Construction of Highly Efficient Expression Systems in Halomonas Based on Orthogonal Transcriptional Machinery

Ouyang, Q., Peking-Tsinghua Joint Centre for Life Sciences

The fact that genetic materials and design principles for synthetic biology have to be studied and formatted in an ad-hoc manner across different genetic context significantly hinders the transformation of achievements from model organisms into real bioengineering practice. One solution is to use orthogonal gene expression machinery to insulate the synthetic circuits and pathways from genetic context. Here we develop a set of orthogonal transcriptional machinery (OTM) that allows insulated transcriptional control in both E.coli and Halomonas TD01, a halophile of potent industrial value. Phage RNAP-promoter pairs as OTM candidates were first identified via genome mining and characterized in both genetic context; three pairs with best performance then engineered to allow genetically stable maintenance and strict inducible control (ON/OFF ratio >1000). Promoter library across a wide range of transcriptional activity was also built for each OTM. Results showed that the performance of OTMs and their libraries in E.coli and Halomonas TD01 are in strikingly linear correlation. OTMs were then used to control bacterial elongation cassette for industrial application. All OTMs enable Halomonas TD01 cells to elongate 100-fold in length compared with wild type, indicating the validity of OTMs