Team:OUC-China/proof1
Proof of concept——Basic fermentation
We first powdered the algae, removed the lignin and then treated remains with the enzyme solution to produce cellobiose and xylose.
In order to engineered the yeast for utilizing cellobiose and xylose as carbon resources, we cloned relative genes to a shuttle plasmid in E. coli cell, then transformed it to yeast. We prepared SC medium containing xylose and cellobiose as the only carbon resources, so that we can verify that we have already cultivated the strain which can grow with merely cellobiose and xylose as their carbon sources. If our novel strains grow significantly better than the negative control (which does not have the gene GH-1, CDT-1, or XYL1, XYL2) we can prove that our ideas are feasible and the genetic circuit works well.
At the same time, we used HPLC to detect the concentration changing of cellobiose and xylose, and got more data to support our idea that the engineered yeast can utilize cellobiose and xylose as carbon sources. If the concentration of xylose/cellobiose decrease with time. SBA biosensor can detect the reaction process of substrate by immobilized enzyme as electrochemical signal, which supports us to describe a curve reflecting the ethanol generation under culture condition. Here, we prove that two engineered yeast strains can utilize cellobiose and xylose to produce ethanol directly.
Pretreatment
1.Enteromorpha physical pretreatment.
2.Treat the residue with 0.2% H2O2 to remove the lignin.
3.Preparation of the enzymatic hydrolysis solution of Enteromorpha fiber.
4. The validation of xylose and cellobiose content after pretreatment.
Fig 1.1 Enteromorpha Powder .
Fig 1.2 Treat the residue with 0.2% H2O2.
Fig 1.3 Enzymatic hydrolysis solution of Enteromorpha fiber.
Yeast A--xylose fermenting strain
Circuit construction
We used pYC230 provided by our PI, as the backbone and constructed xylosidase gene XYL1 and XYL2 by Gibson Assembly. Gene XYL1 encoding xylose reductase (XR) which reduces D-xylose into xylitol, and facilitates xylose assimilation in yeast. Gene XYL2 encoding xylitol dehydrogenase (XDH). Xylitol is oxidized to xylulose under the action of xylitol dehydrogenase, which relies on Nicotinamide adenine dinucleotide (NAD+).
Selective Medium Assay
We transformed the pYC230nto S. cerevisiae EBY100 and got the xylose-utilizing strain successfully. We measured the growth rate of both our engineered strain and the negative control. If there is an obvious superiority of our new strain in xylose utilization, we can prove that the circuit works as our design. As a result, the curve well demonstrates that the strain with xylose-utilizing circuit has a higher OD600 than the negative control strain during the entire cultivation period. The culture of engineered yeast (EBY100 with XDH-XR) reaches stationary phases after 40 hours cultivation with an OD600 of around 2.35. While the control strain(EBY100) has an almost horizontal OD curve.
Fig 1.4 Growth curve of strains of our recombinant strain and negative control.Error bars represent standard deviation of four biological replicates, so do the other charts in this section.
Proof of function
We use HPLC to detect the concentration changing of xylose, getting extra data to support our idea directly that our engineered yeast can utilize the xylose as carbon sources and the concentration of xylose decrease with time in our culture system.
The following chart shows the dynamic curve of xylose content of both the recombinant strain and negative control.
Fig 1.5 Xylose content of both our recombinant strain and negative control.
It is obvious that for our xylose-utilize strain, xylose content decreases as time goes, by while for the negative strain, the xylose content stays steady, indicating the disability of using xylose.
For the final goal of our project, we ultimately detected the production of ethanol. Here, we choose to use the SBA-Biosensor. Because it can detect the reaction of substrate catalyzed by the immobilized enzyme as an electrochemical signal, which supports us to describe a curve reflecting the ethanol change in the culture condition, and is able to give the output instantly and conveniently.
The following chart shows the ethanol content from both our recombinant strain and negative control.
Fig 1.6 Ethanol content of both our recombinant strain and negative control.
Gladly, the curve of xylose-consuming strain goes gradually up along with cultivating hours, and reach the plateau at around 90 hours, which is consist with the xylose consuming curve, indicating that our strain does produce ethanol on the basis of xylose, thus realizes our design and proves the concept of basic fermentation part.
This three-in-one chart demonstrates our evidence. The left chart belongs to recombinant strain that can utilize xylose and the right chart belongs to the negative control.
Fig 1.7 Fermentations of biomass(red), xylose(blue) and ethanol(black) by XDH-XR strain.
Fig 1.8 Fermentations of biomass(red), xylose(blue) and ethanol(black) by control strain.
Yeast B--cellobiose fermenting strain
Circuit construction
We use pYC230 provided by our PI as our backbone and integrate cellubiose related gene CDT and GH-1 through Gibson. CDT encoding a cellubiose transporter, which assimilate cellubiose into intracellular,and GH-1 encoding β-glucosidase, which capable of hydrolyzing cellubiose into glucose.
Fig 1.9 from lane 1 to 7 respectively:pYC230-GH1-CDT1 single enzyme digestion; pYC230-XYL1-XYL2 single enzyme digestion;marker; pYC230-GH1-CDT1 circular plasmid;pYC230-XYL1-XYL2 circular plasmid; marker
The bright band in the AGE result shows that we successfully get the fragments of GH-1,CDT-1 through PCR. The following experiment includes splicing the fragments onto the backbone pYC230.
Strain construction
We transformed the plasmid into S.cerevisiae EBY100 and got the cellubiose-utilize strain successfully then test the growth rate of the of both our recombinant strain and negative control.
Proof of function
Using HPLC we detect the changing concentration of cellubiose. The result shows that the concentration of cellubiose decrease with time, indicating a well utilization of cellubiose.
The following chart shows of both our recombinant strain(left) and negative control(right).
To examine the production of ethanol, we use the SBA-Biosensor, the same as in the xylose pathway.
The following chart shows the cellubiose content, ethanol content, and growth rate of both our cellubiose recombinant strain (left) and negative control (right).
Fig 1.10 Concentration of cellobiose,ethanol as well as OD600 of biomass change in 140h for cellobiose-recombinant strain.
Fig 1.11 Concentration of cellobiose,ethanol as well as OD600 of biomass change in 140h for negative control.
When characterizing the growth rate of recombinant cells utilizing cellubiose with OD600, we found a platform stage in the middle of the curve suggesting probably a secondary growth. We figure out a hypothesis that the accumulation of the glucose when making use of cellubiose might be the reason underlying this phenomenon because a substitution process of the carbon sources can exactly lead to such a plateau in the growth curve. [1]Therefore, we detect the glucose content of the medium after that to validate our assumption.
Fig 1.12 The glucose concentration change in 140h for recombinant EBY100(CDT-1-GH-1) and WT EBY100.
Fig 1.13 The ethanol concentration change in 140h for recombinant EBY100(CDT-1-GH-1) and WT EBY100.
The result (left) shows that glucose content in the medium reaches a peak at about 40 hours and then decrease rapidly. At around 70 hours, the content reaches the minimum and stay steady ever since. Considering the ethanol content (right) that also present a largest amount at around 80 hours, we believe that the glucose in yeast may be transformed gradually into acetic acid and ethanol participated in further metabolic procedure with acetic acid called Glucose Glycolysis, thus was turned into lipid.
And for a more visualized display, we use plate streaking to show the growth condition of our two recombinative strains and negative strains in different carbon source. We make three plates with either xylose, cellobiose or xylose plus cellobiose as sole or compound carbon source, respectively and monitor the growth of each strain in three different plate after cultivating for 32 hours at 30℃.
As a result,yesat with pYC230-XYL1-XYL2 grows faster than the rest of two strains in 2% xylose as sole carbon plate, and cellobiose strain with pYC230-CDT-GH1 has a much faster growth rate than that of the other two strains in 2% cellobiose as sole carbon plate. These two recombinant strains also show a better growing condition than WT EBY100 with a medium of 2% xylose and 2% cellobiose as compound carbon source.
This phenomenon indicates that we can actually utilize xylose and cellobiose from algae residue and produce ethanol with our engineered yeasts!
Figure 2.1 recombinant strains with pYC230-XYL1-XYL2,pYC230-CDT-GH1 and WT EBY100 in different medium
Protocol about xylose pathway
Experimental purpose1. Tested the growth difference between engineered strains which were introduced into the xylose pathway and negative strains. The utilization ability of xylose and the difference of ethanol content. Verify the availability of the line.
2. Calculated the growth curve，xylose change curve and ethanol change curve.of positive strains and negative strains in the xylose as the only carbon source medium.
Materials
1.SC：0.67%YNB（2.814g+362ml）+ complete aa（42ml） （404ml）
2.xylose：17ml water +8.5g （0.5g/ml）
3.complete aa：10ml/100ml mother liquor
4.HPLC: C18 Pillars (80% PBS and 20% water as mobile phase)
5.BioTek Synergy H1 Automatic enzyme-linked immunosorbent assay systems;and 96-hole plate
6.SBAbiosensor
The determination of xylose content
1.Use YPD to activate yeast, prepare xylose medium (2% xylose +sc=10ml+40ml)
2. Test OD600, the content of the xylose and the change of ethanol content 30°c 180rpm (negative, positive 4 bottles, initial od=1) 96 Hole Plate, 200ul sample/hole, 3 hole/sample; Sample, 2340rpm, 5min bacteria, 620u/times, save -80°c. Time of measurement and retention time See timetable
3. Using HPLC to detect the change of xylose in medium, the change of ethanol content was detected by SBA Biosensor.
Method of adjusting OD value
1. measure the OD600 value of the bacterium liquid
2. Maintain the same volume as the original medium
3. 2340rpm, 5min, discard supernatant, repeat with sterile water or aseptic PBS to wash yeast two times
4. Suspension the cell with appropriate medium, then move to the appropriate medium.
Protocol about cellobiose pathway
Experimental purpose1. Tested the growth difference between engineered strains which were introduced into the cellobiose pathway and negative strains. The utilization ability of cellobiose and the difference of ethanol content. Verify the availability of the line.
2. Calculated the growth curve，cellobiose change curve and ethanol change curve.of positive strains and negative strains in the cellobiose as the only carbon source medium.
Material preparation
1、SC：0.67%YNB（3.35g+350ml）+ complete aa（50ml） （400ml）
2、cellobiose：83ml water +8.3g （0.1g/ml）
3、complete aa：10ml/100ml mother liquor
4、HPLC：Mobile phase Na2SO4
0.1M Na2SO4 ， 0.6 mL/min ， 35℃ ，Shodex OHpak SB-804 HQ and Shodex OHpak SB-802.5 HQ column (8.0mm×300mm)
5、BioTek Synergy H1 Automatic enzyme-linked immunosorbent assay systems and 96-hole plate
6、SBAbiosensor
Solubilty
cellobiose= 0.12 g/mL
The determination of cellobiose content
1.Use YPD to activate yeast, prepare cellobiose medium (2% fiber two sugars +sc=10ml+40ml)
2. Test OD600, the content of the cellobiose and the change of ethanol content 30°c 180rpm (negative, positive 4 bottles, initial od=1)
96 Hole Plate, 200ul sample/hole, 3 hole/sample;
Sample, 2340rpm, 5min bacteria, 620u/times, save -80°c.
Time of measurement and retention time See timetable
3. Using HPLC to detect the change of cellobiose in medium, the change of ethanol content was detected by SBA Biosensor.
Method of adjusting OD value
1, measure the OD600 value of the bacterium liquid.
2. Maintain the same volume as the original medium.
3, 2340rpm, 5min, discard supernatant, repeat with sterile water or aseptic PBS to wash yeast two times.
4. Suspension the cell with appropriate medium, then move to the appropriate medium.
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