Research Goal & Overview

Our project aimed to engineer Synechocystis sp. PCC 6803, a unicellular freshwater cyanobacteria that produces polyhydroxybutyrate (PHB) by consuming CO₂ from the atmosphere. Specifically, we enhanced the production of PHB and reduction of CO₂ by exposing bacteria under high pH conditions, addressing climate change and plastic pollution. We conducted a series of experiments involving adaptive laboratory evolution (ALE), PHB production screening, and RNA sequencing (RNA-seq) analysis to achieve this goal.

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

Adaptive Laboratory Evolution (ALE) is a method for naturally selecting suitable phenotypes to cultivate desired characteristics in the laboratory. This method is highly based on Darwin’s theory of natural selection, stating that evolution, or change in genotype and phenotype over generations, can occur to increase the chance of survival. We simulated natural selection by increasing the pH of the growth medium in laboratory conditions, letting the cyanobacteria adapt to high pH conditions and produce a high amount of PHB.

1.1. Incremental pH Increase and Selection

We initiated our experiment by cultivating Synechocystis sp. under optimal conditions at pH 7.5, a pH level mainly of H₂O. To promote adaptation to higher pH levels, we employed a pH increment strategy, gradually increasing the pH of the growth medium by 0.1 pH units per cycle. After each incremental adjustment, the cultures were maintained at the new pH level until stable growth was observed. This stepwise approach allowed the cells to adapt gradually to the increasing alkalinity, applying selective pressure to favor strains capable of thriving under those conditions. This gradual pH elevation minimized shock to the cells and facilitated the selection of a phenotype that can produce PHB efficiently, effectively harnessing ALE principles to enhance pH tolerance.

1.2. Monitoring Growth and Adaptation

These results illustrate that while Synechocystis sp. is highly adaptable and capable of sustaining growth in a wide pH range, there are limits to this adaptability. The optimal growth occurs between pH 7.5 and 9.5, with substantial growth still observed up to pH 11.0. However, at pH levels beyond 11.0, the extreme alkalinity adversely affects cellular functions, leading to significant growth inhibition. Understanding these pH effects is crucial for optimizing cultivation conditions for applications such as sustainable bioplastic production, where maintaining cell viability and productivity is essential.

Our study demonstrates that Synechocystis sp. exhibits robust growth and maintains consistent cell morphology in alkaline conditions up to pH 11.0, with only a slight reduction in growth rate compared to neutral pH levels. Brightfield microscopy images of cells grown at pH levels 7.5, 9.5, 11.0, and even 11.5 revealed no significant changes in cell shape, size, or distribution. Figure 2 indicates that increasing the pH does not affect the Synechocystis sp. cell morphology, suggesting that the cells are resilient to high pH stress and can maintain their structural integrity even under more alkaline conditions.

The preservation of cell morphology is crucial as it reflects the cells' ability to sustain essential physiological processes, including photosynthesis and metabolic activities related to polyhydroxybutyrate (PHB) production. Maintaining consistent morphology ensures that the cells remain functional and efficient in converting atmospheric CO₂ into valuable bioplastics.

2. Screening for Enhanced PHB Production

The values of fluorescent intensity and intracellular PHB content were plotted on a scatter graph, and a linear regression line was drawn to determine the strength of the correlation. The R² value of 0.9091 of the linear trendline indicates a strong, positive correlation between intracellular PHB content and the fluorescence intensity of Nile red-stained PHB granules (Figure 3A). This confirms that fluorescence intensity serves as an accurate proxy for quantifying PHB levels in Synechocystis cells.

Fluorescence microscopy images captured under varying pH conditions from pH 7.5 to 11.5 further illustrate the distribution and intensity of Nile red-stained PHB granules in PHB-producing Synechocystis cells (Figure 3B). PHB content, normalized to cell dry weight, was quantified using the chloroform-methanol extraction method. As shown in Figure 3B, pH conditions that resulted in higher PHB content – pH 9.5 and 11.0 – displayed fluorescence of visibly greater intensity than those with lower PHB content. Such correlation indicates the reliability and efficiency of Nile red staining in visualizing and quantifying PHB accumulation under different environmental conditions in Synechocystis sp.

Figure 4 demonstrates the effect of varying pH levels on PHB content (wt%) in Synechocystis cells. The PHB content increased as the pH level rose from 7.5 to 11.0, with a significant peak observed at pH 11.0, where the PHB content reached 31.3 wt% (p = 0.002). At pH 7.5 and 9.5, the PHB content was measured at 20.0 wt% and 24.7 wt%, respectively. However, a further increase in pH to 11.5 resulted in a notable decrease in PHB content to 17.3 wt%. These results indicate that moderately alkaline conditions (pH 11.0) optimally enhance PHB production in Synechocystis, whereas more extreme pH conditions (either lower or higher) reduce the accumulation of PHB.

Based on the results from Figure 4, we observed that increasing the pH levels significantly affected PHB production in Synechocystis cells, with a notable peak at pH 11.0, where PHB content reached 31.3 wt%. This suggests that moderately alkaline conditions enhance PHB biosynthesis, likely due to the cells' improved metabolic activity and increased availability of bicarbonate ions for carbon fixation under these conditions. However, at pH levels beyond 11.0, such as 11.5, the extreme alkalinity resulted in a marked reduction in PHB content, dropping to 17.3 wt%. This indicates a threshold beyond which further increases in pH become detrimental to PHB accumulation, likely due to cellular stress that disrupts normal metabolic functions. Overall, these findings support the hypothesis that Synechocystis sp. can be engineered for enhanced PHB production under high pH conditions, but they also highlight the importance of maintaining pH within an optimal range to maximize bioplastic yield.

The cytotoxicity of Polyhydroxybutyrate (PHB) on Detroit 551 human fibroblast skin cells was evaluated using the Prestoblue assay. The standard curve generated (left graph) displayed a high linear correlation (R² = 0.991) between absorbance values at 570/600 nm and the number of cells, indicating a robust relationship for measuring cell viability. The right graph presents absorbance measurements across various PHB concentrations (0% to 10%) over different incubation periods. Across all concentrations, absorbance increased up to 72 hours, suggesting cell proliferation, followed by a slight decline at 96 hours. Statistical analysis (one-way ANOVA with Tukey’s post hoc test) indicated no significant differences (ns) in cell viability between PHB concentrations, demonstrating that PHB exhibited no cytotoxic effects at concentrations up to 10%.

3. RNA Sequencing (RNA-seq) Analysis

3.1. Sample Preparation

• Selection of Strains: Duplicate samples of Synechocystis grown in pH 7.5 and pH 11 were used for the RNA-seq analysis (Total four samples).
• RNA Extraction: Extracted total RNA using Trizol.

3.2. Transcriptome Sequencing

• Library Construction: Prepared Illumina TruSeq RNA library for RNA-seq.
• Sequencing Platform: Utilized Illumina TruSeq RNA technology for high-throughput sequencing.

Total RNA is first isolated from the sample of interest, such as cells or tissue. If DNA contamination is present, genomic DNA is removed using DNase. The RNA's integrity and total quantity are then assessed. Two main methods are available for library preparation: mRNA selection and rRNA depletion. Next, the purified RNA is fragmented into smaller pieces suitable for sequencing and reverse transcribed into cDNA. Sequencing adapters and sample-specific index sequences are attached to the ends of the cDNA fragments to allow the identification of individual samples in multiplexed sequencing. The resulting products are purified and amplified by PCR to create the final cDNA library. The library is then quantified and qualified to ensure readiness for sequencing. Typically, the insert size for RNA-Seq libraries ranges between 200-400 base pairs, and in paired-end sequencing, both ends of the fragment are sequenced to the desired read length.

After sequencing, the raw reads undergo quality control, with basic statistics such as overall read quality, total bases, and total reads calculated to ensure the data is suitable for analysis. If the sequencing cycle exceeds the insert size of the library, adapter sequences may appear at the 3' end of the reads. To reduce biases, these adapter sequences and low-quality bases are trimmed, and the trimmed data's basic statistics are recalculated. For transcriptome resequencing, the trimmed reads are aligned to the reference genome using HISAT2, a splice-aware aligner. Gene and transcript-level expression profiling is quantified with StringTie in reference-guided mode, considering strand-specific libraries when necessary. The expression profiling matrix provides read counts and normalized values like FPKM and TPM, which account for transcript length and coverage depth. Differentially expressed genes (DEGs) are identified by statistical testing between comparison groups, filtered by fold change and p-value criteria. Gene-set enrichment analysis is performed on significant gene lists to gain biological insights using Gene Ontology and KEGG pathway databases to identify relevant biological functions and pathways.

3.3. Differential Gene Expression Analysis

The heatmap displays the results of hierarchical clustering analysis using the Euclidean distance metric and complete linkage method, clustering genes and samples based on expression levels from the significant gene list (filtered by |fold change| ≥ 2 and exactTest raw p-value < 0.05). The heatmap shows that the duplicate samples from pH 11 and pH 7.5 exhibit highly similar gene expression patterns within their respective conditions, indicating minimal variation between replicates. This confirms that the samples were correctly prepared and processed, ensuring the reliability of the RNA-seq data for subsequent analyses.

The volcano plot illustrates the log2 fold change versus -log10 p-value obtained from comparing the average gene expression levels between the initial (pH 7.5) and adapted (pH 11) Synechocystis strains. The x-axis represents the log2 fold change, indicating the magnitude of gene expression differences, while the y-axis represents the -log10 p-value, reflecting the statistical significance of those differences. Blue dots represent the 67 genes that are significantly underexpressed in the adapted strains, while yellow dots represent the 40 significantly overexpressed genes. This visualization highlights the key changes in gene expression as a result of adaptation to the high pH environment.

4. Discussion

4.1. Enhanced CO₂ Capture and PHB Production

• High pH Environment: In our experiment, PHB content peaked at pH 11.0 with a PHB content of 31.3 wt%, a significant increase compared to a PHB content of 20.0 wt% observed at pH 7.5. This is due to facilitated efficient CO₂ capture as bicarbonate ions, increasing availability for photosynthesis and PHB synthesis. However, higher pH conditions past the pH threshold may lead to decreased PHB content, most likely due to disruptions in cellular metabolism caused by extreme pH stress. As a result, optimizing pH at 11 is the best in facilitating PHB synthesis and CO₂ production.
• Synergistic Effect: Adaptation to the high pH environment not only improved tolerance but also enhanced metabolic pathways involved in PHB production. This may be due to shifts in resource allocation or rerouting of metabolic pathways in response to increased CO₂ availability and pH stress. However, the exact mechanisms underlying these changes remain unclear and warrant further investigation.

4.2 Key Upregulated Genes with Potential Relevance to PHB Synthesis:

The upregulation of genes related to sugar metabolism (mannose, transketolase), energy storage (polyphosphate kinase), and other metabolic pathways suggests that Synechocystis may be rerouting its carbon and energy resources toward PHB synthesis in response to the high pH environment. The hypothetical proteins and sensory kinases may also play a role in this regulation, but their exact functions would need further study.

1. Alkaline Phosphatase: This enzyme could be involved in providing phosphate groups necessary for cellular processes. Increased phosphate availability may impact the synthesis of key intermediates in the PHB production pathway.
2. Mannose-1-phosphate Guanyltransferase: Mannose metabolism is associated with nucleotide sugars like GDP-mannose, which could link to carbohydrate metabolism. Sugars and sugar phosphates are essential in generating acetyl-CoA, the precursor for PHB synthesis.
3. Transketolase: This enzyme is a part of the pentose phosphate pathway, which produces NADPH and precursors for biosynthesis. Increased NADPH production is essential for PHB synthesis, as it provides reducing power for the biosynthesis of fatty acids that lead to PHB accumulation.
4. Polyphosphate Kinase: This enzyme is involved in polyphosphate metabolism, which could influence cellular energy storage. An increased energy pool might favor the diversion of carbon into storage polymers like PHB.
5. GDP-D-Mannose Dehydratase: This enzyme could participate in carbohydrate metabolism by modifying sugar nucleotides. This could affect carbon flux towards acetyl-CoA, which feeds into PHB biosynthesis.
6. Alpha-Mannosidase: This enzyme, involved in carbohydrate degradation, may help regulate the flow of sugar substrates, potentially influencing PHB synthesis by balancing carbon flow toward storage polymers.

Hypothetical Proteins:

Many of the hypothetical proteins might play a role in either sensing the high pH environment or directly or indirectly regulating metabolic pathways related to PHB synthesis, but further functional characterization would be required to clarify their roles.

Hybrid Sensory Kinase:

This kinase could be involved in environmental sensing and might help the cells adapt to the high pH environment, possibly triggering pathways that lead to increased storage polymer PHB as a stress response.

4.3 Key Downregulated Genes and Potential Impact on PHB Synthesis

The downregulation of genes related to photosynthesis, protein synthesis, stress response, and carbon metabolism may indicate a general shift in cellular energy and resource allocation. The cell might be conserving energy and redirecting carbon toward the production of PHB as an energy and carbon storage mechanism under the stress of high pH conditions. This metabolic rerouting likely helps Synechocystis survive in unfavorable environments by building up storage compounds like PHB, which can later be metabolized when conditions improve.

1. tRNAs and Ribosomal Proteins (tRNA-Ser, tRNA-Val, tRNA-Gln, 16S Ribosomal RNA, 50S Ribosomal Protein L16, 30S Ribosomal Protein S1, Elongation Factor TS, Elongation Factor EF-G, Peptidyl-tRNA Hydrolase):
   • Role: These genes are involved in protein synthesis and translation machinery.
   • Impact: A decrease in translation and protein synthesis may suggest that the cell is conserving energy and resources, possibly redirecting carbon and energy toward storage molecules like PHB instead of producing new proteins.
2. Phycobilisome Proteins (Phycobilisome LCM Core-Membrane Linker Polypeptide, Phycobilisome Rod-Core Linker Polypeptide CpcG, Allophycocyanin a Chain, Phycocyanin b Subunit, Phycocyanin-Associated Linker Protein):
   • Role: These are involved in light harvesting in photosynthesis.
   • Impact: Reduced activity in light-harvesting complexes may indicate a shift in energy use away from photosynthesis. In Synechocystis, this could reroute cellular energy and carbon from photosynthetic processes toward the biosynthesis of storage compounds like PHB, especially under stress.
3. Photosystem Proteins (Photosystem I Subunit II, Photosystem II PsbT Protein, Photosystem II Manganese-Stabilizing Polypeptide, Photosystem I Subunit III):
   • Role: Involved in photosynthetic electron transport and energy production.
   • Impact: Downregulation of these components may limit photosynthetic capacity. In a high-pH environment, cells may reduce photosynthesis and focus on survival strategies, including the accumulation of energy storage molecules like PHB.
4. Oxidative Phosphorylation and Carbon Metabolism (OxPPCycle Protein, Carbonic Anhydrase, Inorganic Pyrophosphatase, N-Acetyl-Gamma-Glutamyl-Phosphate Reductase):
   • Role: These proteins are involved in energy metabolism and carbon assimilation.
   • Impact: Reduced carbon fixation and energy production through oxidative phosphorylation may conserve resources for alternative carbon storage, including PHB synthesis.
5. Stress-Response Proteins (Rehydrin, OmpR Subfamily, Heat Shock Protein GrpE, DnaK Protein):
   • Role: These are stress response proteins that help the cell manage environmental stress.
   • Impact: Lower expression of stress-response proteins may indicate that the cells are adapting to the pH stress by entering a storage mode rather than a defense mode, conserving resources and enhancing PHB production as a protective mechanism.
6. Carbon Storage and Fatty Acid Biosynthesis (Gamma-Glutamyl Phosphate Reductase, Biotin Carboxyl Carrier Protein of Acetyl-CoA Carboxylase):
   • Role: Acetyl-CoA carboxylase is crucial for fatty acid synthesis, which is a precursor for PHB synthesis.
   • Impact: Interestingly, downregulation of acetyl-CoA carboxylase subunits might seem counterintuitive, but it could indicate a regulatory shift where fatty acid synthesis is redirected or slowed, allowing more carbon flux into PHB synthesis.
7. ABC Transporters and Other Regulatory Proteins (ABC1-Like Protein, Ferric Uptake Regulation Protein, Negative Aliphatic Amidase Regulator, PxcA):
   • Role: These proteins are involved in nutrient transport and regulation.
   • Impact: Reduced nutrient uptake (e.g., iron via ferric uptake regulation) could trigger a shift in metabolism, favoring internal resource storage. PHB, being a carbon and energy storage polymer, could be synthesized as a response to decreased availability of essential nutrients.
8. Hypothetical Proteins:
   • While their exact functions are unknown, they may play a role in the regulation of metabolic pathways, environmental stress response, or cellular adaptation mechanisms.

4.4 Key Downregulated Genes and Potential Impact on PHB Synthesis

Next, we performed KEGG pathway analysis to identify which pathways were involved in the upregulated and downregulated DEGs. The KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html) integrates various types of omics data, including molecular information (e.g., genome sequences, structures), chemical information (e.g., metabolism of glycans and lipids), and molecular interaction data (e.g., physical interactions and co-expression). An enrichment test based on KEGG Pathways was conducted using the significant gene list. The results indicated that downregulated genes were primarily associated with pathways such as aminoacyl-tRNA biosynthesis, photosynthesis-antenna proteins, and photosynthesis. In contrast, only one pathway was associated with upregulated genes: fructose and mannose metabolism.

5. Conclusion

Our results support the modified hypothesis that culturing Synechocystis sp. under high pH conditions selects strains with enhanced PHB production. However, when the pH exceeds a critical threshold, PHB synthesis and bacterial growth cease. The adaptive laboratory evolution (ALE) approach successfully developed strains capable of thriving in alkaline environments while efficiently converting atmospheric CO₂ into valuable bioplastics, PHB. Gene enrichment and functional annotation analysis using KEGG pathways were consistent with the key genes identified in earlier sections of the study. Notably, photosynthesis-related genes were significantly downregulated, suggesting that high pH levels impair the photosynthetic machinery of Synechocystis.

Unexpectedly, we found that fructose and mannose metabolism pathways were upregulated in high pH conditions. This upregulation likely plays a key role in enhancing PHB production, as fructose and mannose metabolism increase the flux of carbon intermediates, particularly acetyl-CoA, which is a direct precursor for PHB synthesis. Additionally, the heightened activity of these metabolic pathways could provide excess reducing power (NADH, NADPH), which is essential for driving the biosynthesis of PHB. This metabolic shift towards carbohydrate processing, while photosynthesis is suppressed, allows the cells to channel carbon and energy towards PHB production as a stress response to the alkaline environment. Overall, these findings highlight a metabolic reprogramming in Synechocystis sp. that favors bioplastic production under alkaline stress, offering insights into optimizing PHB biosynthesis in cyanobacteria.