PHB Production Through Synechocystis sp.

The Problem: Climate Change & Plastic Pollution

Our planet's atmosphere is increasingly burdened by carbon dioxide (CO₂) emissions, predominantly from burning fossil fuels for energy and transportation. The consequence of climate change is multifaceted, including but not limited to:

• Negative impact on the agricultural sector due to significant fluctuations in patterns of rainfall, seasons, and temperature, as well as frequent droughts [11]
• Detrimental effects on marine ecosystems and biodiversity due to rising temperatures and sea levels, ocean acidification, and increase in extreme weather patterns [4]

At the same time, plastic pollution has emerged as one of the environmental crises of our time. Millions of plastic waste are in our oceans, landscapes, and landfills yearly. As such, the environmental toll is visible: tremendous harm to wildlife, deaths to marine species, and interruption in the food chain [7].

While distinct at a glance, the issues of climate change and plastic pollution are interconnected. Accordingly, addressing one issue helps mitigate the other, urging the need for integrated solutions that combat both crises simultaneously:

Set of Problems

• Carbon-intensive production of plastics that contribute to global climate change
• Greenhouse gas emissions from plastic degradation that increase atmospheric CO₂ level [3-4, 7]
• In 2023, over 40 billion tons of CO₂ emissions from fossil fuels were estimated to be over 40 billion tons, with nearly 37 billion tons coming from fossil fuels. This is a record high and a 1.1% increase from 2022 levels [9]

Our Vision: Harnessing Nature’s Ingenious Process

At the heart of our vision is photosynthesis, the natural process by which plants and certain microbes convert light energy into chemical energy, absorbing CO₂ and releasing O2. This process is a model of efficiency and sustainability, converting natural resources – sunlight and CO₂ – to produce organic compounds.

Our focus is on Synechocystis sp., a photosynthetic cyanobacterium. This bacterium can be engineered to consume atmospheric CO₂ and convert it into biodegradable plastics, directly tapping into the carbon cycle [12].

This project aims to amplify this natural ability, turning Synechocystis into a bio-factory for producing polyhydroxybutyrate (PHB).

What is Synechocystis?

Synechocystis is a type of cyanobacteria, commonly referred to as blue-green algae, that holds promise in the field of synthetic biology with the following features:

• Relatively simple genetic makeup and the ease of cultivation [6]
• Capable of photosynthesis, capturing CO₂ for its growth and survival [12]
• Naturally transformation-competent and engineering versatility [3]

What is polyhydroxybutyrate (PHB)?

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate (PHA), a biodegradable bioplastic produced by certain bacteria as an energy storage material [1].

PHB is a polyester with properties similar to polypropylene, a petroleum-derived plastic, but with the crucial advantage of being biodegradable and derived from renewable resources:

Advantages of PHB

• Produced naturally under nutrient-limited conditions with excess carbon sources [10]
• Readily biodegradable, reducing pollution with no toxicity [8]
• Wide range of applications, being a potential substitute for conventional plastics [1]

In essence, we envision a sustainable production model that addresses the multifaceted question of climate change and plastic pollution.

The production of PHB from Synechocystis represents a circular approach to plastic manufacturing, converting CO₂ into a biodegradable product, then back into CO₂ at the end of its life cycle, with no net increase in atmospheric carbon levels.

Reasoning Behind Selecting Synthetic Biology

• Problem: Inefficiency of the overall project in cost, time, and output
• Solution: Adaptive Laboratory Evolution including incremental increase of pH and serial transfer allows effective CO₂ capture, Synechocystis accumulation, and PHB production
• Benefit: Generates a novel, cost-efficient, and productive method to mitigate climate change's detrimental impact

Our Hypothesis: Envisioning the Sustainable Circular Approach

The PHB biopolymer is stored as granules in the cytoplasm of Synechocystis. It accumulates in bacterial cytoplasm when there is an excess of carbon source in relation to other nutrients like nitrogen and phosphorus. Furthermore, PHB serves as fan energy and carbon reserve and can be broken down by bacteria when external carbon sources are scarce. In short, culturing Synechocystis in a carbon-excess, nitrogen/phosphorus-deficient medium is optimal.

The direct collection of atmospheric CO₂ would be the most intuitive solution to expedite PHB accumulation. However, this procedure leads to high costs due to the energy-expensive concentration step. Taking this into account, we modified our hypothesis as follows:

• Original Hypothesis: Culturing Synechocystis under increased atmospheric CO₂ levels with liquid culture media without phosphorus/nitrogen-deprived media will select the surviving bacteria that may present increased PHB storage.
• Modified Hypothesis: Culturing Synechocystis under high pH conditions with liquid culture media without phosphorus/nitrogen-deprived media will select the surviving bacteria that may present increased PHB storage.

Why high pH conditions?

In general, Synechocystis species:

• Are commonly grown in a pH range of 7 to 9 [12]
• Are photosynthetic cyanobacteria [12]
• Have a versatile metabolism, allowing them to adapt to various environmental conditions at different pH levels [12]

In specific:

At higher pH, atmospheric CO₂ initially reacts with hydroxide ions to form bicarbonate. This reaction is favored at high pH due to the presence of hydroxide ions. Soon, hydroxide ions are regenerated through further fractions involving water molecules, maintaining a high pH necessary for the continued, efficient capture of carbon dioxide:

• Step 1: CO₂ + OH- → HCO₃-
• Overall: CO₂ + 2H₂O → 2HCO₃- + 2OH-

Two Benefits Over the Initial Hypothesis:

1) Can convert into CO₂ by Synechocystis through the carbonic anhydrase enzymatic pathway because bicarbonate remains as a dominant form of dissolved inorganic carbon.

2) Maintaining a high pH prevents the reverse reaction, in which bicarbonate releases CO₂ back into the atmosphere, which would be undesirable for efficient carbon capture.

Our Approach: A Three-Step Experimentation

Step 1: Adaptive Laboratory Evolution - Increased Tolerance

First, we will perform adaptive laboratory evolution (ALE) to select the Synechocystis species that can grow in very high pH (10.5-11.2) to capture atmospheric CO₂ efficiently.

In ALE, microbes are cultured in a desired growth environment for an extended period, allowing natural selection to enrich mutant strains (altered phenotype) with improved fitness:

• Start with Baseline: Begin with a pure culture of Synechocystis known for its PHB production capabilities, ensuring a genetically stable and well-characterized strain.
• Initial Cultivation: Grow the culture under optimal conditions to establish a healthy, robust population, using a standard growth medium optimized for Synechocystis.
• Incremental Increase of pH: Gradually increase the medium's pH in small increments (by 0.1 units per cycle), allowing the culture to adapt to new pH levels.
• Apply Selective Pressure: Apply and maintain selective pressure through a high pH environment, removing cultures that do not adapt or fail to grow properly.
• Isolate Adapted Colonies: Isolate individual colonies that thrive at higher pH levels using streak plating or dilution methods using selective dyes or pH indicators.
• Serial Transfer and Monitoring: In serial passages, transfer adapted colonies to fresh medium at the new pH level and monitor growth kinetics to assess adaptation and fitness.

Why adaptive laboratory evolution (ALE)?

In microbial engineering, ALE accelerates natural evolution processes to obtain strains with enhanced features, leading to novel mutations that confer advantages under specific growth conditions. ALE yields robust strains suitable for industrial processing and generates comprehensive genetic changes not easily achievable by targeted genetic engineering alone [5].

Step 2: Screening for PHB Production

We will periodically test cultures for PHB production using appropriate assays during and following ALE. This ensures that increased pH tolerance does not come at the expense of PHB synthesis. Here, we will perform Nile Staining for PHB.

Why Nile Staining for PHB?

Nile Staining, in our experiment, visually detects and quantifies the presence of PHB granules within microbial cells. This occurs as the fluorescent dye (Nile red) selectively binds to PHB, allowing for the identification of these biopolymer inclusions under a fluorescence microscope.

Step 3: Analyze the RNA Transcriptome using RNA-seq analysis

Finally, we performed RNA sequencing (RNA-seq), a next-generation sequencing (NGS) method, to analyze the RNA transcriptome of both the initial and final Synechocystis strains that adapted to high pH conditions and exhibited increased PHB production.

Why Next Generation Sequencing (NGS)?

Compared to conventional methods, such as Sanger sequencing, NGS is a high-throughput, cost-effective method with improved speed, versatility, and accuracy [2]. Through RNA-sequencing (RNA-seq), we can focus on the regions of interest relevant to the production of PHB and thereby uncover the specific molecular mechanism by which we amplified the production of PHB.

How does RNA-seq differ from more traditional synthetic biology methods?

RNA sequencing (RNA-seq) offers a distinct approach compared to standard methods in synthetic biology, particularly when focusing on understanding and optimizing the production of compounds like polyhydroxybutyrate (PHB).

Discovery vs. Design: RNA-seq is more exploratory, aimed at discovering how the Synechocystis's natural regulatory networks respond to adopting high pH and high PHB production, whereas standard methods are more design-oriented, focusing on constructing and optimizing predefined genetic circuits.

Global vs. Targeted: RNA-seq provides a global view of gene expression, capturing the broader cellular response, while standard methods often target specific genes or pathways for manipulation based on prior knowledge.

Mechanistic Insight vs. Functional Output: RNA-seq helps elucidate the mechanistic basis of adopting high pH and high PHB production by revealing changes in gene expression. In contrast, standard synthetic biology methods primarily focus on achieving the desired functional output, such as higher PHB yield, through direct genetic manipulation.

Overview of the Project: Climate Change & Plastic Pollution Mitigation

This year, our project tackled three biological hallmarks relevant to the advancement of both the natural world and synthetic biology academia:

1) CO₂ reduction through biological innovation: Our project is to demonstrate a biological approach to CO₂ reduction. Our measure of success will be quantifiable: the amount of CO₂ converted into PHB, demonstrating a tangible impact on carbon levels.

2) Sustainable bioplastic production: We aim to establish a proof-of-concept for producing biodegradable plastics through an environmentally friendly and renewable method. We aim to optimize the production process to make it scalable and cost-competitive with traditional plastic production methods.

3) Optimization of biological systems: A key objective is to optimize the PHB synthesis pathway in Synechocystis to achieve the highest yield possible. This involves tweaking genetic and environmental conditions to push the boundaries of biological production. We plan to explore various bioprocessing technologies that could further enhance efficiency and reduce costs, making our method a viable commercial option.

References

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2. Canadian Agency for Drugs and Technologies in Health. (2014, February 6). Summary of findings – cost-effectiveness of next generation sequencing. Next Generation DNA Sequencing: A Review of the Cost Effectiveness and Guidelines - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK274079/.

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6. Jelena Spasic, Oliveira, P., Pacheco, C., Kourist, R., & Tamagnini, P. (2022). Engineering cyanobacterial chassis for improved electron supply toward a heterologous ene-reductase. Journal of Biotechnology, 360, 152–159. https://doi.org/10.1016/j.jbiotec.2022.11.005.

7. Kida, M., Ziembowicz, S., & Koszelnik, P. (2023). Decomposition of microplastics: Emission of harmful substances and greenhouse gases in the environment. Journal of Environmental Chemical Engineering, 11(1), 109047. https://doi.org/10.1016/j.jece.2022.109047.

8. McAdam, B., Fournet, M. B., McDonald, P., & Mojicevic, M. (2020). Production of polyhydroxybutyrate (PHB) and factors impacting its chemical and mechanical characteristics. Polymers, 12(12), 2908. https://doi.org/10.3390/polym12122908.

9. Stanford Doerr School of Sustainability. (2023). Global carbon emissions from fossil fuels reached record high in 2023. Stanford University. https://sustainability.stanford.edu/news/global-carbon-emissions-fossil-fuels-reached-record-high-2023.

10. Sirohi, R., Lee, J. S., Yu, B. S., Roh, H., & Sim, S. J. (2021). Sustainable production of polyhydroxybutyrate from autotrophs using CO₂ as feedstock: Challenges and opportunities. Bioresource Technology, 341, 125751. https://doi.org/10.1016/j.biortech.2021.125751.

11. Yoro, K. O., & Daramola, M. O. (2020, January 1). Chapter 1 - CO₂ emission sources, greenhouse gases, and the global warming effect (M. R. Rahimpour, M. Farsi, & M. A. Makarem, Eds.). ScienceDirect; Woodhead Publishing. https://www.sciencedirect.com/science/article/abs/pii/B9780128196571000013.

12. Zhu, J., Liu, J., Huang, Y., Lin, S., & Jiang, Y. (2023). Genome editing of Synechocystis sp. PCC 6803 for enhanced production of bioplastics polyhydroxyalkanoates. Microbial Cell Factories, 22(1), 1–13. https://doi.org/10.1186/s12934-023-02003-x.