered a Synechocystis cell factory that is able to produce fumarate around the clock, using two different production strategies - both stable - one for day and another for night. On top of this, since we used only knock-outs and did not resort to the cloning of heterologous genes, the Δ fumC Δ zwf will be a stable production strain for many generations to come. As a bonus, the higher nighttime production of the Δ fumC Δ zwf compared to the Δ fumC does imply that by knocking out the Δ zwf , we force flux to the TCA cycle. This is an important finding, as it opens up opportunities for the nighttime production of valuable TCA cycle intermediates in Synechocystis . To our knowledge such a diurnal, dual strategy, photoautotrophic cell factory has never been reported before.

Methods

Strain construction: Δ zwf and Δ fumC Δ zwf and segregation

The zwf gene encodes glucose-6-phosphate 1-dehydrogenase, which catalyses the first step in the Pentose Phosphate Pathway. We knocked out the zwf gene in the Wild Type and the Δ fumC background, to construct the Δ zwf and the Δ fumC Δ zwf mutants. We used the <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Methods" target="_blank"> Markerless knock out method </a> . The homologous regions of the zwf gene were amplified from the Synechocystis genomic DNA, with Herculase polymerase using primers BP1, BP2, BP3 and BP4. The <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/Methods" target="_blank"> biobrick T vector </a> used was the pFL-AN. Resulting in plasmid in zwf knockout plasmids, which were used for the first and second round of transformation.

Characterising Δ zwf , Δ fumC and Δ fumC Δ zwf

In order to characterise the different Synechocystis strains, we performed different cultivation experiments. We simultaneously performed a batch and a turbidostat experiment in a modified Multi-Cultivator under a photonfluxostat light regime, as described in Du et al. 2016[4] and outlined on our methods page. Our Synechocystis strains were cultivated in BG-11 medium which contained 10 mM TES KOH buffer. For the batch experiment, we had 4 vessels that contained Δ fumC and 4 vessels that contained Δ fumC Δ zwf . In the turbidostat set up, we cultivated four strains i) Wild Type, ii) Δ zwf , iii) Δ fumC Δ zwf , and iv) Δ fumC all in duplicates. The light intensity per OD followed a sinusoidal regime to simulate day/night cycles yielding 16 hours of darkness (0 μE s -2 OD -1 ) and 8 hours of light (peaking at 120 μE s -2 OD -1 ), calculated by equation 2.1, where t is the time in hours.

\[2.1 \frac{\mu E}{s^{2}}=240\sin (2\pi\cdot (\frac{t}{24}+\frac{1}{4}) )-120\]

This equation returns negative values during the period, so they are clamped at a minimum value of 0. All cultures are inoculated at an initial OD 720 of 0.05 and were grown at a constant light intensity of 20 μE until all vessels reached an OD 720 of 0.6. At this point, we switched the light output to the designated light regime.

Instead of the more commonly adopted 12h day/12h night, we chose a 8h day/ 16h night as indicated above. While longer days would have probably allowed us to reach higher levels of production in the lab, after visiting an actual <a class="in-text-link" href="https://2017.igem.org/Team:Amsterdam/HP/Gold_Integrated" target="_blank"> production facility </a> , we were convinced that this would not be representative of a real-world scenario. The structures surrounding the greenhouses in many production plans provide shading during dawn and dusk. This makes the sun rise somewhat later, and set somewhat sooner, for production photoautotrophs. Our light regime in the lab mimics this, and is yet another factor that confers credibility to the actual production numbers that we report..

Sampling and fumarate measurements

During the course of the experiment, we took samples at every perceived dawn and dusk. After sampling we had to determine the concentration of fumarate. 1 ml of sample was centrifuged at 15.000 rpm for 10 min. Then 500 μl supernatant was taken and filtered (Sartorius Stedin Biotech, minisart SRP 4, 0.22 μm) for sample preparation. Fumarate concentration was measured by HPLC-UV/VIS (LC-20AT, Prominence, Shimadzu), with ion exclusion Rezex ROA-Organic Acid column (250x4.6 mm; Phenomenex) and UV detector (SPD-20A, Prominence, Shimadzu) at 210 nm wavelength. 50 μL of the HPLC samples were injected through an autosampler (SIL-20AC, Prominence, Shimadzu), with 5 mM H 2 SO 4 as eluent at a flow rate of 0.15 ml min -1 and column temperature of 45 ℃. Fumarate retention time was determined as 18.16 and 18.36 min and fumarate samples were normalised by a correction factor composed of 10 mM divided by the measured TES concentration.

Production calculations

To calculate the fumarate production during the day and the fumarate production during the night. We made the following assumptions: i) Synechocystis does not grow in the absence of sunlight, so does not grow during the night. ii) The production of fumarate during the day is growth coupled. We calculated the fumarate yield during the day, yield day by Δ fumarate/ Δ OD in mmol OD -1 over the course of 1 day in the batch culture. By dividing this number by 8 hours, we could calculate Qp day in mmol OD -1 h -1 for the day. During the night, no cell growth was assumed, therefore we expected no change in OD, however as Synechocystis physiology changes at night, this can influence the scattering of the light and thereby the OD measurement can change during the night. To account for this effect, we determined the night time fumarate yield, yield night as Δ fumarate/mean OD. By dividing this number by 16 hours we could calculate Qp night in mmol OD -1 h -1 . The overall 24h fumarate production could be determined by knitting together the nighttime production and the daytime production. we determined the yield daily as yield day plus yield night . Dividing this number by 24 hours, we could determine Qp daily in mmol OD -1 hour -1 . To transform these QPs to a more familiar unit, we multiplied all QP's by a conversion factor that converts OD 720 to gram dry weight (148 mg L -1 OD -1 [16] ) . We then receive fumarate QPs in mM gDW -1 hour -1 .

2.2 \[yield_{day} =\frac{\triangle [fumarate]}{\triangle OD}\] 2.3 \[yield_{night} =\frac{\triangle [fumarate]}{mean OD}\] 2.4 \[Qp_{day} =\frac{yield_{day}}{8 \ Hours}\cdot148\ mg \cdot L^{-1} \cdot OD^{-1}\] 2.5 \[Qp_{night}=\frac{yield_{night}}{16 \ Hours}\cdot148\ mg \cdot L^{-1} \cdot OD^{-1} \] 2.6 \[Qp_{daily} =\frac{yield_{day}+yield_{night}}{24 \ Hours}\cdot148\ mg \cdot L^{-1} \cdot OD^{-1}\]

References

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