Results

1. Test results of tolerance of engineered strains to ethanol and acetaldehyde

In this study, two engineering strains E.coli pSB-AA and E.coli pSB-AN were constructed to test their performance in degrading ethanol and acetaldehyde.

2. The activity of ethanol and acetaldehyde dehydrogenase and the content of coenzyme NAD+

Fig.1 Engineering strains and control strains express the activity of related enzymes. (A) The enzyme activity of alcohol dehydrogenase and acetaldehyde dehydrogenase; (B) NAD+ content in different strains; (C) The enzyme activity of NADH oxidase.

In order to reflect the expression of functional genes of engineered strains E.coli pSB-AA and E.coli pSB-AN more directly, we detected the activities of various exogenous enzymes and the content of coenzyme NAD+ in different strains cultured in vitro. Results as shown in Figure 1, E.coli 1917 showed almost no activities of alcohol dehydrogenase, acetaldehyde dehydrogenase and NADH oxidase in the crude enzyme extracts of each bacterium, and the content of coenzyme NAD+ was very low. The ADH activity of E.coli pSB-AA was 47.91±3.12 U/mL, and the ALDH activity was 33.57±2.59 U/mL. No NADH oxidase activity was detected. However, the ADH activity of E.coli PSP-AN was 81.41±3.64 U/mL, the ALDH activity was 57.56±1.48 U/mL, and the nox activity was 14.4±2.29 U/mL. The content of coenzyme NAD+ was also significantly higher than that of E.coli pSB-AA. These results indicated that the expression of NAD synthetase gene nadE and NADH oxidase gene nox could contribute to the increase of the content of dehydrogenase coenzyme NAD in bacterial cells, thus improving the degradation ability of alcohol dehydrogenase and acetaldehyde dehydrogenase.

3. Detection results of the tolerance of engineering strains to ethanol and acetaldehyde

Fig.2 The tolerance of different strains to different concentrations of ethanol. (A) The growth curve of E. coli1917; (B) The growth curve of E. coli 1917 with pSB-AA; (C) The growth curve of E. coli 1917 with pSB-AN.

Fig.3 The tolerance of different strains to different concentrations of acetaldehyde. (A) The growth curve of E. coli 1917; (B) The growth curve of E. coli 1917 with pSB-AA; (C) The growth curve of E. coli 1917 with pSB-AN.

The growth curves of engineered strains E.coli pSB-AA and E.coli pSB-AN in the medium containing different concentrations of ethanol and acetaldehyde were drawn to show the tolerance of engineered strains to ethanol and acetaldehyde. As shown in Figure 2 and Figure 3, when ethanol concentration> 6%, acetaldehyde concentration> 0.3% would seriously inhibit the growth of E.coli 1917; When ethanol concentration> 8%, acetaldehyde concentration> The growth of E.coli pSB-AA was inhibited at 0.4%, and the tolerance of E.coli pSB-AA to ethanol and acetaldehyde was improved to a certain extent. And when the concentration of ethanol> 10%, acetaldehyde concentration> At 0.5%, E.coli pSB-AN began to inhibit the growth of engineered strain E.coli pSB-AN, indicating that the tolerance of E.coli pSB-AN to ethanol and acetaldehyde was significantly improved.

4. Test results of the ability of engineering strain to degrade ethanol and acetaldehyde

Fig.4 The degradation ability of different strains to different concentrations of ethanol and acetaldehyde. (A) Ethanol content of different strains after 14 h; (B) Acetaldehyde content of different strains after 14 h.

In order to alleviate the damage caused by excessive drinking, the ability of the engineered strain to degrade ethanol and acetaldehyde is crucial. Here, we detected the content changes of ethanol or acetaldehyde in the culture medium of different strains after growing in different concentrations of ethanol and acetaldehyde for 14 h, so as to measure the degradation ability of the strain to ethanol and acetaldehyde. As shown in Figure 3.3, the degradation efficiency was the highest when the ethanol content was 2% and the acetaldehyde content was 0.1%. The degradation rates of ethanol and acetaldehyde of the engineered strain E.coli pSB-AA were 54.3% and 41.4%, respectively. The degradation rates of ethanol and acetaldehyde of E.coli pSB-AN were 61.5% and 53.5% respectively. However, with the increase of ethanol and acetaldehyde concentration, the growth and metabolism of bacteria are also inhibited, so the degradation ability of bacteria to ethanol and acetaldehyde is gradually reduced.