Part:BBa_K5175011

rhlA

RhlA encoded by the rhlA gene is a phthalyltransferase responsible for the synthesis of β-hydroxy fatty acids

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 720
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 720
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 69
  Illegal BamHI site found at 629
  Illegal XhoI site found at 805
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 720
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 720
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Rhamnolipid is a biosurface activator first isolated from Pseudomonas aeruginosa by Jarvis and Johnson, Rhamnolipid emulsifies, disperses, and solubilizes hydrophobic organic pollutants and improves their bioavailability, facilitating the removal of hydrophobic organic pollutants. Rhamnolipids also have some metal chelating ability and are used to remove heavy metals from soil, sewage and other liquids. In agricultural applications, rhamnolipids can be used to improve soil, enhance the effects of pesticides and fertilizers, and inhibit agricultural diseases.

Rhamnosyltransferase I is a key enzyme necessary for the synthesis of rhamnolipids, which is a complex enzyme containing 2 subunits, RhlA and RhlB, encoded by the rhlAB gene on the same manoeuvre rhlABRI. Among them, RhlA encoded by the rhlA gene is a phthalyltransferase responsible for the synthesis of β-hydroxy fatty acids, whereas RhlB encoded by the rhlB gene is responsible for catalyzing the synthesis of mono-rhamnolipids containing one rhamnose group from dTDP-L-rhamnose and β-hydroxy fatty acids.

Fig 1. Engineered rhamnolipid synthesis pathway in P. putida

Molecular cloning

Initially, we transformed the company-synthesized plasmids containing designed sequences into E. coli DH5α for amplification, allowing us to obtain a sufficient quantity of plasmid DNA for subsequent experiments. Following this, colony PCR was performed to confirm successful transformation, and the required plasmids were subsequently extracted for further experimentation.Subsequently, we employed PCR to obtain the target fragments, which were then integrated into the requisite plasmids for our study.

We constructed three plasmids for P. putida KT2440: pTerephthalate-A, pTerephthalate-B, and pRhamnolipid. We verified the size of each plasmid as well as all the fragments involved in constructing the plasmids .

Fig.2 The schematic diagram of pRhlmnolipid

Unfortunately, due to time constraints, we were unable to successfully construct the complete plasmids. Consequently, we opted to construct T7-rhlA-rhlB-T7 and T7-phaZ-T7 as alternative plasmids for individual validation of their functionalities.

Fig.3 The bands of rhlA-rhlB（2000+ bp）from PCR

The bands of rhlA-rhlB（2000+ bp）from PCR are identical to the theoretical lengths of 2452bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.