Team:OUC-China/proof1

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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 make good use of cellobiose and xy-lose, we cloned relative genes to a shuttle plasmid and then transformed it to our engi-neered yeast. We prepared culture medium that contains xylose and cellobiose as the only carbon resources, so that we can verify that we have already cultivated the cell that can grow with merely cellobiose and xylose as their carbon sources. If our novel strains grow better than the control, we can prove that our ideas are feasible and the genetic circuit could work. At the same time, we use HPLC to detect the changing concentration of cellubiose and xy-lose, getting more data to support our idea that our cells can utilize the carbon sources as long as the concentration of xylose/cellobiose decrease with time. SBA biosensor can con-vert the reaction of immobilized enzyme to electrochemical signal, which supports us to de-scribe a curve reflecting the ethanol change in the culture condition. Here, we can testify that two recombinant 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 cellubiose 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 use pYC230 provided by our PI as our backbone and integrate xylosidase gene xyl1 and xyl2 through Gibson. Gene XYL1 encoding xylose reductase (XR),which reduces D-xylose into xylitol, facilitated xylose assimilation in yeast, and gene XYL2 encoding xylitol dehy-drogenase (XDH) .Xylitol is oxidized to xylulose under the action of xylitol dehydrogenase, which relies on NAD.

Strain construction

We introduce the plasmid into S.cerevisiae EBY100 and construct the xylose-utilize strain successfully. We measured the growth rate of both our recombinant strain and nega-tive control. If there is an obvious superiority of our new strain in xylose utilization, we can prove that the strain is constructed correctly. As a result, the curve can well demonstrate that the strain that carries our plasmid grows much better than the strain that not and reach the stationary phases after 40 hours’cultivation.

Fig 1.4

Proof of function

We use HPLC to detect the changing concentration of xylose, getting more data to support our idea directly that our cells can utilize the carbon sources as long as the concentration of xylose decrease with time.

The following chart shows the xylose content of both our recombinant strain and negative control.

Fig 1.5

It is obvious that in our xylose-utilize strain, xylose content decrease as time goes by while for the negative strain the xylose content stays steady, indicating the disability of using xy-lose.

For the final goal of our project, we will ultimately detect the production of ethanol. Here we choose to use the SBA-Biosensor. Because it can convert the reaction of immobilized enzyme to electrochemical signal, which supports us to describe a curve reflecting the etha-nol change in the culture condition, and is able to give the output instantly and convenient-ly.

The following chart shows the ethanol content of both our recombinant strain and negative control.

Fig 1.6

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 do produce ethanol on the basis of xylose, thus realizes our de-sign 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

Fig 1.8