Difference between revisions of "Part:BBa K5165002"

Revision as of 03:57, 2 October 2024

PHB synthesis and depolymerization to BHB

Polyhydroxybutyrate (PHB) can be depolymerized to form the high-value product beta-hydroxybutyrate (BHB). This construct accomplishes BHB formation through two steps in E. coli: 1) PHB synthesis 2) PHB depolymerization The construct is designed so that the host cell prioritizes the production of the enzymes involved in PHB synthesis. Quorum sensing mechanisms, regulated by the lux system, are responsible for transitioning the cell into stage two for PHB depolymerization. Such a two-part system allows for optimized production in each step separately, which is essential for BHB synthesis as the initiation of step two depends on the products of step one.

The first part is regulated by the repressible promoter pTet. PHB synthesis is driven by three enzymes that are characterized in the part BBa_K934001: phaC, phaA, and phaB. The phaCAB enzymes, accompanied by the strong RBS BBa_B0034, constitute the main component of part one. BBa_K2260002 codes for a protein tagging the PHB for secretion, and BBa_K4998027 codes for LuxI, an Acyl-Homoserine Lactone (AHL) synthase enzyme. LuxR, working in tandem with AHL to regulate quorum sensing, is constitutively expressed in the construct. As time progresses, the concentration of both phaCAB enzymes and AHL-luxR complexes increase.The AHL-luxR complexes in turn induce the downstream lux promoter pLux, initiating the start of stage two.

In stage two, the pLux promoter regulates the synthesis of the PHB depolymerase, encoded in the part BBa_K5165000, and the TetR repressor. The TetR repressor inhibits the upstream pTet promoter, ceasing the production of stage one proteins, focusing the cellular processes on depolymerizing the PHB molecules produced from stage one.

Sequence and Features