Part:BBa_K4083007

rhlA gene with SalI and SacI sites at ends

rhlA gene codes for RhlA which is 3-(3-hydroxydecanoyloxy) decanoate synthase in Pseudomonas aeruginosa.

Usage and Biology

The P. aeruginosa is gram-negative bacillus, and opportunstic pathogen. It secretes rhamnolipids - the rhamnose containing glycolipid biosurfactant. This biosurfactants are used by P. aeruginosa to emulsify the oil sunstances for easy digestion. Thus, rhamnolipids can increase the availability of fats wchich can be important in many different areas like petrleum, bioremediation, cosmetics, food, agroculture, etc. [1] However, due to the toxicity and infectiousness of P. aeruginosa, other alternative organisms are tested. Currently, genetically engineered Pseudomonas putida has more proising results than others. P. putida only lacks two enzymes for mono-rhamnolipid production: RhlA and RhlB. These enzymes are encoded by rhlA and rhlB coding regions in rhlAB operon. It was previously thought that rhlA and rhlB forms heterodimer, however, further research showed that they act independently from each other [2].

Our team planned to extract rhL and rhlB genes from P.aeruginosa and to insert them into pRGPDuo2 plasmid obtained from Gauttam, R. [2] We developed the new approach to increase the P. putida's rhamnolpid synthesis by adding nadE gene which encodes NAD synthetase. This way, we hoped to see more rhamnolipid production in engineered P. putida.

The RhlA catalyzes the production of 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) from β-hydroxydecanoyl-ACP. HAA is the precursor for rhanolipid production which is catalyzed by RhlB to form mono-rhamnolipids [1].

Figure 1. RhlA and RhlB metabolic pathway

Reference

[1] Chong, H., & Li, Q. (2017, August 5). Microbial production of rhamnolipids: opportunities, challenges and strategies. Microbial Cell Factories. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0753-2

[2] Wittgens, A., Kovacic, F., Müller, M. M., Gerlitzki, M., Santiago-Schübel, B., Hofmann, D., Tiso, T., Blank, L. M., Henkel, M., Hausmann, R., Syldatk, C., Wilhelm, S., & Rosenau, F. (2016). Novel insights into biosynthesis and uptake of rhamnolipids and their precursors. Applied Microbiology and Biotechnology, 101(7), 2865–2878. https://doi.org/10.1007/s00253-016-8041-3

[3] Gauttam, R., Mukhopadhyay, A., & Singer, S. W. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110. https://doi.org/10.1016/j.plasmid.2020.102514

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 94
  Illegal BamHI site found at 654
  Illegal XhoI site found at 830
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]