Synthetic and Systems Biotechnology (Dec 2021)

Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides

  • Tong Shi,
  • Lu Zhang,
  • Mindong Liang,
  • Weishan Wang,
  • Kefeng Wang,
  • Yue Jiang,
  • Jing Liu,
  • Xinwei He,
  • Zhiheng Yang,
  • Haihong Chen,
  • Chuan Li,
  • Dongyuan Lv,
  • Liming Zhou,
  • Biqin Chen,
  • Dan Li,
  • Li-Xin Zhang,
  • Gao-Yi Tan

Journal volume & issue
Vol. 6, no. 4
pp. 335 – 342

Abstract

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The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q10 (Q10) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, Prsp_7571 and Prsp_6124, showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a Prsp_7571-derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q10 was modulated by the selected promoter T334* in JDW-710; the Q10 yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs.

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