Part:BBa_K5165002
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.
The results indicate a high level of BHB in the extracellular fluid of cells containing Plasmid 1 (self-sustaining system) indicating endogenously produced AHL was upregulating the expression of PHAZ_TALFU in comparison to trace amounts found the extracellular space of cells transformed with plasmid 2. Using the standard curve of BHB concentration (y = 0.4651x - 00221) we determined the average concentration of BHB in the extracellular space of cells transformed with plasmid 1 to be 1.347 umol/mL compared to just 0.138 umol/mL in the extracellular space of cells transformed with plasmid 2 (which needed to be induced for expression).
The concentration of BHB was determined using a standard curve for BHB as seen below.(y = 0.4651x - 00221)
The image below qualitative data from β-Hydroxybutyrate colorimetric assay. Tube 1 contains kit reagents + a known quantity of standard solution (2 umol/mL). Tubes 2, 3, & 4 contains kit reagents + supernatant of cell environment for BL21 cells transformed with plasmid 1, plasmid 2, and wild type (untransformed cells) respectively.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 998
Illegal BglII site found at 1823
Illegal BglII site found at 5410
Illegal BamHI site found at 6606 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 304
Illegal NgoMIV site found at 375
Illegal NgoMIV site found at 975
Illegal NgoMIV site found at 1287
Illegal NgoMIV site found at 1566
Illegal NgoMIV site found at 2218
Illegal NgoMIV site found at 2240 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4084
Illegal BsaI site found at 5220
Illegal BsaI site found at 6571
Illegal BsaI.rc site found at 5851
Illegal BsaI.rc site found at 5907
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