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Fine-tuning native promoters of Synechococcus elongatus PCC 7942 to develop synthetic toolbox for heterologous protein expression Annesha Sengupta, Avinash Vellore Sunder, Sujata V. Sohoni, and Pramod P. Wangikar ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00066 • Publication Date (Web): 11 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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ACS Synthetic Biology

Fine-tuning native promoters of Synechococcus elongatus PCC 7942 to develop synthetic toolbox for heterologous protein expression

Annesha Sengupta1, Avinash Vellore Sunder1, Sujata V. Sohoni1, Pramod P. Wangikar1,2,3*

1Department

of Chemical Engineering, 2DBT-Pan IIT Centre for Bioenergy, 3Wadhwani Research

Centre for Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076 India

*Corresponding author Prof. Pramod P. Wangikar, Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India Email: [email protected]; Tel: +91 22 25767232; Fax: + 91 22 25726895

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ABSTRACT The cyanobacterium Synechococcus elongatus PCC 7942 is a potential photosynthetic cell-factory. In this study, two native promoters from S. elongatus PCC 7942 driving the expression of abundant cyanobacterial proteins phycocyanin (PcpcB7942) and RuBisCO (Prbc7942) were characterized in relation to their sequence features, expression levels, diurnal behaviour and regulation by light and CO2, major abiotic factors important for cyanobacterial growth. PcpcB7942 was repressed under high light intensity, but cultivation at higher CO2 concentration was able to recover promoter activity. On the other hand, Prbc7942 was repressed by elevated CO2 with a negative regulatory region between 300 and 225 bp. Removal of this region flipped the effect of CO2 with Rbc225 being activated only at high CO2 concentration, besides leading to the loss of circadian rhythm. The results from this study on promoter features and regulation will help expand the repertoire of tools for pathway engineering in cyanobacteria. Keywords CpcB promoter, rbc promoter, promoter strength, light-CO2 effect, diurnal rhythm INTRODUCTION Cyanobacteria are increasingly being explored as photosynthetic cell factories for the conversion of CO2 to biofuels and platform chemicals1–6. However, currently reported product yields with engineered cyanobacteria are below those needed for a commercially viable process. Pathway engineering efforts in cyanobacteria are often dependent on the use of inducible promoters such as PtetR, Ptrc or PlacO derived from heterotrophic model organisms2,7,8. However, the use of chemical inducers such as IPTG or anhydrotetracyclin in large scale cultivation might be prohibitive owing to their cost or light sensitivity9. Recent reports have explored the application of native cyanobacterial promoters,


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terminators and ribosome binding sites (RBS) and their engineered variants10–13. Nonetheless, the genetic toolkit for cyanobacteria is substantially less developed compared to heterotrophs. Characterization of native promoters from cyanobacteria has been largely limited to the model strains Synechocystis sp. PCC 680310–16 or Synechococcus sp. PCC 700217,18. Synechococcus elongatus PCC 7942 is another well-established cyanobacterium with significant potential for metabolic engineering19–21. A few fast growing strains of Synechococcus elongatus closely related to S. elongatus PCC 7942 are also emerging as potential cell factories22,23. However, only a few promoters like PpsbA124or PnirA25 have been functionally characterized from S. elongatus PCC 7942. Further, S. elongatus PCC 7942 is part of an isolated clade distant from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 in the cyanobacterial phylogenetic tree26, making direct translation of promoter information obtained from these organisms to S. elongatus PCC 7942 difficult. The presence of the Highly Iterated Palindromic sequence (HIPs) 5’-GCGATCGC-3′ in most cyanobacteria27,28 also makes prediction of promoter elements using bioinformatics tools such as MEME29 challenging7. In addition, the effects of cultivation conditions in modulating heterologous expression are often overlooked in promoter engineering and synthetic biology studies. Development of a cyanobacteria based biorefinery will necessitate their cultivation in large outdoor ponds or photobioreactors30 that are exposed to bright light and diurnal variations. A continuous supply of CO2 would also be required to ensure sufficient cyanobacterial growth and pH control, thereby necessitating the development of expression elements that can maintain protein expression levels in the presence of high light and CO2. It is therefore imperative to characterize native promoters from S. elongatus PCC 7942 for their expression and response to environmental stimuli. Here we describe the characterization of



native promoters driving the expression of two major proteins responsible for light harvest (Phycocyanin, PcpcB7942) and CO2 sequestration (RuBisCO, Prbc7942) in S. elongatus PCC 7942. The primary objective was to identify the sequence elements and understand the regulation of the promoters with respect to light and CO2 under outdoor simulated diurnal light cycles.

RESULTS AND DISCUSSION Cultivation and reporter systems: PcpcB7942 and Prbc7942 were characterized using two reporter proteins eYFP (enhanced yellow fluorescent protein) and luciferase under different cultivation conditions. Due to the high stability (t1/2 >24 h) of eYFP31,32, its expression was independent of fluctuations in light intensity and provided an estimate of cumulative protein production. In contrast, luciferase (t1/2 ~ 2 h) expression reflected a real-time response to environmental perturbations33, with the bioluminescence pattern following diurnal rhythm (Figure S1). The use of reporters with varying half-lives helps study the promoter strength and regulation across growth and diurnal cycles. Besides promoter strength, gene expression is also controlled by other factors including the growth stage and the action of regulatory factors at the gene and protein level34. To study the effect of growth and carbon availability, S. elongatus PCC 7942 was cultivated in (i) a shake flask (SF) on a shaker incubator and (ii) in a Multi-Cultivator (MC) photobioreactor where CO2 is bubbled into the medium. Cultivation in MC mimicked a carbon-saturated state even at ambient levels of CO2 (0.04%), in contrast to the growth regime of S. elongatus cultivated in a shaker. While the growth rate in SF increased from 0.02 to 0.075 h-1 upon increasing CO2 to 1%, bubbling CO2 in MC ensured a comparably high growth rate even at ambient condition due to better carbon availability


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(Figure S1). The growth rate also doubled in response to an increase in light intensity from 150 to 400 µE (Figure S1). Protein expression driven by PcpcB7942 and Prbc7942 was studied in relation to length of the promoter sequences under several combinations of light and CO2 conditions to evaluate their strength and regulation by abiotic factors. Characterization of cpcB promoter: A 560 bp “super-strong” PcpcB has been recently identified in Synechocystis sp. PCC 6803 containing two promoter elements and 14 transcription factor binding sites (TFBSs), and capable of driving high levels of protein expression11. PcpcB7942 has been reported to possess two functional promoter regions with a conserved -10 element35. We truncated the promoter sequence successively by 100 bp from 5’ end while retaining the native RBS to study the features of PcpcB7942. Expression of eYFP driven by the promoter peaked at 300 bp (Figure 1a). The enhancement in promoter strength could be attributed to the removal of a binding domain for NtcA, a global transcription regulator of nitrogen metabolism (Figure 1f); deletion of this region has been reported to relieve negative regulation and contribute to the increased strength of Pcpc56011,36. PcpcB7942 was weakened by further truncation with a significant reduction in expression levels at 200 bp and negligible expression at 100 bp, probably due to the sequential loss of both promoter elements (Figure 1a). PcpcB7942 was repressed by ~50% under high light intensity (400 E), similar to PcpcB6803 and PcpcB700214 (Figure 1a). Since all truncated PcpcB7942 variants exhibit the same behavior under high light, the putative light-responsive region in PcpcB7942 might be located in the 5’ UTR (Figure 1f). This extended 5’UTR region in cyanobacterial promoters has been reported to contain elements capable of responding to environmental cues11,37. A truncated variant of PcpcB6803 containing only the 89 bp core promoter region has been



reported to be relieved of repression by high light intensity, although protein expression was reduced by 50%14. PcpcB7942 recorded a substantial increase in eYFP expression under elevated CO2 levels in SF compared to the carbon-limited ambient condition (Figure 1b), although the enhancement was not significant beyond 0.5% CO2. In contrast, when air was bubbled in a MC, high eYFP fluorescence was reached even at ambient level of CO2 (Figure 1c). The repression under high light was apparent in both modes of cultivation. Nevertheless, this negative regulation was alleviated by cultivation under 0.5% CO2 (Figure 1c), a characteristic also shared by PpsbAI (Figure S2). While the expression of luciferase was also repressed under high light, changing CO2 levels did not appear to alter the observed promoter strength (Figure 1d). The discrepancy observed in the expression levels of these reporter proteins under similar cultivation conditions indicates that besides the promoter elements, the apparent promoter strength is also dependent on the nature of the protein expressed, regulation by environmental factors and phase of cell growth. PcpcB6803 has been reported to exhibit maximum activity during the exponential growth phase with a decline in stationary phase, probably on account of cross-talk with native regulatory mechanisms38. Therefore, we hypothesize that the higher activity of Pcpcb7942 observed under elevated CO2 levels may be a result of a higher growth rate39, rather than assume the presence of a CO2-responsive element in the promoter sequence. We suggest that the cpcB300 variant could be employed as a short promoter supplemented by cultivation under favourable light and CO2 conditions for efficient heterologous expression. Reporter strains bearing variants of the full-length PcpcB7942 were also subjected to diurnal light cycles to evaluate promoter regulation via circadian rhythms (Figure 1e). The analysis revealed that expression reached peak intensity twice a day, typically at the start and end of the light phase.


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Characterization of rbc promoter: Prbc drives the expression of the Ribulose 1,5-bisphosphate carboxylase (RuBisCO) enzyme in photosynthetic organisms, and is responsible for controlling carbon availability to the cell. By analyzing truncated forms of the promoter, we observed that Rbc400 drove the highest level of eYFP expression across all CO2 concentrations (Figure 2a). Rbc225 was the shortest functionally active promoter and further truncation to Rbc200 led to negligible eYFP expression. The reported transcription start site is 157 bp upstream of the translational start site40, indicating the presence of -10 and -35 elements of the promoter between 157 and 225 bp upstream of the start codon. The maximum expression that could be achieved by the optimal promoter variant Rbc400 was observed to be 50% of that with CpcB300 under carbon-limited state (Figure S3). Under high light intensity (400 E), Prbc variants did not show significant change in bioluminescence (Figure S4); though a slight increase was observed in eYFP expression, probably on account of increased growth rate (Figure S4, S1b). Interestingly, all the truncated variants except Rbc225 were negatively regulated by elevated (0.5%) CO2. However, increasing the CO2 concentration to 1% did not further regulate expression levels (Figure 2a, S5). On the other hand, Rbc225 exhibited a reduced translational efficiency under carbon-limitation but was activated at higher CO2 concentrations (Figure 2a,c), indicating the presence of a CO2-responsive regulatory region between 300 and 225 bp from the translational start site (Figure 2b). A 30 bp CO2responsive regulatory element has been previously identified in the 5’UTR of Prbc7002 along with its trans-acting protein factor18. While this region is AT-rich and homologous to the cis-acting elements of rbcS promoter in higher plants, the probable negative regulatory region identified in Prbc7942 contains an equal AT/GC ratio. The regulatory region was more conserved across cyanobacterial clades compared to other promoter elements (Figure



S6), and its removal led to depleted expression levels under ambient CO2 suggesting an essential role in modulating Prbc strength under carbon limitation. While the dynamics of Rbc225 under diurnal light was similar to Rbc500 with sufficient CO2, the truncated variant appeared to have lost its regulation by the circadian rhythm and instead displayed a steady increase in expression levels under continuous light (Figure 2d). Promoters in Synechocystis sp. PCC 6803 have been observed to lose freerunning bioluminescence circadian rhythm in continuous light even after being entrained in 12:12 LD diurnal cycles41; and minimal mutation in the promoter sequence caused a change in phase from dusk to dawn or vice versa in S. elongatus PCC 794242. The lack of circadian control coupled with enhanced promoter activity in the presence of high CO2 and continuous light makes Rbc225 a favourable candidate for the constitutive expression of heterologous genes in photobioreactor cultivations.

CONCLUSION Besides adding to the library of native cyanobacterial promoters, this study provides a better understanding of the promoter dynamics under environmental parameters typically experienced during cyanobacterial cultivation. Knowledge about promoter regulation opens up options to modulate expression levels during pathway engineering not only by modifying the expression elements, but also by taking advantage of CO2 levels, light intensity and diurnal rhythm. This would help design specific production and harvesting protocols aimed at increasing production efficiency and reducing the toxic effect of byproducts in the commercial cultivation of engineered cyanobacteria. SUPPORTING INFORMATION Materials and methods, Supplementary Tables and Supplementary figures are provided in the Supporting Information.


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ACKNOWLEDGEMENT The authors are grateful to Dr. Shinjinee Sengupta for reviewing the manuscript. CONFLICT OF INTEREST STATEMENT AS, AVS, SVS and PPW declare no conflict of interest. CONSENT FOR PUBLICATION All authors have reviewed the manuscript and have agreed to the order of the authorship. ETHICAL APPROVAL This article does not contain any studies with human participants or animals performed by any of the authors. AVAILABILITY OF DATA AND MATERIAL The sequences of the promoter regions used in this study have been obtained from NCBI and Cyanobase. The datasets are either included in the main manuscript or in its Supplementary Information. FUNDING This work was supported by a research grant provided by Department of Biotechnology, Government of India (Grant No: BT/EB/PAN IIT/2012). AUTHOR’S CONTRIBUTION AS, SVS and PPW designed the research. AS and SVS performed the research. AS, AVS and PPW analyzed the data. AS, AVS and PPW wrote the paper. REFERENCES (1)

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Figure captions: Figure 1. (a) eYFP expression levels in CpcB-eYFP strains grown on low (150 µE) and high (400 µE) light in shake flask (SF) under 0.04% CO2 . (b) eYFP expression in CpcB-eYFP strains grown under carbon-limited ambient CO2 (0.04%) and high CO2 (0.5%, 1%) conditions with 150 µE light intensity in SF. (c) Effect of the combination of light and CO2 on PcpcB7942 driving the expression of eYFP in Multi-Cultivator (MC). Promoter strength was measured as relative eYFP fluorescence. Error bars correspond to standard deviations from three biological replicates. The letters a-c denote statistically different values of μ for each category (P < 0.05). Data points are normalized with respect to wild type. (d) Bioluminescence from luciferase expression driven by CpcB500 promoter under varying light and CO2 conditions performed in SF. (e) Diurnal and circadian rhythms of CpcB500 promoter studied in MC under 14:10 h light-dark cycles and subsequent continuous illumination at 150 µE. (f) Characteristic features and regulatory regions of PcpcB7942 (full length and truncated variants). TSS denotes transcription start site40 represented by black line; P1 and P2 (purple box) denote the two promoter elements35. Red block represents the light-responsive negative regulatory region; the brown box represents the NtcA binding domain.

Figure 2. (a) Effect of CO2 levels on eYFP expression driven by truncated variants of Prbc7942. The Rbc-eYFP strains were cultivated on shake flask (SF) with light intensity 150 E. (b) Characteristic features and regulatory regions in Prbc7942 of the full length and truncated promoter. TSS is the transcription start site40 (black line), P represents the promoter elements (-10 and -35, purple box) predicted using Virtual Footprint43. The CO2-regulatory region is denoted in red. (c) Effect of CO2 on the bioluminescence produced from luciferase expressed under the control of Rbc225 promoter when Rbc-lux strains were grown under diurnal light dark-cycle of 14:10 h with maximum intensity of 150 µE in SF. (d) Bioluminescence from luciferase expression driven by Rbc500 and Rbc225 promoters, depicting diurnal and circadian rhythm. Rbc-lux strains were cultivated in Multi-Cultivator (MC) under 0.04% CO2 with a 14:10 h diurnal light-dark cycle and subsequent continuous illumination at 150 E. Error bars correspond to standard deviations from



three biological replicates. The letters a, b denote statistically different values of μ for each category (P < 0.05). Data points are normalized with respect to wild type.


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Figure 1 59x47mm (300 x 300 DPI)



Figure 2 83x47mm (300 x 300 DPI)


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Graphical Abstract 84x38mm (300 x 300 DPI)


Fine-tuning native promoters of Synechococcus ... - ACS Publications

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