Energy Crisis and Solar Energy

Humanity grapples with a multitude of challenges stemming from climate change and the unsustainable rise in energy demands. Despite the global implementation of efficient energy policies, the demand for energy is anticipated to surge by 32% from 2013 levels, reaching an astounding 17,934 million tons of oil equivalent by 2040¹. This trajectory not only propels oil production by over 15% but also exacerbates global CO2 emissions. To mitigate these effects, a pivotal step involves the decarbonization of the economy, necessitating the formulation of innovative energy policies and, crucially, a reduction in reliance on fossil fuels within the energy and materials supply chain². The key lies in the development of novel, sustainable technologies.

At present, addressing the escalating consumption and demand for energy stands out as a primary concern³. A solution lies in the adoption of renewable and alternative sources of electrical energy. Solar energy systems have emerged as the predominant player in renewable energy technologies (see Figure 1). Their appeal lies in requiring less maintenance than wind turbines, being visually less disruptive, causing minimal alterations to ecosystems compared to hydropower stations, and being more cost-effective and versatile in construction. In contrast to biomass, solar energy boasts much higher efficiency (19% vs 1% for biomass)⁴. In essence, solar panels lend themselves to easier installation in urban settings and on residential rooftops compared to other renewable energy technologies.

However, solar is not a perfect technology, and numerous issues can stem from the unsustainable way current silicon-based solar panels are manufactured and used. The vast majority (95%) of current solar panels use silicon as a semiconductor. However, mining transporting, and processing of the raw material is extremely energy and labor-intensive. Temperatures of up to 2000 degrees Celsius are required to purify the silicon and for every 50,000 tons produced, 500,000 tons of carbon dioxide gas are released into the atmosphere. Additionally, solar cells require plenty of space, often necessitating the destruction of fields for large-scale installations, incurring a heavy ecological burden on their surroundings. Further to this are issues pertaining to recycling and the life cycle of current panels being 20-25 years and non-circular. There are few current or planned efforts to deal with the significant volumes of silicon waste from degraded panels, and this issue will pose significant ecological burdens with toxic heavy metal leakage.

Bio-photovoltaics (BPVs) represent a groundbreaking approach that harnesses the capabilities of photosynthetic microorganisms, such as cyanobacteria or algae, to generate electricity from CO2 and sunlight. Within a bio-photovoltaic panel, electrons produced during photosynthesis are utilized to generate electrical current. Cyanobacteria reside in an anodic chamber, where oxidation occurs, and as photosynthesis generates electrons, these excited electrons traverse the anode, moving towards the reductive electrode (cathode) in the cathodic chamber. This electron flow propels an electric current through an external circuit (see figure 2). In comparison to inorganic counterparts like conventional photovoltaic systems (PVs), such as silicon-based solar panels, BPVs offer several advantages:

1. Renewable and Sustainable: Genetically engineered bacteria in BPVs can be sustainably grown, establishing them as a renewable energy source. Conversely, PVs necessitate the extraction and processing of raw materials, contributing to environmental degradation and resource intensiveness.
2. Biodegradable and Environmentally Friendly: Engineered bacteria in BPVs are inherently biodegradable, minimizing their environmental impact. In contrast, PVs have a limited lifespan and pose challenges in disposal and recycling due to their complex composition and toxic materials.
3. Versatility: BPVs offer a level of personalization not found in traditional PV systems. Engineered bacteria can be modified to optimize performance based on the installation environment, using different strains for specific conditions and energy requirements.
4. Carbon Neutral: BPVs can achieve carbon neutrality as photosynthetic microorganisms consume CO2 to produce electricity. On the other hand, the manufacturing of PV systems involves energy-intensive processes that generate greenhouse gases during production.

Despite their potential, bio-photovoltaics face challenges with low conversion efficiencies primarily attributed to inefficient extracellular electron transfer (EET) across the outer cell membrane⁸. Furthermore, the precise mechanisms governing electron transfer between microorganisms and the electrode remain poorly understood⁹.

Oxygenic photosynthetic organisms initiate a complex series involving charge separation and the delivery of electrons and photons through a phosphorylation cascade. This intricate process incorporates three membrane-bound protein complexes: the cytochrome bf complex, photosystem I (PSI), and photosystem II (PSII) (see figure 3). Working in tandem, these proteins sequentially transfer electrons from water (H2O) to nicotinamide adenine dinucleotide phosphate (NADP+), generating oxygen as a by-product and assimilating CO2. Mobile carrier molecules, plastocyanin (PC), and plastoquinone (PQ) facilitate the movement of electrons within these protein complexes. PQ, a hydrophobic molecule, plays a central role in the electrogenic pathway in cyanobacteria, acting as an energy shuttle between PSII electron flow and cytochrome b6f.

Putative routes for electron transfer from thylakoid membrane constituents (photosystems) to an anode. The routes described in the diagram are both mediated electron transfer and potential transfer mechanisms through pili or other membrane configurations. The dashed red routes are conjectured electron transfer pathways. Top right: a schematic depiction of a prototypical BPV arrangement.

Addressing the challenge of low conversion efficiencies entails exploring avenues such as engineering optimized carbon sequestration mechanisms in microorganisms or devising a transport complex in the outer membrane to facilitate electron transport from the microorganism to the electrode.

Various cyanobacterial species, including Leptolyngbia, Synechococcus, Anabaena variabilis M-2, Oscillatoria limnetica, Nostoc, and Arthrospira platensis, have been employed in bio-photovoltaic research. The highest reported power density to date was 610 mW/m2 using Synechococcus sp. BDU 140432. A comparative study in 2010 evaluated the electrogenic activity of different wild-type cyanobacteria genera and an undefined phototrophic consortium¹⁰. The highest electrogenic yield was observed in the microbial consortium from a freshwater pond, although its exact species composition remains unknown. Notably, a pure culture of Synechocystis sp. PCC 6803 exhibited only a quarter of the highest activity and half the performance of seven other pure cultures tested in the study.

However, Synechocystis sp. PCC 6803 emerged as the most widely used cyanobacterium in bio-photovoltaic research, likely owing to its status as a model organism in photosynthesis research. It is well-characterized with a fully sequenced genome and abundant tools for genetic engineering compared to other species, such as the CyanoGate assembly system, which was adapted and designed for this organism¹¹.

The MTR Complex - Powering Our Solar Cells

Previous studies have consistently demonstrated low photocurrent production in wild-type cyanobacteria and algae¹²,¹³,¹⁴,¹⁵. The challenge in enhancing this process lies in the incomplete understanding of extracellular electron transfer (EET) mechanisms. In contrast, non-photosynthetic microorganisms with metal-reducing capabilities, such as Shewanella oneidensis MR-1 and Geobacter metallireducens, exhibit an inherent EET pathway that positions them as ideal candidates for bioelectricity applications¹⁶. Among these, S. oneidensis MR-1 boasts a well-characterized EET pathway involving the MtrCAB complex¹⁷,¹⁸. Despite unsuccessful attempts to solve its crystal structure, a homologous complex from Shewanella species, Shewanella baltica OS185, was successfully crystallized, and its crystal structure has been elucidated (see Figure 4)¹⁹.

The electron transfer pathway, depicted in Figure 5, remains an area requiring comprehensive understanding. Current knowledge suggests that electrons, derived from the substrate's oxidation, ultimately find their way to the periplasmic decaheme cytochrome MtrA during anaerobic respiration. In the subsequent step, electron transfer to the outer membrane β-barrel protein MtrB establishes direct contact between MtrA and MtrC. MtrC, in turn, undergoes re-oxidation through the reduction of terminal electron acceptors, such as electrodes, achieved via either direct or mediated electron transfer. The former demands physical contact between the electrode and outer membrane redox proteins, while the latter involves a soluble electron shuttle capable of being reduced by outer membrane cytochromes and ferrying the electrons to the electrode in a reduced state.

Metal-reducing microorganisms exhibit heightened electron transfer efficiencies compared to photosynthetic bacteria. However, their bioelectricity generation relies on the oxidation of specific and limited organic substrates. Unlike photosynthetic microorganisms, they lack the CO2 sequestering mechanism, and crucially, the light-harvesting machinery essential for water-splitting. Without this machinery, the reaction becomes thermodynamically unfavorable, presenting a pivotal challenge for the development of the next generation of renewable energy technologies dependent on hydrogen production and a clear advantage for biophotovoltaics.

The MtrCAB complex has already been successfully expressed in E. coli for applications in microbial fuel cells. While MtrA has been heterologously expressed in Synechocystis sp. PCC 6803, MtrB and C are yet to be expressed in a cyanobacterial system. Notably, cells expressing MtrA did not exhibit significant improvements in conductivity under dark conditions compared to the wild type. However, there was a noteworthy 30% increase in the peak oxidation current, despite the cells expressing MtrA at a low level. The heightened photocurrent observed in cells expressing MtrA was directly attributed to MtrA expression, prompting the hypothesis that further enhancements in photocurrent could be achieved by expressing outer membrane proteins, such as MtrB, MtrC, OmcS, and CymA. This expression would facilitate direct electron transfer to the electrode surface and is the focus of this subproject.

Utilizing Biophotovoltaic Cell Culture Waste as Artificial Pollen Substitute for Circular Economy

In the face of the multifaceted challenges posed by the climate crisis, our commitment extends beyond addressing human-related effects, recognizing that ecosystems and vulnerable species are on the front lines of impending environmental changes. With an understanding that disruptions to food chains and ecosystems are inevitable consequences, our approach embraces the complexity of the crisis. To mitigate these impacts, we are engineering our Cyanobacteria strain Synechocystis sp. PCC 6803 for a pollinator-friendly solar panel—a subproject intricately woven into the fabric of a circular economy. This endeavor goes beyond merely producing clean energy; it involves devising a solution for the internal recycling of residual waste generated during the energy production stage. Such an initiative aligns seamlessly with our overarching goal of minimizing the ecological footprint of solar panels on land use and the environment. By repurposing panel cell culture waste into artificial pollen, we not only contribute to a more ecologically friendly life cycle but also address the critical need for sustainable practices in the broader context of the climate crisis. This holistic approach recognizes the interconnectedness of environmental challenges and endeavors to forge solutions that benefit both humanity and the delicate balance of our ecosystems.

Rising global temperatures and climate change have disrupted flowering seasons, affecting the synchronization of pollinators and posing a threat to biodiversity. Conventional solutions involve planting more flowers, but the evolving impact of climate change challenges the efficacy of this approach. To ensure a sustainable food source for pollinators, independent of climate fluctuations, we explore the possibility of artificially producing the nutritional content found in natural pollen within a host organism.

The foundation for this project draws heavily from the research conducted by Ricigliano et al. in 2021. Their work explores the potential use of Cyanobacteria as a substitute for natural pollen in pollinator diets, providing a conceptual framework for our endeavor. The objective is to enhance two crucial amino acids, histidine and lysine, in Cyanobacteria to make it a more viable alternative to natural pollen.

Studies indicate that pollinators resort to unconventional methods, such as damaging flower leaves, to accelerate floral pollen production. Traditional alternatives like sucrose solutions lack the nutritional richness of natural pollen. The Cyanobacteria genus Spirulina has emerged as a potential substitute due to its photosynthetic activity, offering a sustainable approach. However, the deficiency of histidine and lysine in Cyanobacteria compared to natural pollen necessitates further research to optimize its nutritional content.

Histidine Biosynthesis

The biosynthesis pathway of histidine (BPH) in bacteria is a complex and highly regulated process that involves several enzymatic reactions, being a model system for studying relationships between enzymes and intermediate flow in bacteria. The BPH typically starts with the conversion of 5-phosphoribosyl-1pyrophosphate (PRPP) and ATP to phosphoribosyl-ATP (PR-ATP) through the enzyme phosphoribosyl pyrophosphate synthetase (PRPP synthetase), regulated by the protein HisG. Subsequent steps involve several enzymatic reactions, including the transformation of PR-ATP to imidazole ribotide, followed by the formation of histidine monophosphate (HMP) and histidinol. The final conversion of histidinol to histidine is catalyzed by histidinol dehydrogenase. This pathway is tightly regulated by feedback inhibition, ensuring that the production of histidine is balanced with the cellular requirements.

Lysine Biosynthesis

In bacteria, the biosynthesis pathway of lysine (BPL) is a crucial metabolic route that leads to the formation of this essential amino acid. The pathway begins with aspartate, which undergoes a series of reactions involving enzymes such as aspartokinase, dihydrodipicolinate synthase, and dihydrodipicolinate reductase, ultimately leading to the formation of diaminopimelate (DAP). DAP serves as a key intermediate in the pathway, and its subsequent conversion involves several enzymatic steps to yield lysine. The last step in BPL is catalysed by lysine racemase, which is responsible for producing the biologically active L-lysine isomer. BPL is intricately regulated through feedback inhibition and other regulatory mechanisms to also ensure a balanced supply of this amino acid for various cellular processes. The gene dapA, encoding the enzyme dihydrodipicolinate synthase (DapA), was specifically a central gene involved in increasing lysine production which could be involved in the first stages of lysine biosynthesis. Even individual expression of dapA increased the production of lysine within this bacterium and its for this reason we chose to overexpress DapA.

Biosafety

It is important to consider how our GMO-driven solar panel could fit in the real-world and what potential challenges in relation to biosecurity and GMO regulations it could face. First, given that GM cyanobacteria would be grown outdoors, although within the solar panel, the potential for these cells to escape into the environment is elevated beyond that of a typical industrial microbial cultivation in bioreactors within designated facilities. Thus, to receive the government approval of the product, it might be essential to implement additional containment strategies into our system.

One approach to reduce the probability of survival of genetically engineered organisms outside of the laboratory or industrial setting is genetic biocontainment. Many strategies have been developed that include active strategies like killing of escaped cells by expression of toxic proteins and passive strategies that use knockouts of native genes to reduce fitness outside of the controlled environment of labs and industrial cultivation systems (Sebesta et al., 2022).

For genetic containment of our system, we designed and built a kill switch with the purpose to prevent the escape and survival of GMO Synechocystis in the environment. The kill switch is based on the constitutive expression of a toxin, NucA nuclease (Ghosh et al., 2005), from a different cyanobacterium Anabaena sp. PCC 7120 together with its inhibitor NuiA (Meiss et al., 1997), which we put under the control of a zinc-inducible promoter. We chose to use zinc as the inducer because it was previously shown that Synechocystis tolerance to Zn2+ ions is relatively high in comparison to other heavy metals with IC50 (half growth inhibitory concentration) ranging between 8 and 16 µM Zn2+ (Blasi et al., 2012). Moreover, it was also found that Synechocystis had a native promoter regulating the copMRS operon involved in copper response, and that this promoter could also be induced by zinc (Čelešnik et al., 2016).

NucA is a non-specific DNA/RNA nuclease from the cyanobacterium Anabaena sp. PCC 7120 that can cut single-stranded and double-stranded DNA and RNA (Ghosh et al., 2005). Nucleases of this type are present in several bacterial species and are believed to have evolved to serve for nutritional purposes and sometimes as bacteriocides (Meiss et al., 1998). NucA requires divalent metal ions like Mn2+ or Mg2+ as cofactors, the optimal concentration for these being around 5 mM (Meiss et al., 1998). NucA activity was shown to decrease with increasing concentration of monovalent salt (Meiss et al., 1998). NucA contains a ββα metal finger motif and a hydrated divalent metal ion at the active site (Ghosh et al., 2005). Ghosh et al. (2005) proposed that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state. NucA forms a 1:1 complex with its specific inhibitor NuiA (Figure 2) from the same bacterium Anabaena sp. PCC 7120, leading to complete inhibition of NucA (Meiss et al., 1998). The inhibition involves an unusual divalent metal ion bridge between the nuclease and its inhibitor (Ghosh et al., 2007). The C-terminal Thr-135 hydroxyl oxygen in NuiA interacts directly with the catalytic Mg2+ in the nuclease active site, while Glu-24 in NuiA extends into the active site, mimicking the charge of a scissile phosphate (Ghosh et al., 2007). NuiA residues Asp-75 and Trp-76 contribute to the strength and specificity of the interaction (Ghosh et al., 2007).

Our idea is that within the solar panel, the growth medium would be supplemented with Zn2+ ions to induce NuiA antitoxin expression, thus counteracting NucA nuclease effects. In the environment, Zn2+ concentration would usually be too low for the efficient NuiA induction, thus NucA would cut cellular DNA and RNA, killing escaped cells. One of the reasons why we chose this specific nuclease as a toxin in our system is because it is non-specific, meaning that it can cut all nucleic acids, including single-stranded and double-stranded, DNA and RNA (Ghosh et al., 2005). Thus, it has the potential not only to kill the escaped GM bacteria but also prevent the horizontal gene transfer. Importantly, we designed and built the kill switch on a plasmid due to time constraints of the iGEM competition; however, bacteria can easily lose plasmids, thus ideally in a real-world scenario, the kill switch would have to be incorporated into the genome. Moreover, it would be necessary to remove all antibiotic resistance genes that are generally used for selection in cloning experiments to avoid the potential spread of these genes to other organisms in the environment.

Despite the neat design of many genetic containment systems, including kill switches, there are multiple problems associated with them. First, random mutations in any component of the kill switch can inactivate it, for example, in our case, mutations in the zinc promoter could impair its inducibility and lead to constitutive expression of antitoxin allowing cell survival in the environment, or mutations in the nuclease could impair its structure inactivating it or reducing its toxicity. In addition, when testing for the elimination of bacteria, it should be noted that available methods for monitoring cell survival, like optical density measurements of bacterial growth and colony-forming unit counting for measuring cell viability, are typically done in carefully controlled experiments, which may fail to capture mechanisms of escape that may arise in the more complex natural environment, and thus cannot prove complete eradication of genetically engineered strains, as there is always a possibility that the cells could be surviving in a dormant state.

References

• Blasi, B., Peca, L., Vass, I. and Kós, P.B., 2012. Characterization of stress responses of heavy metal and metalloid inducible promoters in Synechocystis PCC6803. J. Microbiol. Biotechnol, 22(2), pp.166-169.
• Čelešnik, H., Tanšek, A., Tahirović, A., Vižintin, A., Mustar, J., Vidmar, V. and Dolinar, M., 2016. Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biology Open, 5(4), pp.519-528.
• Ghosh, M., Meiss, G., Pingoud, A., London, R.E. and Pedersen, L.C., 2005. Structural insights into the mechanism of nuclease A, a ββα metal nuclease from Anabaena. Journal of Biological Chemistry, 280(30), pp.27990-27997.
• Ghosh, M., Meiss, G., Pingoud, A.M., London, R.E. and Pedersen, L.C., 2007. The nuclease a-inhibitor complex is characterized by a novel metal ion bridge. Journal of Biological Chemistry, 282(8), pp.5682-5690.
• Meiss, G., Franke, I., Gimadutdinow, O., Urbanke, C. and Pingoud, A., 1998. Biochemical characterization of Anabaena sp. strain PCC 7120 non-specific nuclease NucA and its inhibitor NuiA. European journal of biochemistry, 251(3), pp.924-934.
• Sebesta, J., Xiong, W., Guarnieri, M.T. and Yu, J., 2022. Biocontainment of genetically engineered algae. Frontiers in Plant Science, 13, p.839446.

Cyanogate

CyanoGate is a versatile genetic engineering system developed to address the synthetic biology gap in cyanobacteria. Leveraging the Golden Gate MoClo kit, CyanoGate unites cyanobacteria with plant and algal systems, offering a standardized modular cloning approach. Developed by the McCormick lab at the University of Edinburgh, CyanoGate facilitates the generation of knock-outs, integrations, and multigene expression systems in cyanobacteria.

Cyanobacteria, crucial for their ecological importance and potential biotechnological applications, have been underutilized in synthetic biology. CyanoGate aims to bridge this gap by providing a suite of genetic parts and acceptor vectors, enabling integrative or replicative transformation. Tested in model cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus elongatus UTEX 2973, CyanoGate demonstrates its applicability for efficient genetic modification.

The importance of cyanobacteria in biotechnology, from pharmaceuticals to biophotovoltaic devices, underscores the need for robust synthetic biology tools. Despite progress, cyanobacteria lag behind in synthetic biology compared to other systems. CyanoGate addresses this challenge by offering an easy-to-use system that integrates with existing standards, enhancing scalability and accelerating the "design, build, test, and learn" cycle.

The syntax of level 0 parts was adapted for prokaryotic cyanobacteria, addressing typical cloning needs in cyanobacterial research. New level 0 parts were assembled from various sources. Level 1, M, and P acceptor vectors from the MoClo Plant Tool Kit facilitated the assembly of level 0 parts in a level 1 vector, with up to seven level 1 modules in level M. Level M assemblies could be further combined into level P and cycled back into level M for larger multimodule vectors if needed.

Vectors exceeding 50 kb in size, assembled by MoClo, have been reportedModules from level 1 or level P could be assembled in new level T vectors designed for cyanobacterial transformation. Both UTEX 2973 and Synechocystis produced recombinants through electroporation or conjugation methods with level T vectors, with a preference for the conjugation approach in the outlined work.

Level 0 (Lv0) Cyanogate vector assembly from either genomic extraction or synthesized G-Block. A Lv0 Acceptor vector containing a prokaryotic selection marker (pink) and LacZ gene insert (yellow) flanked by restriction sites and the G-block of DNA are digested and ligated with BbsI. The reaction result is a transformed vector containing the DNA sequence of interest in the position of LacZ (blue).

Level 1 (Lv1) Cyanogate vector assembly from three Lv0 vectors containing a promoter, GOI (gene of interest), and terminator. A Lv1 Acceptor vector containing a prokaryotic selection marker (pink) and LacZ gene insert (cyan) flanked by restriction sites and the G-block of DNA are digested and ligated with BsaI. The reaction result is a transformed vector containing the DNA sequence of interest in the position of LacZ (blue).

Level T (LvT) Cyanogate vector assembly from two Lv1 vectors containing a Lv1 Cassette and Lv1 End Linker. A LvT Acceptor vector containing a prokaryotic selection marker (pink) and LacZ gene insert (green) flanked by restriction sites and the G-block of DNA are digested and ligated with BbsI. The reaction result is a transformed vector containing the DNA sequence of interest in the position of LacZ (blue).

LvT Cassettes contain origins of replication for both E. coli and Cyanobacteria, allowing for assembly and testing in E. coli strains before transforming via triparental conjugation or electroporation into a cyanobacteria host.

Early Projects

These following projects were discontinued due to time constraints and a desire to consolidate the project.

Conventional photovoltaic panels can only capture light energy but currently have no scalable means to store this energy. In contrast, living biophotovoltaic systems which use photosynthetic cyanobacteria are capable of both capturing and storing energy in the form of carbohydrates and other polymers. This process can further sequester or cycle carbon dioxide, helping to make our solar panel carbon negative.

Consequently, we pursued increasing the biosynthesis of polyhydroxybutyrate (PHB), a thermoplastic polyester with excellent biodegradability that is capable of being degraded by various microorganisms living in soil and saltwater. PHB acts as a carbon sink, funnelling excess metabolic flux into its production whilst sequestering CO2. PHB is native to Synechocystis sp PCC. 6803 but is only expressed in small granules in small concentrations.

A two-pronged approach for PHB was explored: Overexpression of phaAB and directed evolution of phaB. By undergoing error-prone PCR on phaB of the phaCAB operon involved in PHB biosynthesis, we hoped to generate a mutant library for the rate-limiting step to increase PHB production in E. coli initially and then transfer to Synechocystis sp PCC. 6803.

PHB Biosynthesis

Examining the biosynthesis pathway of Polyhydroxybutyrate (PHB) in Synechocystis sheds light on the pivotal role of acetyl-CoA in a three-step reaction leading to PHB production (Fig 2). The process initiates with 3-ketothiolase catalyzing the Claisen condensation of two acetyl-CoA molecules, yielding acetoacetyl-CoA. Subsequently, acetoacetyl-CoA transforms into R-3-hydroxybutyryl-CoA under the influence of acetoacetyl-CoA reductase. Finally, PHA synthase orchestrates the polymerization of R-3-hydroxybutyryl-CoA. Genes phaA (3-ketothiolase), phaB (acetoacetyl-CoA), phaC, and phaE (PHA synthase subunits) encode the enzymes for these critical steps.

In the pursuit of enhancing PHB production, attention has been directed towards the phaB gene. This avenue was explored by overexpressing the PHA synthase using the Ralstonia eutropha operon in Synechocystis sp. PCC6803. While the PHA synthase witnessed a two-fold increase in expression, the resulting PHB content only exhibited marginal improvement. This observation suggests that the polymerization reaction catalyzed by PHA synthase may not be the primary rate-limiting step in the PHB synthesis pathway.

Overexpressing the native pha operon in Synechocystis PCC6803 led to the highest PHB productivity. Notably, the genes phaA and phaB were located in the same operon, and focusing on the phaB gene contributed to the success of this approach. This strategy, which emphasizes the phaB gene, holds promise for further optimizing PHB production through targeted genetic modifications in the biosynthesis pathway.

Directed Evolution

Error-prone PCR is a commonly used approach to generate mutant libraries using low-fidelity DNA polymerases such as Taq M0267 lacking 3'-5' exonuclease proof-reading abilities. Increasing MgCl­2 concentrations further decrease fidelity, increasing mutation rates.

To generate a mutant library for phaB, the rate-limiting gene involved in PHB biosynthesis, a plasmid construct was designed with the complete phaCAB operon. This construct was derived from the iGEM part BBa_K1149051, which contained a hybrid constitutive and native promoter and the phaCAB operon. This part was optimized for bioplastic production in E. coli MG1655, and for directed evolution, the strong promoter would enable easier quantification of PHB production using Nile red fluorescence. We modified the part to contain BsaI overhangs, which would enable us to digest and ligate the ordered G-block into a level 1 plasmid backbone (DVK_AE) from the Cyanogate parts kit we were provided with thanks to the MacCormick lab. Additionally, we included a double terminator (BBa_B0015) in the G-block design.

Two primer pairs, one for replicating the plasmid backbone and phaCA, and a second pair for phaB were designed. Two PCR reactions would have been run, one high-fidelity reaction to replicate a linearized backbone for phaCA, and a separate epPCR reaction with low-fidelity polymerase to generate a library of phaB mutants. The two reaction products would then be digested and ligated together to form a circularized level 1 plasmid for phaCAB* where * denotes a mutagenized gene. This would have been transformed into E. coli, and expression of PHB would have been quantified by a fluorescent plate reader-based assay using Nile red. Unfortunately, we planned to synthesize the G-block as a level 1 insert from IDT but our level 1 insert was 4.43 kb. IDT only synthesizes G-blocks up to 3 kb in size, and so we were forced to either redesign or abandon this project. Due to the added complexity of having a dual plasmid system with one containing phaCA and another containing phaB, we decided to focus efforts on salinity resistance. The main issue with dual plasmids was that one would likely be in level 1, and another in level T (containing origins of replication for cyanobacteria as well as E. coli) to prevent plasmid loss from the same origin of replication. Additionally, generating a mutant library with both plasmids by co-transformation would have added extra complexity to the project. Due to time pressures and a desire to focus on one directed evolution project, we didn't follow through with this project.

Figure 2: Designed level 1 G-Block for digestion and ligation into level 1 plasmid backbone DVK_AE. Part colors correspond to Figure 1.

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