Results

1. Plasmids construction

As indicated in Figure 1, the synthesis principle of ethyl caproate is that acetyl coenzyme A is generated from acetic acid, and malonyl coenzyme A is generated under the action of ACC1, and then converted into fatty acid, fatty alcohol and ethyl caproate under the action of FAS1 and FAS2. Therefore, the resultant quantity of ethyl caproate can be increased by ACC1, FAS1 and FAS2.

Fig.1 Synthesis principle of ethyl caproate

Firstly, we construct three plasmids, which are YPA1k-ACC1, YPA1k-FAS1 and YPA2k-FAS2. PCR is the abbreviation of polymerase chain reaction, which is a method of enzymatic synthesis of specific DNA fragments in vitro. PCR is an examination of DNA or RNA of various pathogens such as bacteria, viruses and fungi by using in vitro amplification technology. Therefore, the gene fragments of ACC1 (cut into ACC1-1 ACC1-2 because ACC1 is too long) and FAS1 and FAS2 can be replicated and amplified by PCR and extended until it can be observed. The results (Figure 2) indicated that FAS1, FAS2, ACC1-a and ACC1-b DNA strands are correctly replicated.

Figure 2 FAS1, FAS2, ACC1-a and ACC1-b DNA strands are correctly replicated
Identification of FAS1, FAS2, ACC1-a and ACC1-b DNA strands by agarose gel electrophoresis.

Secondly, we used double enzyme digestion to construct YPA1k-ACC1, YPA1k-FAS1 and YPA2k-FAS2 plasmids. Then, the constructed YPA1K-ACC1, YPA1K-FAS1 and YPA2k-FAS2 plasmids were added to the competent cells and mixed well. After 30min ice bath, the plasmid was subjected to 42℃ heat shock for 45s. 1 mL of antibiotic free LB medium was added to the EP tube of the mixture of plasmid and competent cells and mixed well. After shaking for 1 h at 600 rpm at 37°C, 100 mL of bacterial solution was coated on the solid medium containing ampicillin or kanamycin, and inverted in the 37°C bacterial incubator for 12 h, Finally, the monoclonal colonies shown in the figure were successfully obtained.

Figure 3 FAS1, FAS2, ACC1-a and ACC1-b DNA strands are correctly replicated

The constructed plasmids are added to the transformation reagent and prepared yeast competent cells. The mix is then applied onto the YBP culture plates and left under 37℃ overnight to ensure colony growth.

Thirdly, PCR was used to verify the monoclonal colony of the strain. As indicated in Figure 4, the plasmids were successfully constructed.

Figure 4 YPA1k-FAS1, YPA2k-FAS2 and YPA1k-ACC1 plasmids are correctly replicated
Identification of YPA1k-FAS1, YPA2k-FAS2 and YPA1k-ACC1 plasmids by agarose gel electrophoresis.

Fourthly, gene sequencing is used to double verification. As indicated in Figure 5, YPA1k-FAS1 and YPA2k-FAS2 sequences were correct.

Figure 5 Sequencing of YPA1k-FAS1 and YPA2k-FAS2 plasmids
Identification of YPA1k-FAS1 and YPA2k-FAS2 by gene sequencing.

2. Strain functional test

To ensure the growth of the cells in the culture medium, we determined the optical density at 600nm (OD₆₀₀) (Table 1).

After guaranteeing the growth in cells, the culture medium is sent to a professional company to detect the concentration of FAEEs. Results show that the concentration also significantly increased compared with the control group.

Figure 6 Fermentation growth curve
Concentration values at different time points.

We collected samples that have been fermented for 60 hours and send them to the company to measure the content of ethyl hexanoate. We first used conventional gas chromatography (GC) methods to detect ethyl hexanoate in the sample, and found that the peaks of the ester and alcohol crossed and could not be completely separated or the content of ethyl hexanoate could not be detected. Subsequently, through interviews with experts and research literature, we learned that alcohols and esters can be separated using the gas chromatography-mass spectrometry (GC-MS) method. So we re-fermented, collected 60 hours of fermentation samples, and sent them to the company to measure GC-MS.

Figure 7. The ethyl caproate peak of YEP-FAS1 detectedby GC-MS.

Figure 8. Partial enlarged view of ethyl caproate peak of YEP-FAS1 detected by GC-MS.

According to the results of detection of ethyl caproate content(fig.9), compared with the control group, YEP-FAS1 and YEP-FAS2 engineering bacteria can produce more ethyl caproate, and the fermentation content of YEP-FAS1 engineering bacteria is significantly higher. From the peak graph of ethyl caproate, our engineering bacteria really ferment to produce ethyl caproate. This is also a supplement to the wiki results. Our experiment can provide reference for other iGEM teams, and provide some guidance for subsequent industrial production, which verifies the engineering success.

Figure 9. Relative value of ethyl caproate fermented of three strains by GC-MS detection.