Development and Application of Transcription Terminators for Polyhydroxylkanoates Production in Halophilic Halomonas bluephagenesis TD01

Abstract

Halomonas bluephagenesis TD01 is one of the ideal chassis for low-cost industrial production based on "Next Generation Industrial Biotechnology," yet the limited genetically regulatory parts such as transcriptional terminators, which are crucial for tuned regulations on gene expression, have hampered the engineering and applications of the strain. In this study, a series of intrinsic Rho-independent terminators were developed by either genome mining or rational design, and seven of them proved to exhibit higher efficiencies than the canonical strong T7 terminator, among which three terminators displayed high efficiencies over 90%. A preliminary modeling on the sequence-efficiency relationship of the terminators suggested that the poly U sequence regularity, the length and GC content of the stem, and the number and the size of hairpin loops remarkably affected the termination efficiency (TE). The rational and de novo designs of novel synthetic terminators based on the sequence-efficiency relationship and the "main contributor" engineering strategy proved to be effective, and fine-tuned polyhydroxylkanoates production was also achieved by the regulation of these native or synthetic terminators with different efficiencies. Furthermore, a perfectly positive correlation between the promoter activity and the TE was revealed in our study. The study enriches our knowledge of transcriptional termination via its sequence-strength relationship and enables the precise regulation of gene expression and PHA synthesis by intrinsic terminators, contributing to the extensive applications of H. bluephagenesis TD01 in the low-cost production of various chemicals.

Keywords: Halomonas bluephagenesis; RNA-Seq; intrinsic terminator; polyhydroxylkanoates; rational design; synthetic biology; termination efficiency.

Figure 1

The typical structure (A) of an intrinsic terminator and its canonical mechanism for transcription termination (B), as well as the schematic design of this study (C). (B) Rho-independent termination does not require any protein factors while it will be enhanced by an accessory proteins NusA. (C) The native or engineered terminators were employed to regulate the PHB production, one of the environmentally friendly biomaterials accumulated intracellularly from glucose by three key enzymes: β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). The phaC gene in the genome of H. bluephagenesis TD01 was deleted by CRISPR/Cas9, and functionally rescued by phaC expression vectors regulated by terminators with different efficiencies.

Figure 2

The validation of termination efficiencies of 10 native intrinsic terminators in H. bluephagenesis TD01. (A) The distribution of the free energies ΔG_W of the 256 native intrinsic terminators mined by RNA-seq data in H. bluephagenesis TD01. The top 10 with potential high efficiencies were selected via a reported primary rating system. (B) The validation plasmids constructed by Golden Gate assembly (up) and the recombinant H. bluephagenesis TD strains harboring different terminators showed a variety of colors reflecting different termination efficiencies (down). S1-S5 indicates five Bsa I sites. (C) The normalized fluorescence intensity of the GFP and RFP in the recombinant H. bluephagenesis TD strains harboring the 10 terminators. (D) The termination efficiencies of the 10 terminators in both H. bluephagenesis TD01 and E. coli DH5α. Errors were calculated from three parallel measurements.

Figure 3

The analysis of the main contributors to the termination efficiencies of terminators. All of the hairpin structures were predicted by Kine Fold using only the stem-loop sequence, without the poly A and poly U sequence nor the upstream and downstream text. The main contributors to termination efficiency were analyzed individually: the contribution of the poly U sequence (A), loop numbers (B), loop size (C), and stem length (D). Taken together, A preliminary model was established to predict termination efficiency based on the parameters indicated in (E). TE, the termination efficiency. ΔG_H, the free energy for stem-loop structure formation without the upstream and downstream context. ΔG_H/n_H, the normalized free energy for the stem-loop structure formation.

Figure 4

Verification of the termination efficiency of engineered terminators based on the “main contributors” strategy in H. bluephagenesis TD01. (A,C): The normalized fluorescence intensities of the GFP and the RFP in the recombinant H. bluephagenesis TD strains harboring six rationally designed terminators based on A04 scaffold (A) and three de novo designed terminators (C). (B,D): The termination efficiencies of rationally designed terminators based on A04 scaffold (B) and de novo designed terminators (D).

Figure 5

Determination of the effects of promoter activity on the terminator efficiency. (A) Validation plasmids harboring promoters with different activities (Porin, H19060, H18740, and ML03285) to promote the expression of sfGFP and mRFP genes, with the same T7 terminator inserted between the two fluorescent genes. (B) The normalized fluorescence intensities of the GFP and the RFP of the recombinant H. bluephagenesis TD strains harboring different promoter-T7 terminator pairs. (C) The relationship of promoter acitivty and terminator efficiency. The promoter activity was presented by fluorescence intensity of GFP measured in the recombinant H. bluephagenesis TD (pMCS-P_lib-sfGFP) previously constructed.

Figure 6

PHB production regulated by terminators with different efficiencies. (A) The phaC expression vectors were constructed with a series of native or engineered terminators, to rescue the deletion of phaC gene in H. bluephagenesis TD01 genome. (B) The relationship of the relative intracellular mRNA level of phaC gene and the termination efficiency. (C) Cell dry weight and PHB content in the recombinant H. bluephagenesis TD strains harboring different phaC expression vectors. (D) TEM observations of PHA granules in the recombinant H. bluephagenesis TD strains with PHB production regulated by the terminator A02.