Part:BBa_K3971000

Strong constitutive cyanobacterial PcpcB-7942 mutant promoter.

Usage and Biology

PcpcB is a native cyanobacterial 'superstrong' promoter (1) for the phycocyanin β-1 subunit (cpcB) gene, whose gene product is responsible for light harvest (2).

This part, called PcpcB-m6 is a mutant promoter of the cpcB gene in the cyanobacterium Synechococcus elongatus PCC 7942 (hereby referred to as PcpcB-7942), created by error-prone PCR by Sengupta et. al (2020) (3).

However, PcpcB-7942 is known to be repressed under high-light conditions (2), however the phycocyanin gene product. Although cyanobacteria are increasingly being used for biomanufacturing of various chemicals, reported productivities have been low (3). Developing cyanobacteria-based cell factories will require their cultivation in outdoor ponds or photobioreactors (4) and a continuous CO2 supply to maintain sufficient growth and pH (2). There it is crucial to have strong cyanobacterial promoters that maintain high levels of expression at high CO2 and high light levels.

PcpcB-m6 exhibited high expression under both low (100 μE.m−2.s−1) and high light (300 μE.m−2.s−1) intensities and in high carbon concentrations (1% CO2) (3) (shown to have higher phycocyanin content under carbon saturated conditions in Synechococcus elongatus PCC 11801 (5)).

In a previous study (2), truncated versions of PcpcB-7942 were studied to find the optimal promoter length and identity key regulator elements. The authors truncated the PcpcB-7942 sequence by 100 bp successively from the 5' end and tested the expression of a downstream eYFP gene with the native RBS of the promoter intact. They found that the eYFP expression peaked when PcpcB-7942 was truncated to 300 bp, which they attributed to the removal of the NtcA binding domain (a global transcription regulator involved in nitrogen metabolism).

Sengupta et. al (2020) (3), performed error-prone PCR on the 300bp sequence described above, with the native RBS and tested the strengths of the mutant promoters by cloning an eYFP gene downstream, and integrating the construct into the neutral site 1 (NS1) of S. elongatus PCC 7942.

The PcpcB-m6 mutant promoter was observed to have a 1.32 fold change in expression levels with respect to PcpcB-7942 under high light and a 0.94 change under low light conditions. It also had a point mutation at the 24 G position which was mutated to A.

We used the PcpcB-m6 promoter to express the cscB (sucrose permease) transporter to enable Synechococcus elongatus UTEX 2973 to secrete sucrose into the media when placed under salt stress.

References

1: Zhou, Jie, et al. "Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria." Scientific reports 4.1 (2014): 1-6.

2: Sengupta, Annesha, et al. "Fine-tuning native promoters of Synechococcus elongatus PCC 7942 to develop a synthetic toolbox for heterologous protein expression." ACS synthetic biology 8.5 (2019): 1219-1223.

3: Sengupta, Annesha, Swati Madhu, and Pramod P. Wangikar. "A Library of tunable, portable, and inducer-free promoters derived from cyanobacteria." ACS Synthetic Biology 9.7 (2020): 1790-1801.

4: Seth, Jyoti R., and Pramod P. Wangikar. "Challenges and opportunities for microalgae‐mediated CO2 capture and biorefinery." Biotechnology and bioengineering 112.7 (2015): 1281-1296.

5: Mehta, Kanika, et al. "Elevated carbon dioxide levels lead to proteome-wide alterations for optimal growth of a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801." Scientific reports 9.1 (2019): 1-14.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]