Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas

Optimization of intracellular biosynthesis process involving regulation of multiple gene expressions is dependent on the efficient and accurate expression of each expression unit independently. However, challenges of analyzing intermediate products seriously hinder the application of high throughput assays. This study aimed to develop an engineering approach for unsterile production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or (P3HB4HB) by recombinant Halomonas bluephagenesis (H. bluephagenesis) constructed via coupling the design of GFP-mediated transcriptional mapping and high-resolution control of gene expressions (HRCGE), which consists of two inducible systems with high- and low-dynamic ranges employed to search the exquisite transcription level of each expression module in the presence of γ-butyrolactone, the intermediate for 4-hydroxybutyrate (4HB) synthesis. It has been successful to generate a recombinant H. bluephagenesis, namely TD68-194, able to produce over 36 g/L P3HB4HB consisting of 16 mol% 4HB during a 7-L lab-scale fed-batch growth process, of which cell dry weight and PHA content reached up to 48.22 g/L and 74.67%, respectively, in 36 h cultivation. HRCGE has been found useful for metabolic pathway construction.