주메뉴 바로가기 본문 바로가기
Engineering

PHB Production Through Synechocystis sp.

Design

Engineering Synechocystis sp. for Enhanced PHB Production under High pH Conditions

Our engineering strategy aimed to enhance polyhydroxybutyrate (PHB) production in Synechocystis sp. PCC 6803 under high pH culture conditions. This was achieved through an iterative approach involving Adaptive Laboratory Evolution (ALE), high-throughput PHB production screening, and RNA sequencing (RNA-seq) for transcriptional profiling.

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

ALE was implemented to evolve Synechocystis towards greater tolerance of alkaline conditions. The experimental design began by gradually increasing the pH from 7.5 to a target of 11.5 in small increments of 0.1 pH units per cycle. Each pH adjustment was followed by a stabilization period, allowing the culture to adapt to the higher pH while maintaining growth. Selection pressure was applied as the pH increased, favoring strains that showed robust growth under alkaline conditions.

This method leveraged natural selection to optimize phenotypes, enhancing both growth and PHB production in alkaline environments. Regular monitoring of growth rates and cell morphology confirmed that Synechocystis adapted to these conditions with minimal impact on cellular structure, retaining its physiological integrity even at pH 11.0. However, growth was significantly inhibited at pH levels of 11.5, which defined an upper threshold for successful adaptation.

2. PHB Production Screening and Optimization

PHB production was quantified across a range of pH levels using a fluorescence-based assay. Synechocystis cells were stained with Nile red, a fluorescent dye that binds to PHB granules, allowing for the visualization and quantification of PHB accumulation. Fluorescent intensities correlated with PHB content, establishing an efficient screening process to identify high-PHB-producing strains.

Results indicated that PHB production was highest at pH 11.0, reaching 31.3 wt%, significantly higher than the 20.0 wt% observed at pH 7.5. This improvement was attributed to enhanced metabolic activity and increased availability of bicarbonate ions under moderately alkaline conditions. At pH 11.5, however, PHB content dropped to 17.3 wt%, suggesting a stress-induced reduction in biosynthesis at extreme pH levels.

3. RNA-seq Analysis for Transcriptional Insights

To further understand the genetic basis for enhanced PHB production, RNA sequencing was conducted on Synechocystis strains grown at pH 7.5 and pH 11.0. Differential gene expression analysis revealed significant changes in metabolic pathways linked to PHB synthesis. Specifically, genes involved in sugar metabolism (e.g., mannose-1-phosphate guanyltransferase), energy storage (e.g., polyphosphate kinase), and the pentose phosphate pathway (e.g., transketolase) were upregulated in high pH conditions.

Conversely, genes associated with photosynthesis and protein synthesis were downregulated, indicating a shift in cellular resource allocation toward energy storage (PHB production) as a stress response to alkaline conditions. These findings suggest that high pH induces metabolic reprogramming in Synechocystis, favoring bioplastic production as a survival mechanism under stress.

Building Process

The development of our engineered Synechocystis sp. PCC 6803 for enhanced polyhydroxybutyrate (PHB) production under high pH conditions was a multi-phase process that integrated experimental evolution, synthetic biology techniques, and molecular biology methods.

1. Strain Preparation and Cultivation

We began by sourcing a wild-type strain of Synechocystis sp. PCC 6803, a unicellular freshwater cyanobacterium known for its natural ability, to produce PHB and capture atmospheric CO2 via photosynthesis. The initial cultures were grown in a modified BG-11 medium at pH 7.5 under standard laboratory conditions (light intensity, temperature, and CO2 concentration). The goal was to establish a baseline growth curve and PHB production levels under neutral pH conditions before subjecting the cells to evolutionary pressure for high pH adaptation.

[Figure 1. A modified BG11 medium without phosphorus and nitrogen was used to enhance the PHB production.]

2. Adaptive Laboratory Evolution (ALE) Setup

To drive the evolutionary adaptation of Synechocystis to high pH environments, we designed a gradual pH-incremental system. The process started by cultivating cells at pH 7.5 and then gradually increasing the pH of the growth medium by 0.1 units per cycle. After each pH increase, the cultures were allowed to grow and adapt until stable growth was observed at the new pH level. This approach mimicked natural selection in a controlled laboratory environment, favoring phenotypes that could survive and thrive in progressively alkaline conditions.

We regularly monitored cell growth using optical density (OD750), and cell morphology was assessed using brightfield microscopy. Growth curves were plotted to determine the optimal pH range for both growth and PHB production. Over time, this process resulted in Synechocystis strains that could tolerate pH levels up to 11.5, although optimal PHB production was observed at pH 11.0.

[Figure 2. Synechocystis sp. growing at pH 10.]

3. PHB Production Assay

After the ALE process, we shifted our focus to identifying the evolved strains that exhibited enhanced PHB production. We used a fluorescence-based quantification assay to screen for PHB-producing strains. Synechocystis cells were stained with Nile red dye, which selectively binds to PHB granules, enabling visualization under a fluorescence microscope. This assay allowed us to quantify intracellular PHB content by measuring fluorescence intensity, which was then correlated with actual PHB production via chloroform-methanol extraction and gravimetric analysis.

Using this screening method, we identified the most promising strains capable of producing high levels of PHB at pH 9.5 to 11.0. The results demonstrated a significant increase in PHB content under moderately alkaline conditions, validating the success of the ALE approach.

[Figure 3. PHB extracted (white particles) from the Synechocystis sp. pH7.5 (left) and pH11 (right)]

4. Molecular Engineering and Genetic Analysis

To further understand the genetic and molecular mechanisms underlying the enhanced PHB production, we performed RNA sequencing (RNA-seq) on Synechocystis strains grown at both pH 7.5 and pH 11.0. This step involved:

  • RNA Extraction: We used Trizol reagent to extract total RNA from the cultured cells.
  • Library Preparation: The RNA samples were prepared using the Illumina TruSeq RNA library kit, and the quality and quantity of the libraries were verified before sequencing.
  • Sequencing and Data Analysis: The libraries were sequenced using the Illumina platform, and the raw data was processed for quality control and alignment to the Synechocystis reference genome. Differentially expressed genes (DEGs) were identified, and pathway analysis was conducted to link gene expression changes to PHB biosynthesis.

The RNA-seq analysis revealed significant upregulation of genes involved in carbon metabolism, energy storage, and biosynthetic pathways, providing insights into the metabolic reprogramming of Synechocystis in high pH conditions. Also, the downregulation of genes involved in photosynthesis indicates the high pH induced the cellular stress in Synechocystis sp.

[Figure 4. Gene-Enrichment and Functional Annotation Analysis using KEGG pathway identifies four significant pathways in both downregulated and upregulated genes.]

Test

To validate the effectiveness of our engineered Synechocystis sp. PCC 6803 for enhanced polyhydroxybutyrate (PHB) production under high pH conditions, a series of tests were performed across three key areas: growth tolerance, PHB production, and genetic response.

  1. Growth Tolerance Under High pH Conditions
    • Objective: Assess the evolved Synechocystis sp. strains' ability to grow in increasingly alkaline environments.
    • Method: Cultures were grown in modified BG-11 medium, and the pH was gradually increased from 7.5 to 11.5 in 0.1-unit increments, following the Adaptive Laboratory Evolution (ALE) method.
    • Measurements: Growth was monitored by measuring optical density at 750 nm (OD750), with stabilization times recorded at each pH increment.
    • Results: Synechocystis strains showed robust growth up to pH 11.0, with a significant reduction in growth observed at pH 11.5. Optimal growth and stability were achieved at pH 11.0, confirming the successful adaptation of the strains to high pH environments.
  2. PHB Production Screening
    • Objective: Quantify PHB production in Synechocystis strains across different pH levels.
    • Method: Nile red dye was used to stain the cells, allowing for fluorescence-based quantification of PHB granules. Fluorescence intensity was correlated with PHB production, confirmed through chloroform-methanol extraction and gravimetric analysis.
    • Results: Strains grown at pH 11.0 produced the highest PHB content, reaching 31.3 wt%, a significant improvement over the 20.0 wt% observed at pH 7.5. PHB content decreased to 17.3 wt% at pH 11.5, indicating that extreme pH levels induced stress, reducing biosynthetic capacity.
  3. RNA-seq Transcriptional Analysis
    • Objective: Identify genetic changes contributing to enhanced PHB production under alkaline conditions.
    • Method: RNA sequencing was performed on Synechocystis strains grown at pH 7.5 and pH 11.0. Total RNA was extracted, sequenced using the Illumina platform, and analyzed for differential gene expression.
    • Results: RNA-seq analysis revealed upregulation of genes related to carbon metabolism, energy storage, and PHB biosynthesis under high pH conditions. In contrast, downregulation of genes involved in photosynthesis suggested a shift in resource allocation towards PHB production, likely as a survival response to stress.

These tests confirm that our engineered Synechocystis sp. PCC 6803 not only thrives in high pH environments but also exhibits significantly enhanced PHB production. The combination of phenotypic adaptation and genetic reprogramming underscores the effectiveness of the engineering approach.

Learn

Through the 'Build' and 'Learn' phases of our Engineering Cycle, we actively engaged with the core principles of iGEM and Synthetic Biology, iterating and optimizing our approach to enhance PHB production in Synechocystis sp. under high pH conditions.

Learn #1: Address project challenges using synthetic biology tools and experimental techniques to meet expected outcomes.

Our goal was to enhance polyhydroxybutyrate (PHB) production in Synechocystis sp. PCC 6803 while ensuring its ability to thrive under high pH conditions. We addressed key project challenges and achieved our objectives through the following:

  • Build: Implemented Adaptive Laboratory Evolution (ALE) by gradually increasing the culture pH from 7.5 to 11.5 to select for strains with enhanced alkaline tolerance.
  • Build: Regularly monitored cell growth (via OD750) and assessed cell morphology using brightfield microscopy to ensure physiological integrity during the adaptation process.
  • Build: Used Nile red staining and fluorescence microscopy to screen for high PHB-producing strains under varying pH conditions.
  • Test: Quantified PHB production through fluorescence-based assay, confirmed with chloroform-methanol extraction and gravimetric analysis.
  • Test: Performed RNA sequencing (RNA-seq) to identify differentially expressed genes involved in carbon metabolism and PHB synthesis at pH 7.5 and 11.0.

Our experiments validated that the adapted Synechocystis strains exhibited enhanced PHB production at pH 11.0, with the highest PHB content recorded at 31.3 wt%, compared to 20.0 wt% at pH 7.5. Additionally, RNA-seq analysis revealed the upregulation of genes linked to energy storage and PHB biosynthesis, confirming the success of our engineering approach in optimizing PHB production under alkaline stress.

Learn #2: Identify and document improvements for the next iteration of the engineering cycle.

While our first iteration yielded promising results, several limitations emerged:

  • PHB production decline at extreme pH: We observed a decrease in PHB content at pH 11.5, likely due to stress-induced metabolic disruption. In the next iteration, we would explore co-culturing Synechocystis with symbiotic bacteria that could mitigate stress and support PHB biosynthesis at extreme pH levels.
  • Gene expression variability: Although RNA-seq provided insights into differentially expressed genes, we could not quantify the precise contribution of each gene to PHB production. A future approach could involve CRISPR-based gene editing to individually modulate key genes, helping us better understand their role in the metabolic reprogramming of Synechocystis.

These refinements will improve our understanding of how Synechocystis responds to high pH stress and could lead to further optimization of PHB production for industrial applications.

Learn #3: Design and build a new Part, measure its performance, document whether it worked or not, and propose how the results would inform the next design or steps.

In this project, we contributed two new Parts to the iGEM Registry of Standard Biological Parts, specifically focusing on genes that are differentially expressed in Synechocystis sp. PCC 6803 under high pH conditions. These Parts consist of both upregulated and downregulated differentially expressed genes (DEGs) responsible for enhanced PHB production under alkaline stress.

These DEGs were identified through RNA-seq analysis and include genes involved in carbon metabolism, PHB biosynthesis, and energy storage pathways, such as mannose-1-phosphate guanyltransferase and polyphosphate kinase. By documenting these genes as Parts, we provide the iGEM community with potential targets for engineering high PHB production in Synechocystis or other organisms.

Future Implications:

  • Application in Synthetic Biology: The upregulated genes could be used as potential overexpression targets for other cyanobacteria or algae species, broadening the scope of organisms capable of high PHB production. Conversely, the downregulated genes provide insight into metabolic pathways that may need to be repressed to optimize PHB biosynthesis under stress conditions.
  • Further Testing and Iteration: Future iterations will involve validating the specific contribution of each gene to PHB production through CRISPR-based knockouts or overexpression in Synechocystis. This will allow us to fine-tune the genetic network for even higher yields of PHB.
  • Industrial Applications: These Parts pave the way for the development of biotechnological solutions to scale PHB production for industrial applications. The ability to engineer robust, high-PHB-producing strains that can thrive under various environmental conditions, particularly in alkaline environments, could be a game-changer for sustainable bioplastics.

In summary, these newly designed Parts contribute valuable genetic tools for future synthetic biology projects, opening pathways for optimizing PHB production and expanding the use of Synechocystis as a bioplastic-producing organism in industrial contexts.