Synbio engineering of Haloferax mediterranei: creating a next generation haloarchaeal cell-factory for polyhydroxyalkanoate synthesis

Given the negative environmental impact of the production and use of petroleum-based plastics, focus is shifting towards more sustainable alternatives, such as microbial production of biodegradable plastics with as a promising example polyhydroxyalkanoates (PHAs). Despite this promise, commercialization of PHA production is still limited due to low yields and high production costs. The observation of natural PHA biosynthesis in the halophilic archaeon Haloferax mediterranei, which grows optimally at a salt concentration of 20-25% (w/v), is not only very interesting because of the advantages of using a halophile in a PHA bioproduction process, but also because of the evidence that the copolymeric composition of the PHA produced by H. mediterranei is genetically tuneable via paralogs of copolymer-supplying enzymes (PhaC). However, a synthetic biology approach has never been used in Haloarchaea to exploit this.

In this work, I aim to engineer an optimized PHA copolymer composition in H. mediterranei by studying the regulation of phaC expression. I strategically target a putative ‘moonlighting’ regulator, PpsL, which is predicted to be a transcription regulator involved in the regulation of the phaC paralogous genes and shows homology to phophoenolpyruvate synthetase enzymes. Heterologous overexpression of PpsL of H. mediterranei has been attempted in H. volcanii, using the tryptophane inducible system, and will be followed by a detailed characterization of its DNA-binding properties using electrophoretic mobility shift assays and a genome-wide chromatin immunoprecipitation to map its genome occupancy. To go further in engineering the PpsL regulatory pathway for optimizing PHA copolymer composition in H. mediterranei, its genetic toolbox needs to be extended. To this end, the construction of a pyrE knock-out mutant in H. mediterranei is ongoing. This strain will be then used to generate a ppsL deficient strain as well as novel genetic circuits containing phaC paralogues under the control of (an engineered) ppsL promoter. In a broader sense, this will also allow further synthetic biology experiments.