Proof of Concept

Overview

After the outbreak of the COVID-19 pandemic, the demand for alcoholic products such as medical alcohol, alcohol disinfection has increased rapidly. Only in 2020, the output of fermented alcohol in China was 9.24 million kiloliters, with a year-on-year increase of 33.6%. In the meantime, the export volume of fermented alcohol in China also increased in 2020. In that year, the export volume of fermented alcohol was 624.484 million liters, while the import volume was 182.146 million liters.

Faced with COVID-19, alcohol is urgently needed in China, either to satisfy the domestic demand or to enter the global market. Thereafter, our team focus on alcohol production processes. The primary goal of our project is to obtain a yeast functional in making wheat b-starch efficiently ferment and produce alcohol economically.

To be specific, in our Project, we hope to edit pXylan-B, pXylanBD, and pXylaN into yeast through experiments. At the same time, pOdd-1, pOdd-2, and pOdd-3 plasmids were constructed. The expected result is that the gene-edited yeast can decompose wheat B-type starch into monosaccharides by secreting three amylases, and the yeast will then convert the monosaccharides into high-quality alcohol through fermentation.

We consider expressing the preferred codon-optimized xylanase gene AnXlnB, β-xylosidase gene AnXlnD, and acetyl xylan esters derived from Clostridium cellulophilum on the basis of the existing pentosaccharomyces, enzyme gene CcXynA. We plan to obtain a Saccharomyces cerevisiae strain with the ability to decompose and utilize pentosan. The ability of the strain to ferment pentosan to produce alcohol in a specific medium will finally be tested.

Supporting Experiment Results

1. Aspergillus niger derived xylanase expression plasmid construction

Figure 1 pXlnB and pXylan-B plasmids map

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 1).

Figure 2 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 2A, 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 2A lane 4 and Figure 2B 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 2C) was also cut with the same enzyme, these two parts were ligated to make the final plasmid pXylan-B.

2. Aspergillus niger derived xylanase and β-xylosidase expression plasmid construction

Figure 3 pXlnD and pXylan-BD plasmids map

For the effective utilization of the xylan component present in the wheat B starch, the Aspergillus niger derived β-xylosidase (Figure 3A) was also cloned together with the xylanase expression gene. To make the final plasmid pXylan-BD (Figure 3B), the pXlnD plasmid was constructed firstly. The promoter TPI1, codon-optimized AnXlnD CDS, and CYC1 terminator PCR bands were shown in the Figure 2A, lane 5, lane6, and lane 3, respectively. The AnXlnD 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 2A lane 7 and Figure 2B lane 2, respectively. The backbone was cut with Bsa1 restriction enzyme and ligated with the AnXlnD expression cassette to make the plasmid pXlnD.

For the construction of the final plasmid pXylan-BD, the pXlnB and pXlnD were both cut with Sap1 restriction enzyme, and the backbone part (Figure 2C) was also cut with the same enzyme, these three parts were ligated to make the final plasmid pXylan-BD.

Figure 4 Positive colonies verification through the colony PCR. Lane 11 to 20, pXlnB plasmid colony PCR verification. Lane 21 to 30, pXlnD plasmid colony PCR verification.

Figure 4 demonstrated the positive colonies verification of the plasmids pXlnB and pXlnD. The number of 12 to 16, 18 to 20 were the positive colonies of the plasmid pXlnB, the number of 21, 23, 24, 27 to 30 were the positive colonies of the plasmid pXlnD. Number 12 of pXlnB and number 23 of pXlnD were sent for the sequencing.

Figure 5 Plasmids DNA sequencing. A: pXlnB plasmid. B: pXlnD plasmid.

The positive transformants verified using the colony PCR were sent for the DNA sequencing, as shown in Figure 5, both the plasmids pXlnB and pXlnD were constructed successfully.

Figure 6 Positive colonies verification through the colony PCR (A) and DNA sequencing of the plasmid pXylan-B (B) and pXylan-BD (C).

Figure 6A demonstrated the positive colonies verification of the plasmids pXylan-B and pXylan-BD. The numbers 10, 12, and 14 were the positive colonies of the plasmid pXylan-B, the numberss 2, 4, and 6 were the positive colonies of the plasmid pXylan-BD. Number 12 of pXylan-B and number 4 of pXylan-BD were sent for the sequencing. Figures 6B and C showed that both the pXylan-B and pXylan-BD plasmids were constructed successfully.

3. The xylan utilization Saccharomyces cerevisiae strain construction and fermentation test

Figure 7 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 used in 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 7B, 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 experiment results provided us with supporting evidence that our gene-edited strain may be functional in making wheat b-starch efficiently ferment and produce alcohol. However, further study should be made in the future to consolidate the foundation for future possible application.