Part:BBa_K4765118

__NOtinfo>BBa_K4765118 short</partinfo>

Introduction

Mycosporine-like amino acids (MAAs), act as a sunscreen for the biofilm. This composite part contains five enyzmes in the MAA biosynthesis pathway. All the enzymes are constructed into ribozyme-assisted polycistronic co-expression system：pRAP.

Figure 1. The biosynthetic pathway of shinorine, porphyra-334, palythine-Ser, and palythine-Thr

Usage and Biology

Biosynthetic route of MAA initiates with the generation of 4-deoxygadusol (4-DG) from sedoheptulose 7-phosphate, an intermediate within the pentose phosphate pathway. This process is catalyzed by two enzymes: a dimethyl 4-degadusol synthase (DDGS; MysA) and an Omethyltrans-ferase (O-MT; MysB). Subsequently, 4-DG undergoes a transformation into mycosporine-glycine(MG) through an ATP-grasp enzyme MysC, which introduces an amino acid moiety, primarily L-Gly. MAA analogues such as shinorine or porphyra-334 are further derived from MG by the D-Ala-D-Ala ligase-like enzyme MysD. In the final step, the biosynthesis is completed with a nonheme iron-(II)- and 2oxoglutarate-dependent (Fe/2OG) oxygenase MysH, leading to the production of palythines.

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures. From right lane(4) to left lane(1) indicate the successful construction of MysD, MysDH, MysDHB, and MysDHBA.

Anti-UV Survival Assay

We employed the Colony-Forming Unit (CFU) assay. After plasmid transformation and plating, we shielded one/half of the agar plate from UV light using a black paperboard, while the other one/half was exposed to UV irradiation (6W power) with wavelengths of 254 nm and 365 nm for 10 seconds.

Figure 2. Anti-UV Assay.

Our experiments demonstrated that introducing three or four of the five enzymes from the MAA biosynthetic pathway into E. coli did not yield a significant enhancement in the bacterium's UV resistance. We postulate that this lack of effect may arise from two factors: Firstly, the synthetic pathway may not play a pivotal role amidst the numerous routes involved in MAA synthesis. Secondly, to augment MAA levels within E. coli through protein expression in the pathway, additional substrates like ATP and amino acids would likely be required in the reaction.

Figure 3. Plates displaying transformed E. coli after anti-UV assay.

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 1272
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1064
  Illegal BsaI.rc site found at 2666
  Illegal BsaI.rc site found at 3785
  Illegal BsaI.rc site found at 5714