By overexpression of phzM (methyltransferase encoding gene), nadE (NAD synthase gene), rhlA (rhamnosyl transferase gene) in P .aeruginosa PAO1, our project gives an probability of enhancement for bioelectricity generation which is one of the major bottlenecks in practical applications of MFCs.
1 Construction and identification of expression plasmid
1.1 Construction of overexpressed phzM, nadE, rhlA in P. aeruginosa PAO1
We choose shuttle vector pBBR1MCS-5 which used in Pseudomonas aeruginosa PAO1 as expression vector. Then we connect gene phzM, nadE, rhlA respectively to pBBR1MCS-5 by PCR and restriction enzyme digestion. Afterwards, we transform the recombinant plasmid pBBR1MCS-5-phzM, pBBR1MCS-5-nadE, pBBR1MCS-5-rhlA respectively into Pseudomonas aeruginosa PAO1 and obtain the recombination strain PAO1-phzM, PAO1- nadE, PAO1- rhlA．
1.2 Identification of overexpressed phzM, nadE, rhlA vector in P. aeruginosa PAO1
The recombinant expression vector pBBR1MCS-5-phzM, pBBR1MCS-5-nadE, pBBR1MCS-5-rhlA was transformed into the competent state of Pseudomonas aeruginosa respectively, Then, select the positive transformants to expanded culture and extract the recombinant plasmid. Identify the recombinant plasmid by restriction endonuclease digestion. Show as figure.1-1, figure.1-2, figure.1-3.
Figure.1-1 Identification of pBBR1MCS-5-phzM by restriction endonuclease digestion
Marker: DL5000 ; Lane 2: fragments digested by Eco R I and Spe I
Shown as Figure.1-1, there is a band at about 4678 bp and 1000 bp in lane 1 respectively. Combined with the size of empty plasmid pBBR1MCS-5 and gene phzM, it suggests that the two bands are empty plasmid and phzM gene, which ensure the successful recombinant plasmid construction.
Figure.1-2Identification of pBBR1MCS-5-nadE by restriction endonuclease digestion
Marker:DL15000; Lane 1: pBBR1MCS-5- nadE plasmid; 2-5: fragments digested by BsiW I and Kpn I
Shown as Figure.1-2, there is a band at about 4.8kb and 780 bp respectively. Combined with the size of empty plasmid pBBR1MCS-5 and gene nadE, it suggests that these bands are empty plasmid and nadE gene, which ensure the successful recombinant plasmid construction.
Figure.1-3 Identification of pBBR1MCS-5- rhlA by restriction endonuclease digestion
Marker: DL 5000; Lane 1: fragments digested by Kpn I and EcoR I
Shown as Figure.1-3, there is a band at about 5000 bp and 1000 bp in lane 1 respectively. Combined with the size of empty plasmid pBBR1MCS-5 and gene rhlA, it suggests that the two bands are empty plasmid and rhlA gene, which ensure the successful recombinant plasmid construction.
2 Methyltransferase activity assay
2.1 Standard curve for pyocyanin
Prepare standard solutions (0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.08, 0.1 μ mol/mL), and the absorbance was measured at 313 nm. And the enzyme activity (E) of methyltransferase: E = (∆A_313+0.089)/(∆t×0.000578) . (∆t (min) for absorbance changing time, E for the methyltransferase activity (U/mL) contained in each mL of supernatant).
2.2 Determination of Methyltransferase activity
According to the standard curve for pyocyanin E = (∆A_313+0.089)/(∆t×0.000578), and the specific enzyme activity: SE = E/m . (E for the methyltransferase activity (U/mL) contained in each mL of supernatant, SE for the specific enzyme activity of the methyltransferase (U/mg)), the results of enzymatic activities analysis is shown as followed figure.2-1. It can be seen that methyltransferase was highly expressed in engineered cells, which is 2.8~3.04 times higher than the control group.
| strain | Enzyme activity (U/mL) | Total protein concentration (mg/mL) | Specific enzyme activity (U/mg) |
| PAO1 | 33.58 | 21.38 | 1.57 |
| PAO1-nadE | 83.65 | 17.55 | 4.77 |
Figure.2-1 Methyltransferase activity analysis expressed in P. aeruginosa PAO1
3 NAD synthase activity assay
3.1 Standard curve for NADH
Prepare different concentrations of NADH standard solution (0.3~1 mM), and the absorbance was determined at 340 nm at different concentrations. Then the standard curve shown as y = 2.514x + 0.064 (R2=0.994), y for the absorbance of the sample, and x for the concentration of NADH in the corresponding sample (mM).
3.2 Determination of NAD synthase activity
According to the standard curve for NADH： y = 2.514x + 0.064, and SE = (NADH(μmol))/(V·c·∆t) ，SE for the specific enzyme activity of the NAD synthase (U/g)，V for the volume of the enzyme solution(ml),c for the concentration of NADH in the corresponding sample (mM) , t (min) for absorbance changing time. The results of enzymatic activities analysis is shown as followed figure.3-1. It can be seen that NAD synthase was highly expressed in engineered P. aeruginosa PAO1, with 2~3 times higher than that of the recipient bacteria.
| strain | NADH (mM) | Total protein concentration (mg/mL) | Specific enzyme activity (U/g) |
| PAO1 | 0.113 | 66.760 | 2.738 |
| PAO1-nadE | 0.228 | 47.135 | 5.372 |
Figure.3-1 NAD synthase activity analysis expressed in P. aeruginosa PAO1
4 NAD+/ NADH levels Assay
Furthermore, we tested the efficiency of nadE by using the NAD+/ NADH levels Assay.
4.1 Standard curve for NAD+ and NADH
The reaction system was composed of 40 μ L 1.0 mol/L Bicine buffer (pH 8.0), 15 μ L ethanol, 4.2 mmol/L MTT 56 μ L and 16.6 mmol/L PES 40 μ L;, followed by adding 10 μ L standard concentration sample, 151 μ L mixed reaction solution and 1.2 μ L ethanol dehydrogenase (500 U/mL).Mixing well, and the absorbance of different reaction time at 570 nm were measured via enzyme labeling instrument.
Thus:
Standard curve for NAD+ is Y = 2.595X -0.070(R2=0.995,Y for the absorbance at 570 nm, X for the concentration of NAD+ );
Standard curve for NADH isY=2.2150X-0.0002(R2=0.999,Y for the absorbance at 570 nm, X for the concentration of NADH)
4.2 Determination of NAD+ and NADH in PAO1 and PAO1-nadE
As shown in figure.4-1, The amount of recombinant strain NADH and NAD+ overexpressing NAD synthase was 1.38 times and 1.58 times higher than that of Pseudomonas aeruginosa PAO1 respectively, while the overall NAD (H) level was 1.51 times higher than that of the original strain PAO1, while the ratio of NADH/NAD+ was changed from 0.52 to 0.44. These results suggest that the overexpression of NAD synthase gene nadE in Pseudomonas aeruginosa causes the change of NAD+/NADH level, and the increase of NADH as an electronic carrier will directly lead to the increase of electrons that can be released in the cell, which is beneficial to the enhancement of the electricity production capacity of Pseudomonas aeruginosa.
| strain | Concentation of NADH (mg/mL) | Concentration of NAD+ (mg/mL) | The total concentration of NAD(H)(U/mg) |
| PAO1 | 0.016 | 0.031 | 0.047 |
| PAO1-nadE | 0.022 | 0.049 | 0.071 |
Figure.4-1 The concentration of NAD+ and NADH in Pseudomonas aeruginosa
5 Rhamnose Assay
There are hydrophilic group (rhamnose) and hydrophobic group (fatty acid chain) in molecular structure of rhamnolipid contains respectively, and the length of fatty acid chain in different rhamnolipid structure is variable. Thus, we first analyze the content of rhamnose in rhamnolipid hydrolysate by Anthrone -sulfuric acid, and then calculate the content of rhamnolipid.
5.1 Standard curve for Rhamnose
Prepare different concentrations of rhamnose standard solution, and the absorbance was determined at 620 nm at different concentrations. Then the standard curve shown as y = 0.0065 x + 0.0081 (R2=0.9993), y for the absorbance of the sample, and x for the concentration of rhamnose in the corresponding sample(mg/L).
5.2 Determination of rhamnose
According to the standard curve, the results of enzymatic activities analysis is shown as followed figure.5-1. It can be seen that rhamnose was 44.21% higher in PAO1-rhlA than that of the recipient bacteria. This result ensure us that the rhlA gene successfully expressed in the recipient bacteria.
| strain | rhamnose concentration (mg/L) |
| PAO1 | 409.80 |
| P. aeruginosa PAO1 | 590.96 |
Figure.5-1 Rhamnose analysis in overexpression rhlA in P. aeruginosa PAO1
6 HPLC analysis of pyocyanin in P. aeruginosa PAO1 and P. aeruginosa PAO1-rhlA
Then, High-performance liquid chromatography (HPLC) was performed to to analysis the pyocyanin which indicates the rhamnosyl transferase activity.
6.1 Standard curve of pyocyanin
To accurately determine the secretion of pyocyanin from P. aeruginosa PAO1-rhlA and P. aeruginosa PAO1, we first determine the standard curve of pyocyanin. Prepare different concentrations of pyocyanin standard solution(0.5，1，2，5，10，20，25，50，100 μg/mL),and set corresponding parameters, 20 μL injection in turn. Then the standard curve shown as Y = 1.81914 * X + 0.12777 (R2 = 0.9999), Y for peak area(mV) and X for concentrations of pyocyanin standard solution(μg/mL).
6.2 Accurate determination of pyocyanin
P. aeruginosa PAO1 and P. aeruginosa PAO1-rhlA were cultured and sampled at 12 h and 16 h respectively, centrifuged and extracted with chloroform. Dry the lower chloroform phase and dissolve it with acetonitrile under ultrasonic for 5 min. The supernatant was centrifuged and analyzed by HPLC of an injection for 20 μL. Shown as figure.6-1, The tR of standard pyocyanin is about 9 min, while peak of the samples of PAO1 and PAO1-rhlA appeared at about 9 min, suggesting the corresponding substance is pyocyanin. The peak areas of pyocyanin in PAO1 and PAO1-rhlA were 834404 and 1556171 at 12 h and 1183672 and 2797283 at 16 h, respectively.
Figure.6-1 The HPLC map of pyocyanin in P .aeruginosa PAO1 and P .aeruginosa PAO1-rhlA
According to the standard curve, we get the concentration of pyocyanin from PAO1 and PAO1-rhlA. Shown as figure.6-2, the pyocyanin content of PAO1 was 0.926 mg/L, 1.257 mg/L respectively after 12 h and 16 h culture, while that of PAO1-rhlA was 1.746 mg/L,2.995 mg/L, from which we found that the pyocyanin content of PAO1-rhlA at 12 h and 16 h was 1.89 times and 2.38 times higher than that of PAO1. It is suggested that the engineered P .aeruginosa PAO1-rhlA significantly promotes the release of pyocyanin, indicating the higher rhamnosyl transferase activity in P .aeruginosa PAO1-rhlA.
Figure.6-2 Pyocyanin concentration of culture medium of PAO1 and PAO1-rhlA at 12 h, 16h
