Contributions

Our project aimed to engineer Synechocystis sp. , a unicellular freshwater cyanobacterium that produces polyhydroxybutyrate (PHB) by consuming CO₂ from the atmosphere. Specifically, we enhanced PHB production and CO₂ reduction by subjecting the bacteria to high pH conditions, addressing climate change and plastic pollution. To achieve this, we conducted a series of experiments involving Adaptive Laboratory Evolution (ALE), PHB production screening, and RNA sequencing (RNA-seq) analysis to identify the key genetic changes responsible for improved PHB synthesis under alkaline stress.

1. Adaptive Laboratory Evolution (ALE) for High pH Tolerance

Adaptive Laboratory Evolution (ALE) is a process that naturally selects desirable phenotypes by simulating environmental pressures in a laboratory setting. Based on Darwin's theory of natural selection, ALE allows organisms to evolve over successive generations to enhance their survival under specific conditions. In our project, we applied ALE to Synechocystis sp. , gradually increasing the pH of the growth medium to simulate selective pressure for high pH tolerance, thus promoting PHB production under stress.

1.1. Incremental pH Increase and Selection

We began by growing Synechocystis sp. in optimal conditions at pH 7.5, close to the neutral pH of water. To induce adaptation to higher pH levels, we implemented a stepwise strategy by incrementally increasing the pH of the growth medium by 0.1 units per cycle. After each adjustment, the cultures were maintained at the new pH until stable growth was observed. This gradual approach allowed the cyanobacteria to adapt smoothly to the alkaline environment, minimizing cell shock and enabling the selection of phenotypes that thrive in high pH while efficiently producing PHB. By leveraging ALE, we effectively developed strains with enhanced pH tolerance and PHB synthesis.

1.2. Identification of Differentially Expressed Genes (DEGs)

After ALE, we performed RNA-seq analysis to identify differentially expressed genes (DEGs) that were responsible for the enhanced adaptation and PHB production at high pH. Through this analysis, we discovered several upregulated and downregulated genes linked to metabolic pathways involved in stress response, carbon fixation, and PHB biosynthesis. These DEGs are critical for understanding the molecular mechanisms behind high pH tolerance and bioplastic production, offering valuable insights for further optimization of engineered strains.

Upregulated DEGs in Synechocystis sp. Under pH11 Conditions ( Part:BBa K5404000 - parts.igem.org )

The 40 overexpressed differentially expressed genes (DEGs) under pH 11 conditions were identified through RNA sequencing (RNA-seq) analysis, which allowed us to compare the transcriptomes of Synechocystis sp. grown at pH 7.5 and pH 11. By applying statistical analysis, including fold change and p-value thresholds (|fold change| ≥ 2 and p-value < 0.05), we isolated genes that showed significant upregulation in response to the high pH environment. These overexpressed genes include those involved in critical metabolic pathways, such as Mannose-1-phosphate guanyltransferase and Polyphosphate kinase, which enhance carbon flux towards polyhydroxybutyrate (PHB) production, as well as stress response elements like Alkaline phosphatase. Identifying these genes is essential because it reveals the molecular mechanisms that enable Synechocystis to adapt to alkaline conditions, rerouting metabolic resources to optimize survival and bioplastic synthesis. Understanding these upregulated pathways opens the door for future engineering strategies aimed at improving PHB production and CO₂ fixation in cyanobacteria under stress conditions, contributing to more sustainable biomanufacturing approaches.

Differentially expressed genes (DEGs) are genes that show statistically significant differences in expression levels between various experimental conditions. Upregulated DEGs specifically involve genes that reveal increased expression from the original condition [1]. This increased expression can be attributed to various responses, including metabolic adjustments and post-transcriptional modifications. Understanding how organisms influence their gene expression in response to environmental changes is crucial for creating potential biotechnological applications.

In our project, we utilized RNA-sequencing to compare the gene expressions of Synechocystis sp. grown at pH 7.5 and pH 11. We chose this approach over DNA sequencing because RNA sequencing can capture the dynamic state of the transcriptome, which helps reveal which genes are actively expressed under different conditions. By focusing on RNA, we were able to identify specific genes that were upregulated in response to high pH conditions, thus giving information on how Synechocystis adapts to alkaline environments. This information is extremely valuable for developing strategies to enhance PHB production and CO2 uptake in cyanobacteria under stressful conditions.

The RNA sequencing revealed that there were up to 40 upregulated DEGs in Synechocystis under pH 11 conditions when compared to pH 7.5 [Table 1]. The fold changes had a wide range, with fold change values varying from 3.87 (gene-MYO_3720) to 19.09 (gene-MYO_14210). More importantly, all of these changes were statistically significant, as they had an associated p-value of less than 0.05. The top five most upregulated genes were all hypothetical proteins, with fold changes ranging from 9.48 to 19.09, but functional genes, such as alkaline phosphatase and isochorismate synthase showed comparatively lower upregulation. Additionally, transposase genes were typically less upregulated, with fold changes between 4.78 and 6.06. Overall, the data highlighted the diverse range of gene expression when placed in high pH environments, with the hypothetical proteins having the largest increases in expression.

References:

1. McDermaid, A., Monier, B., Zhao, J., Liu, B., & Ma, Q. (2018). Interpretation of differential gene expression results of RNA-seq data: review and integration. Briefings in Bioinformatics , 20 (6), 2044–2054. https://doi.org/10.1093/bib/bby067

Downregulated DEGs in Synechocystis sp. Under pH 11 Conditions ( Part:BBa K5404001 - parts.igem.org )

The 67 downregulated differentially expressed genes (DEGs) under pH 11 conditions were identified through RNA sequencing (RNA-seq) analysis, comparing the transcriptomes of Synechocystis sp. grown at pH 7.5 and pH 11. Using statistical analysis, including fold change and p-value thresholds (|fold change| ≥ 2 and p-value < 0.05), we identified genes significantly downregulated in response to the high pH environment. These downregulated genes include key components of photosynthesis, such as Phycobilisome proteins and Photosystem I and II subunits, indicating a reduced photosynthetic activity under alkaline stress. Additionally, genes related to translation, such as 16S ribosomal RNA and various tRNAs, were suppressed, suggesting a shift away from protein synthesis and cellular growth towards resource conservation. The downregulation of these pathways highlights how Synechocystis adapts to high pH by reallocating energy and carbon resources away from growth and photosynthesis, instead promoting stress survival mechanisms and enhancing PHB production. Understanding these downregulated genes provides insights for optimizing metabolic pathways in cyanobacteria under stress, allowing for more efficient PHB biosynthesis in synthetic biology applications.

Specifically, our team utilized downregulated genes to find optimal strategies to produce PHB under modified Synechocystis growth techniques. Differentially Expressed Genes (DEG) are genes that show dramatically different expressions under lab conditions [1]. The downregulated genes refer to those that have decreased receptor sites, having decreased responsiveness and less transcription abilities.

Our team utilized RNA sequencing to evaluate the effectiveness of pH level modifications because of the distinctive advantage it has. Compared to DNA sequencing, RNA sequencing allows to visually indicate the full catalog of transcripts, define specific structures of genes, and measure gene expression levels [2]. The downregulation of these genes convey how Synechocystis responds to modified pH levels and improve PHB production through revealing stress levels received by genes according to fond change in Table 2.

Accordingly, the results of RNA sequencing for downregulated genes are shown in Table 2. The Fold Change column signifies how much each gene's productivity decreased, quantifying the degree of impact of the modified pH level conditions. The gene that performed highest fold change demonstrated -24.62, and the gene that performed lowest fold change demonstrated -4.02. Overall, the data that our team collected should be considered accurate, since the p-value indicates the probability that the result obtained is due to a random occurrence. Having a range of [0,0.045], the values designate that the fold change values are only attributable from the pH modifications.

References

1. How to employ statistical approaches to identify differentially expressed genes (DEG) . (2023). Novogene. https://www.novogene.com/us-en/resources/blog/how-to-employ-statistical-approaches-to-identify-differentially-expressed-genes-deg/#:~:text=DEGs%20are%20genes%20that%20show

2. Lê, N. (2023, June 13). Explaining DNA vs. RNA sequencing . Dispendix.com; DISPENDIX GmbH. https://dispendix.com/blog/explaining-dna-vs-rna-sequencing#:~:text=RNA%20sequencing%20(RNA%2DSeq)