Promoter engineering for enhanced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production in Haloferax...

Polyhydroxyalkanoates (PHAs) are linear polyesters synthesized by various microorganisms. Due to their excellent physicochemical and material properties, PHAs are considered as a promising and sustainable alternative to petroleum-based plastics. Haloferax mediterranei is a promising and cost-effective producer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In this study, we aimed to improve PHBV yield of H. mediterranei via promoter engineering. Differential RNA-seq (dRNA-seq) was firstly used to examine the genome-wide transcription start sites (TSSs) of H. mediterranei. Based on the TSS profile, 26 constitutive promoters with expression strength spanning ~10³-fold dynamic range were quantitatively characterized using GFP fluorescence reporting system. Furthermore, it was found that the 5' untranslated region (5'-UTR) of phaR encoding PHA synthesis regulatory protein reduced the activity of the promoters derived from phaR, and this regulation occurred at the transcription level. Finally, several suitable promoters were selected to regulate the expression of cimA and bktB to improve the PHBV accumulation and 3HV molar fraction in H. mediterranei, ultimately leading to an increase of 35 % and of 18.4 %, respectively. Taken together, this work established a constitutive promoter library of haloarchaea, which would provide abundant genetic elements for the construction of ideal haloarchaeal chassis cells contributing in biosynthetic biology of extremophiles.