Mutant transporters for bacterial uptake of terephthalic acid

Inventors

Johnson, Christopher W. • BECKHAM, Gregg Tyler • PARDO MENDOZA, Isabel • NEIDLE, Ellen Lee

Assignees

University of Georgia Research Foundation Inc • Alliance for Energy Innovation LLC

Abstract

The present disclosure relates to a non-naturally occurring microorganism that includes a gene encoding a MucK transporter protein, where the microorganism is capable of catabolizing terephthalic acid (TPA). In some embodiments of the present disclosure, the gene encoding the MucK transporter protein may contain at least one mutation, relative to a reference gene encoding a reference MucK transporter protein.

Core Innovation

The disclosure relates to engineering and evolutionary improvement of non-naturally occurring microorganisms for catabolizing terephthalic acid (TPA). The microorganism is described as capable of catabolizing TPA by using a gene encoding a MucK transporter protein and introducing genetic changes affecting regulation of TPA transport and catabolism.

The disclosure identifies mutated MucK transporter variants as responsible for improved TPA transport. In this context, the transcriptional regulator DcaS is described as repressing mucK, and deletion of an endogenous gene encoding this transcriptional regulator or introduction of specific mucK point mutations is described as increasing TPA uptake and related phenotypes.

The disclosure further describes evaluating TPA transport and performance using a transcription-factor-based TPA fluorescent biosensor (TphR/sfGFP) and using adaptive laboratory evolution with chromosomal gene amplification of tph catabolic genes. Evolved microorganisms are reported to show improved growth and TPA consumption rates, including use cases in both native and engineered genetic backgrounds, and the disclosure notes that tph-TTT heterologous transport has minimal role while evolved strains may rely on endogenous MucK/DcaS regulation.

Claims Coverage

The independent claim covers an engineered non-naturally occurring microorganism having a gene encoding a MucK transporter protein together with deletion of an endogenous gene encoding a transcriptional regulator, where the microorganism is capable of catabolizing terephthalic acid (TPA).

Across the dependent claim refinements provided, the coverage emphasizes modifying the MucK transporter gene (including at least one mutation relative to a reference MucK and point-mutation type constraints), specifying reference-sequence similarity, and achieving measurable TPA catabolic performance as characterized by TPA consumption rate constraints, with embodiments including specific bacteria such as Acinetobacter baylyi.

Stated Advantages

Documented Applications