Part:BBa_K2924000

Pcpc560 Promoter

A strong promoter for protein expression in cyanobacteria. It is made up of two predicted promoters and 14 transcription factor binding sites, as well as an RBS. The sequence stems from the cpcB gene from Synechocystis sp. PCC 6803

Usage and Biology

The promoter Pcpc560 was discovered in 2013 by Jie Zhou et al.¹ at the Chinese academy of sciences of Beijing. The name derives from the cyanobacterial gene that it originally expresses, phycocyanin B-subunit (cpcB). Zhou et al. used a region of 560 bases upstream of this gene, which contains two predicted promoters and 14 predicted transcription factor binding sites (TFBSs). Negative transcription TFBSs are usually located between 500-1000 bp upstream of the gene, so this region was left out of the P_cpc560 construct¹. The promoter carries an RBS, so it can be cloned directly in front of the gene of interest. They tested the promoter for the expression of two genes with enzymatic activity, the native enzyme crotonyl-CoA-specific trans-enoyl-CoA reductase (Ter) and D-lactate dehydrogenase from E. coli K-12. In both cases, protein yields of 15 % of soluble protein were reported.

Fig. 1: The constructs which were inserted into the pSDHY vector. mVenus, α-s1-casein and the T1/T7 double terminator are the parts with which the promoter was initially used.

If the promoter found by Zhou et al. makes these high yields possible for any expressed protein, cyanobacteria might become a viable chassis for protein production, especially since they do not need any carbon source and do not produce CO₂ emissions. The promoter has already been used in several publications since its discovery^{2, 3, 4}. Attempts to modify the native RBS have not resulted in higher protein yields, so the native RBS was left in the part⁴.

The promoter is already present in the registry as BBa_K1968001, which was submitted by the team of Edinburgh in 2016, but was not characterized in Synechocystis. However, BBa_K1968001 differs slightly from this promoter in its sequence and since the function of a promoter is strictly sequence-dependent, this P_cpc560 promoter is submitted as a new, characterized part.

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]

Characterization

The characterization data shown on this page is taken from the experimental data of the composite part BBa_K2924036, in which the Pcpc560 promoter expresses mVenus as a reporter gene in the pSDHY plasmid⁵ in Synechocystis sp. PCC 6803.

Fig. 2: Optical density of the cultures at 750 nm, the usual wavelength for cell density measurements of cyanobacteria. Measurements were carried out in triplicates, standard deviations are shown.

The Synechocystis cells were grown in 30 ml BG11 medium at 150 rpm, 1 % CO₂ and 80 µmol photons per second and square meter (80 µE). The empty vector control (EVC) grew faster than the cultures with the heterologous protein, suggesting strong expression leading to a metabolic burden. The cells expressing mVenus had a higher metabolic burden because the gene was codon-optimized for Synechocystis , while the α-s1-casein gene was codon-optimized for E. coli.

Fig. 3: A: Fluorescence of the cultures. B: Fluorescence of the cultures normalized by optical density. Fluorescence was measured at 𝝺_ex/em 527 nm/512 nm. Fluorescence was measured compared to the empty vector control to control for autofluorescence of the cells. Sterile BG11 was used as a blank to measure autofluorescence of the medium. Measurements were carried out in technical triplicates, standard deviations are shown.

The fluorescence per OD₇₅₀ decreased over time, likely due to limitations in light and nutrients, which force the cells to put more energy into photosynthetic pigments. The expression was shown to be strong and constitutive, creating a metabolic burden for the cells, but resulting in very high fluorescence. To gain information about the localization of the expressed protein, some cells were imaged in a fluorescence microscope.

Fig. 4: Fluorescence microscopy image of the mVenus expressing Synechocystis cells. Autofluorescence of the phycocyanin in the phycobilisomes is shown in magenta, fluorescence of mVenus is shown in green.

Fig. 5: SDS-PAGE of soluble fraction of cyanobacterial protein, extracted from the cultures after 2 days. The SDS-PAGE was run at 45 mA for 90 minutes and then stained with Coomassie blue.

The expression of mVenus is strictly intra-cellular and is not localized to any specific location in the cell. The autofluorescence of the cyanobacterial pigments is also visible in the cell membrane, in which light energy is captured.

After protein extraction, the soluble protein of the cells was analyzed by SDS-PAGE.

Between 25 and 35 kDa in the soluble fraction, a band is visible for mVenus, which is not visible in the EVC. mVenus has a molecular weight of 26.9 kDa, so the band is in the correct area. The very intense bands just above 15 kDa is likely cpcB, a protein from the phycobiliprotein complex, which is used to collect light energy. The protein band for the heterologous protein is approximately the second most intense band in the protein solution, suggesting a strong expression and potentially high yields.

More detailed characterrization data can be found in the composite part BBa_K2924036 The promoter was also successfully used to express a bovine milk protein in Synechocystis sp. PCC 6803 in the composite part BBa_K2924034.

cpc560 activity in novel chassis S. elongatus UTEX 2973

IISER-Pune-India 2021:

Shubin Li et al., characterised and compared the activity of various constitutive promoters in S. elongatus UTEX 2973, including cpc560, native to Synechocystis PCC 6803 [6]. The promoters were characterised in cultures grown at 38°C at both high and low light intensities - 500 umol photons/m2/s and 50 umol photons/m2/s respectively - using the reporter gene lacZ, encoding beta-galactosidase.

The activity of beta-galactosidase was calculated using Miller value (Miller = 1000 × OD420 nm/(total volume of the cell culture × reaction time × OD750 nm of the cell)). Transcriptional expression of lacZ was measured using qRT-PCR.

Promoter-lacZ-trbcL constructs, flanked by Neutral Site 1 homology arms, were made on a pBR322 backbone vector (see fig. b). The pBR322 backbone contains an oriT/bom site, allowing it to be introduced into UTEX 2973 by conjugation. The Neutral Site 1 homology arms allow for genomic integration of the Promoter-lacZ-trbcL construct into the Neutral Site 1 via homologous recombination following conjugation.

Constitutive promoters characterised in UTEX 2973:

a. psbA1, psbA2, psbA3, cpcB1, cpcB2 from UTEX 2973

b. cpc560 and psbA2 from Synechocystis PCC 6803

c. lac from E. coli

d. trc, LlacO-1, psbA from the chloroplast of Amaranthus hybridus

Among the constitutive promoters tested at high light conditions, transcriptional activity of lacZ under cpc560 was highest. Beta-galactosidase activity under cpc560 was the second highest, surpassed only by psbA3. The strength of cpc560 was over four times higher at 500 umol photons/m2/s than at 50 umol photons/m2/s.

Characterisation of promoter strength in S. elongatus UTEX 2973. a. beta-galactosidase activity and qRT-PCR analsyis of lacZ under various constitutive promoters at high light condition of 500 umol photons/m2/s. b. diagram of integrative vector containing promoter-lacZ-trbcL construct. c. beta-galactosidase activity under various constitutive promoters at high light condition of 500 umol photons/m2/s and low light light condition of 50 umol photons/m2/s. Figure adapted from Shubin Li et al. (2018) [6]

Vasudevan et al., characterised and compared the strengths of promoters cpc560 and its synthetic variants cpc560+A, cpc560_Dx2, cpc560_Ux2 in S. elongatus UTEX 2973, along with promoters psbA2L and rnpB [4]. This was done by comparing the fluorescence obtained from an eYFP reporter protein expressed under the aforementioned promoters in 10,000 individual cells grown at 40°C and 300 umol photons/m2/s in S. elongatus UTEX 2973.

cpc560+A contains four additional bp at the end of the original promoter's sequence. cpc560_Dx2 contains a duplication of the downstream transcription factors binding sites, and cpc560_Ux2 contains a duplication of the upstream transcription factors binding sites.

The promoters were assembled with the eYFP reporter gene and rrnB terminator into a level 1 expression cassette, which was then cloned into a level T self-replicating vector, pPMQAK1-T for eYFP expression analysis.

cpc560 was found to be stronger than its own variants as well as psbA2L, rnpB promoters. The same result was obtained in Synechocystis PCC 6803 as well, which was grown at 30°C and 300 umol photons/m2/s.

Characterisation of promoter strength in S. elongatus UTEX 2973 and Synechocystis PCC 6803 at 300 umol photons/m2/s light intensity and temperatures 40°C for S. elongatus UTEX 2973 and 30°C for Synechocystis PCC 6803. Figure adapted from Vasudevan et al. (2019) [4]

References

1: Zhou, J., Zhang, H., Meng, H., Zhu, Y., Bao, G., Zhang, Y., ... & Ma, Y. (2014). Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. Scientific reports, 4, 4500.

2: Liu, Deng, and Himadri B. Pakrasi. "Exploring native genetic elements as plug-in tools for synthetic biology in the cyanobacterium Synechocystis sp. PCC 6803." Microbial cell factories 17.1 (2018): 48.

3:Ng, Andrew H., Bertram M. Berla, and Himadri B. Pakrasi. "Fine-tuning of photoautotrophic protein production by combining promoters and neutral sites in the cyanobacterium Synechocystis sp. strain PCC 6803." Appl. Environ. Microbiol. 81.19 (2015): 6857-6863.

4: Vasudevan, R., Gale, G. A., Schiavon, A. A., Puzorjov, A., Malin, J., Gillespie, M. D., ... & Lea-Smith, D. J. (2019). CyanoGate: A modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax. Plant physiology, 180(1), 39-55.

5: Behle, Anna, Pia Saake, and Ilka M. Axmann. "Comparative analysis of inducible promoters in cyanobacteria." bioRxiv (2019): 757948.

6: Li, S., Sun, T., Xu, C., Chen, L., & Zhang, W. (2018). Development and optimization of genetic toolboxes for a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Metabolic engineering, 48, 163-174