Part:BBa_K1031620

NahF-Terminator

NahF-TT

NahF is a 50.8 KDa protein functioning as salicylaldehyde dehydrogenase to transform salicylaldehyde into salicylic acid (salicylate) using NAD+ (Fig. 7). It is encoded in the naphthalene degradation plasmid from Pseudomonas putida, in which the bacterial oxidation of naphthalene has been extensively investigated. Plasmid pDTG1, NAH7 and pND6-1 identified in different P. putida strains all act to degrade naphthalene and share high identity in amino acid sequences.

Fig. 1 Biochemical reaction catalyzed by enzyme NahF: Salicylaldehyde is transformed into salicylic acid (salicylate) accompanied by the reduction of NAD+ to NADH.

NahF from plasmid NAH7 is mostly widely studied. It has a wide range of substrates, including salicylaldehyde, 5-chloro-salicylaldehyde, 3-nitro-benzaldehyde, 2-methoxy-benzaldehyde etc. and can be activated to 140.3% enzyme activity in the presence of Fe2+ [12]. The wide scope of substrates makes it a commendable candidate to be an Adaptor since many aldehydes can be transformed to the corresponding acids that are detected by NahR biosensor (for salicylates) or biosensor XylS (for benzoates).

NahF has been expressed in E. coli and its ability to catalyze the reaction in vitro and in vivo has been proved [13-14]. However, the reaction efficiency of E. coli was only about 3% of that of P. putida possibly due to the difference of expression regulation in these two bacteria [13]. Therefore, it is necessary to fine-tune the expression level of NahF in E. coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 845
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1083
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 981
  Illegal BsaI.rc site found at 1135
  

We built a library of constitutive promoters for tuning the expression of NahF, and NahR biosensor was used to detect the possible salicylates transformed from salicylaldehydes (Fig. 2). Pc Library:

105-NahF-TT[[1]]

106-NahF-TT[[2]]

113-NahF-TT[[3]]

114-NahF-TT[[4]]

117-NahF-TT[[5]]

Fig. 2 Schematic diagrams for the plasmid circuits used as Adaptor: NahF and the Sensor: NahR. A constitutive promoter library for the expression of NahF was constructed to obtain the most appropriate expression level of NahF enzyme in E. coli. The number of the Standard Biological constitutive promoter Parts used in this study and its initiation strength is listed in the left portion of the figure. Promoters are presented in orange, RBS in light green, coding sequence in dark cyan, and terminators in dark red.

The performance of NahF Adaptor was tested. Bacteria carrying NahF enzyme was overnight-cultured in LB containing chloromycetin at 37℃ and then diluted 100 fold into Minimal M9 medium added chloromycetin, growing for 12 hours at 30℃ to transform salicylaldehyde into salicylate. After the Minimal M9 medium was centrifuged, supernatant medium was taken out to further culture NahR biosensor. Induction ratio was then obtained following test protocol 1.

In summary, Adaptors XylC and NahF functioned not only to optimize the response to several originally detectable compounds, but also expand detection profile in a new way besides modification on coding sequence such as mutative selection or DNA shuffling. The advantages of the new concept that using Adaptor to expand detection profile lie in that adding enzymes does not influence the original characteristics of the biosensors they adapt to, and this methodology does not require labor-intensive mutant screening work.

Reference:

[1] Baoli Cai et. al, (2004) Complete nucleotide sequence and organization of the naphthalene catabolic plasmid pND6-1 from Pseudomonas sp. strain ND6, GENE, 336:231–240.

[2] Zhao H, Li Y, Chen W, Cai B. (2007) A novel salicylaldehyde dehydrogenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6. Chin Sci Bull. 52(14):1942—8.

[3] M. A. Schell, (1983) Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7. J. Bacteriol. 153(2):822-829.

[4] R. W. Eaton and P. J. Chapman, (1992) Bacterial metabolism of naphthalene_construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions. J. Bacteriol. 174(23):7542-7554.