Part:BBa_K3776001

DavB

DavB, L-Lysine monooxygenase, catalyzes the reaction of L-lysine to 5-aminovaleramide in the natural 5-aminovalerate pathway in Pseudomonas putida KT2440. This DavB sequence has been codon optimized for expression in Synechococcus elongatus UTEX 2973.

References

Liu, P., Zhang, H., Lv, M., Hu, M., Li, Z., Gao, C., Xu, P., & Ma, C. (2014). Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase. Scientific Reports, 4(1). https://doi.org/10.1038/srep05657

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 239
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1328
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1488
  Illegal AgeI site found at 895
  Illegal AgeI site found at 1144
  
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution From NJTech-China-B 2023

Group:iGEM NJTech-China-B

Author: Yao Yao

Characterization from iGEM23-NJTech-China-B

DavB

5-adminovalerate is produced from L-lysine through the 5-aminovalerate pathway. L-lysine monooxygenase (DavB) plays a key role in the 5-aminivalerate pathway of various microorganisms. In our experiment, DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide.

1. Construct design

1.1 The transformation of palsmid pRSFDuet-DavB into E. coli BL21(DE3)

The gene of DavB was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavB. By sequencing, the correct plasmid pRSFDuet-DavB was then transformed to E. coli BL21(DE3) (Figure 1).

Figure1The transformation of palsmid pRSFDuet-DavB into E. coli BL21(DE3)

1.2 The colony PCR of pRSFDuet-DavB in E. coli BL21(DE3)

The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was between 1000 bp and 2000 bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-DavB was successful obtained (Figure 2).

Figure2. Colony PCR results of recombinant strain that containig the plasmid pRSFDuet-DavB.

M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containg plasmid pRSFDuet-DavB

2. Protein expression of DavB

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. We transformed the plasmid pRSFDuet without DavB gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavB protein was correct, which was consistent with the expected molecular weight of 62.4 kDa (Figure 3).

Figure3. SDS-PAGE analysis of DavB expression in E. coli BL21(DE3)

Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containg pRSFDuet; lanes 3 and 4：supernatant

and precipitation of E. coli BL21(DE3) containg PRSFDuet-DavB

3. Determination of DavB activity

3.1 Determination of DavB whole-cells catalytic activity

DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide. The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB was inoculated and cultured in the LB medium at 37 ℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. Subsequently, we tested its activity by detecting L-lysine consumption with the whole-cells containing DavB enzyme. The results showed that whole-cells containing DavB enzyme can consume L-lysine, indicating that whole-cells containing DavB have active enzyme activity (Figure 4).

Figure4 The enzymatic activity of whole-cells

3.2 Detemination of crude enzyme of DavB

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. Subsequently, we tested its activity by detecting L-lysine consumption with the crude enzyme of DavB. The results showed that crude enzyme of DavB can consume L-lysine, indicating that crude enzyme of DavB has active enzyme activity (Figure 5).

Figure5 The enzymatic activity of crude enzyme