Part:BBa_K3996013

pXylan-B

pXylan-B

Usage and Biology

Wheat B starch is a by-product of wheat starch deep processing, which is often directly used as feed, with low industrial added value. If wheat B starch is used as raw material to produce alcohol, part of the shortcomings of wheat starch alcohol can be avoided and the utilization value of wheat B starch can be improved. After sugar production of wheat B starch by liquid saccharification pretreatment, it can use conventional brewing yeast to produce alcohol, but this process composition of pentosan in wheat B starch did not use, even in the pretreatment stage to join pentosan enzyme, xylose and arabinose (pentose monosaccharides will use by conventional saccharomyces cerevisiae, at the same time the extra pentosan enzyme also increases the cost of production. Therefore, it is ideal to develop saccharomyces cerevisiae strains with the ability of autocrine pentosanase and pentose utilization.

Figure 1. Principle diagram of pentosan fermentation..

Construct design

The plasmid is engineered for further use. (Figure 2).

Figure 2. DNA map of plasmid pXylan-B..

The profiles of every basic part are as follows:

BBa_K3996000

Usage and Biology

The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated.

BBa_K3996003

Usage and Biology

This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems,and can be used for modulating gene expression in yeast.

BBa_K3996004

Usage and Biology

This protein is involved in the pathway xylan degradation, which is part of Glycan degradation. Endo-1,4-beta-xylanase involved in the hydrolysis of xylan, a major structural heterogeneous polysaccharide found in plant biomass representing the second most abundant polysaccharide in the biosphere, after cellulose.

Experimental approach

1. Fragments PCR products Electrophoresis To utilize the xylan component contained in the wheat B starch, we cloned the xylanase expression gene from Aspergillus niger. The xylanase expression cassette contained pXlnB plasmid was constructed firstly to prepare the final plasmid pXylan-B (Figure 2).

Figure 3. Plasmids construction used fragments PCR amplification..

(A) Lane 1: GAP promoter, 695 bp. Lane 2: AnXlnB CDS, 706 bp. Lane 3: CYC1 terminator, 276bp. Lane 4: pXlnB plasmid backbone fragment, 1757 bp. Lane 5: TPI1 promoter, 614 bp. Lane 6: AnXlnD CDS, 2443 bp. Lane 7: pXlnD plasmid backbone fragment, 1804 bp. (B) Lane 1: pXlnB plasmid backbone fragment, 1804 bp. Lane 2: pXlnD plasmid backbone fragment, 1804 bp. (C) Lane 1: pXylan-B plasmid backbone, 5479 bp. Lane 2: pXylan-BD plasmid backbone, 5479 bp. For the pXlnB plasmid construction, the promoter GAP, codon-optimized AnXlnB CDS, and CYC1 terminator PCR bands were shown in the Figure 3A, lane 1, lane2, and lane 3, respectively. The AnXlnB expression cassette was obtained through the overlap PCR. The backbone fragment (kanR with ori) was amplified using two round PCR, the first round and the final fragment band were shown in Figure 3A lane 4 and Figure 3B lane 1, respectively. The backbone was cut with Bsa1 restriction enzyme and ligated with the AnXlnB expression cassette to make the plasmid pXlnB. For the construction of the final plasmid pXylan-B, the pXlnB was cut with Sap1 restriction enzyme, and the backbone part (Figure 3C) was also cut with the same enzyme, these two parts were ligated to make the final plasmid pXylan-B. 2. Verification of the plasmids via colony PCR

Figure 4. Verification of the plasmids via colony PCR..

From the Figure 4, we can figure out that the numbers 10, 12, and 14 were the positive colonies of the plasmid pXylan-B, and Number 12 was sent for sequencing.

Figure 5. sequence information of the final plasmids..

Proof of function

fermentation test

Figure 6. Fermentation performance of the plasmids transformed S. cerevisiae strains in the simulated wheat B starch medium..

A: OD value. B: Sugar concentration. The plasmids pXylan-B and pXylan-BD were transformed into the S. cerevisiae strain, respectively. The resulting positive transformants were undergo the fermentation test. In the simulated wheat B starch medium (YPD20Xylan20), all the strains showed almost the same growth performance during the first 8 h, this is due to the strains preferentially utilized the glucose present in the media. This was verified again in Figure 8B, all the strains showed the comparable sugar utilization capacity, the xylan utilization ability may be covered by the glucose. Therefore, to verify the strains’ xylan utilization capacity, a xylan as the sole carbon source medium was essential in further study. The sugar consumption data showed that starting from 2 hours, the WXA/pXylan-B and WXA/pXylan-BD strain was slightly higher than the WXA control, which could be interpreted as decomposing xylan and producing reducing sugars. Therefore, the engineered bacteria we constructed can decompose the xylan successfully.

Characterization by 2022 iGEM Team Suzhou_Union

https://2022.igem.wiki/suzhou-union/results

Contribution

Our team aims to improve the ability of saccharomyces cerevisiae to produce ester in saccharomyces to improve the lack of flavoring caused by single flavor saccharomyces of l. Our main content is to study a major substance: by yeast culture, mores ethyl hexanoate to enrich the spirit's flavor.

 Ethyl Hexanoate is a colorless liquid, and it has an apple smell. And it is fatty acid ethyl hexanoate. Ethyl Hexanoate is always used to brew spirit. And it can affect the taste of the spirit. Thus, the subject of our team is to use Ethyl Hexanoate to adjust the spirit's flavor.

Our project was inspired by the fact that Ethyl Caproate is one of the most important matters that directly determines the flavor of the Chinese baijiu. During the transition between Acetyl-CoA and Malonyl-CoA, the production rate rises significantly with the increase of Acetyl-CoA carboxylase (ACC1), and during the production of Fatty Acyl-CoAs, Fatty acid synthase (FAS1 FAS2) would improve the production rate as well.

To increase the quantity of Ethyl Caproate in baijiu, we need to transfer both ACC1 and Fatty acid synthase to saccharomycetes through the progress of transformation.

Fatty acid synthase (FAS), as a key enzyme for fatty acid synthesis, has rich enzyme system functions. It exists in different forms in high and low animals, and plays a great role in affecting the energy metabolism of organisms. In recent years, there are more and more research achievements on fatty acid synthase.

Because the fragrant flavor of Baijiu is produced by FAEEs, the concentration of this substance is also the standard for the quality of Baijiu, thus the output of FAEEs from S. cerevisiae is important; However, due to the single strain fermentation process of Baijiu, the production of FAEEs is not enough causing the flavor becoming insufficient. Therefore, if the FAEEs produced by yeast are increased, the flavor of Baijiu can be more abundant and fragrant.

The content of this project is to improve the FAEEs production ability of S.cerevisiae cells by constructing FAS1, FAS2 .

Figure 1. An overview of the experimental principle..

In this experiment, we will construct two plasmids, FAS1 and FAS2. Under the function of Malonyl-CoA, FAS1, and FAS2 synthesize fatty acids, fatty Acyl-CoAs produce FAEEs; Therefore, as long as we successfully construct one of the three plasmids, the production of FAEEs can be significantly increased.

Engineering Success

Firstly, we need to amplify the DNA fragments of FAS1 and FAS2 by using Polymerase Chain Reaction (PCR) technology, then put the amplified fragments into the electrophoresis system, and cut the target DNA fragments after the electrophoresis process is finished.

Secondly, the target DNA fragment was extracted with the quick Gel Extraction Kit, then combined with carrier and ligase to form the recombinant.

Thirdly, after the recombination is completed, transferred it to the competent cells of E. coli. The competent cells with the recombinant products are amplified using an oscillator. Then the amplified competent cells are coated on the solid LB medium and placed in the incubator overnight.

Fourthly, after the bacteria grow out of the culture dish, the plasmid is put into the liquid LB culture medium. In this part, the OD600 value is detected to check whether the concentration of cell expansion is in a suitable range —— about 0.6 ~ 1.0.

Fifthly, prepare the transformation solution using PEG, LiAC, and Single-stranded carrier DNA. The plasmid was transferred into the competent state of yeast cells to increase the FAEEs production capacity of yeast cells. The competent cells were amplified again in the oscillator, then coated the cell on the solid culture medium and placed in the incubator overnight.

Lastly, add the S. cerevisiae to the liquid culture medium, and use PCR to measure the BP value to see whether it is consistent with the expected BP value; If it’s consistent, it means that S. cerevisiae with plasmid can be sent to the brewery to determine the output of FAEEs.

Establish the plasmid of YPF2K-FAS1 and YPF2K-FAS2

We first try to construct the yep plasmid, the plasmid map is shown below Figure 2.

Figure 2. Establish the plasmid of YPF2K-FAS1 and YPF2K-FAS2..

After PCR amplification of the exogenous DNA, the vector and the exogenous DNA fragment were cut by restriction enzyme respectively, and the two were ligated by DNA ligase, and then transferred into the host bacteria. The recombinant clones were obtained by screening and identification. After a series of operations, the plasmid completes the task of presenting the target fragment.

Figure 3. Results of PCR amplification of FAS2 fragments..

DH10B(Yep-FAS1 and Yep-FAS2) were obtained using single-fragment recombination. The sequencing results showed that the constructed Yep-FAS1/2 contained multiple synonymous mutations, but all of them also contained individual mutation sites, which would lead to changes in the corresponding amino acids of the protein. Pick clones with fewer mutations in them (Yep-FAS1/2), The mutation site was then backmutated using site-directed mutagenesis.

Figure 4. Sequencing results for corrected mutations of Yep-FAS1 and Yep-FAS2..

After recombination is complete, it is transferred into competent cells of E. coli. Use a shaker to expand competent cells with recombination products. The expanded competent cells were then coated on solid LB medium and placed in the incubator overnight.

After the bacteria have grown out of the petri dish, place the plasmid into liquid LB medium. This part checks the OD 600 value to check if the cell expansion concentration is in the proper range - about 0.6~1.0.

Prepare transformation solutions using PEG, LiAC, and single-stranded carrier DNA. The plasmid was transferred into competent yeast cells to improve the FAEEs production capacity of yeast cells. Competent cells were re-expanded in a shaker, then cells were coated on solid medium and placed in an incubator overnight.

Finally, S. cerevisiae was added to the liquid medium, and the BP value was measured by PCR to see if it was consistent with the expected BP value; if it was consistent, it meant that S.cerevisiae with plasmids could be sent to the brewery to determine the yield of FAEE.

Fermentation Effect Assay

1. Add 1 mL of yeast (Yep, Yep-FAS1, Yep-FAS2) to 110 mL of LYPD liquid medium

In each group, 2 parallels were set, and the fermentation was carried out at 30°C and 220rpm for 60h;

2. After fermentation, centrifuge at 4000rpm at 4°C for 10min to collect the supernatant, discard the precipitate, and seal it with parafilm.

Avoid alcohol volatilization, send it to the company to measure gc ms, The fermentation time statistics are shown in the table below

Table 1. Fermentation time statistics table..

Figure 5. Fermentation effect comparison..

After the fermentation time of each group continues data processing and drawing, it can be clearly seen from the figure below that after the optimization and improvement of our experiment, through the characterization of OD600, the brewing yield is significantly higher than that of the control group(Figure 5.), indicating that our experiment has been successful and also It is hoped that our experiments can provide reference for other igem teams and provide certain guidance for subsequent industrial production.

Functional Improvement

Compared with iGEM Team 21_ Beijing_ United (Part:BBa_K3996013), which simply increased the content of ethanol. This year, in addition to ethanol production, SuZhou_Union determined the formation and consumption path of fatty acid ethyl ester in Saccharomyces cerevisiae. They constructed a high-yield yeast strain producing ethyl caproate to improve the quality of Luzhou flavor liquor, increasing the content of aroma substances for taste, and lay a foundation for subsequent quality improvement research.

https://2022.igem.wiki/suzhou-union/results

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]