Overview

Our host organism, Synechocystis sp. PCC6803, is a photoautotrophic species of cyanobacteria with natural mercury detoxification pathways that inhabits aquatic environments. In this project we designed a new improved methylmercury detoxification system in Synechocystis using two mercury degrading enzymes: Mercuric reductase (MerA) and Alkylmercury lyase (MerB). To achieve this, a 7-fragment construct will be assembled with the two genes of interest (merA and merB) regulated by the same lac-based inducible promoter with a “leaky transcription”. Since there are currently no valid in silico tools for selecting RBS’s (ribosomal binding sites) in Synechocystis we aim to optimize this system by testing three different RBS’s and also two homologs for each of our genes.

The different variations of constructs will be assembled with a new Golden Gate based assembly system which offers an efficient way of changing the RBS’s in a simple one-pot reaction. The success of the assembly is confirmed by multiplying the insert in E. coli with colony-PCR and separating them by size with gel-electrophoresis. We will then transform our constructs to Synechocystis and verify the result with the same method as in E. coli before moving on to further mercury experiments. The most promising approach to test our modified constructs with mercury is an experimental setup inspired by team Minnesota 2014 which is based on a qualitative assay on mercury resistance.

Literature research on mercury detoxification pathways in Synechocystis sp. PCC6803

The design of our project is supported by thorough and extensive literature research as well as contacting experts around this topic. We chose Synechocystis sp. PCC6803, a genus of cyanobacteria, as our model organism for its photosynthetic abilities which makes it self-sustaining and cost-effective. As a photoautotrophic organism it uses light as an energy source and is able to convert carbon dioxide into organic material. Both light and carbon dioxide are readily available for large scale systems. Synechocystis naturally inhabits aquatic environments which makes it a well-suited candidate for bioremediation of mercury-contaminated waste water (Franco et al., 2018). Genetic modifications can be engineered to its naturally existing mercury detoxification pathways. In the University of Turku (UTU) there has been a lot of research and experiments using Synechocystis so we had a lot of resources and data available for us.

Synechocystis possesses native genes that encode mercury reducing and degrading enzymes, such as MerA. The merA -gene codes for mercuric reductase (EC 1.16.1.1) that catalyzes the reduction of toxic ionic mercury (Hg2+) to less volatile and toxic elemental mercury (Hg0). The merB -gene is found in Escherichia coli (E. coli) and it codes for alkylmercury lyase (EC 4.99.1.2) that plays a vital role in the degradation of complex organic mercury compounds, such as methylmercury (CH3Hg) (Priyadarshanee et al., 2022). The combined enzyme activities of MerA and MerB and the unique metabolic abilities of Synechocystis offer a promising approach to tackling the issue of mercury pollution in waters (Fig. 1). Methylmercury is one of the most commonly occurring forms of mercury bioaccumulation within the aquatic food web (Carrasco et al., 2011).

Reaction mechanisms of mercury degrading enzymes: MerA and MerB

MerB mediates the first steps of degradation in various organomercurial salts. It catalyzes the protonolysis reaction where a proton (H+) attacks the organic mercury compound, breaks the carbon-mercury bond and leads to the formation of ionic mercury (Hg2+) as well as a respective organic compound. In the case of methylmercury the addition of carbon-mercury bond breaking protons (H+) leads to the formation of methane and ionic mercury (Hg2+). MerA subsequently detoxifies ionic mercury (Hg2+) with the help of a NADPH substrate. (Fig. 2) (Priyadarshanee et al., 2022). With many reactions in heterotrophic organisms, NADPH is often a limiting factor, making the autotrophic Synechocystis an optimal candidate to catalyze this reaction.

Detailed MerB and MerA enzyme mechanisms may require further research and validation. Based on experimental data and computational calculations, the enzyme mechanism of MerB has been proposed to follow a two-step process where a cysteine residue gives a proton to Asp99. This allows two sulfur-containing groups to interact with the organic mercury compound while Asp99 helps to break the carbon-mercury bond releasing the end product. (Parks et al.,2009).

MerA is a homodimer with each subunit composed of two domains: nMerA and an active site. nMerA contains two N-terminal cysteine residues (Cys11/Cys14) in charge of receiving and transporting Hg2+ into the active site. The active site contains buried Cys136/Cys141 which form the site of catalysis. On the surface there are two cysteine residues in charge of transporting electrons. The electron transfer happens through NADPH substrate from cytoplasm and FAD cofactor embedded in the active site. (Fig. 3) (Peng et al., 2014) .

1) MerA binds Hg2+ and transports it to the active site by forming different mercury-cysteine adducts.

2) NADPH transfers a hydride to FAD producing FADH−, which subsequently interacts with Hg2+ in the mercury-cysteine adducts.

3) Hg0 is released from the cell.

Designing the homologous merA and merB genes for enhanced efficiency

Optimizing the most efficient mercury degradation system included finding and testing different homologs for merA and merB . Homologous genes from different species, despite similarities in their gene sequences, can affect the efficiency in many different ways. Subtle variations can influence gene expression, function, regulation and compatibility with different engineered systems etc.

The first merA was chosen to be from Synechocystis and the first merB was chosen to be from E. coli since they are both native genes in their respective organisms. They offer a secure option for testing our system in both of these host organisms. Genes from E. coli are also often compatible with Synechocystis and tend to offer reliable results. The other alternatives for both genes were chosen from Pseudomonas aeruginosa (P. aeruginosa) based on evidence of its potency we found from literature (Hirak et al., 2017). The genes were harmonized using a codon optimization tool “CodonWizard” (Rehbein et al., 2019) to enhance the translation of the genes.

Designing the plasmid construct for efficient gene integration

Introducing new genes into cyanobacteria requires the assembly of a plasmid construct that serves as a vector transporting and controlling the genes of interest. Plasmids are small circular pieces of DNA acting independently from the chromosomal DNA of the bacterium and they are autonomously replicated during cell division. By modifying the plasmid to carry the genes of interest, we can produce the desired proteins within bacterial cells without the need for integration into the chromosome.

For the two genes of interest, merA and merB , we designed a 7-fragment plasmid construct model with the basic components of P0 Plasmid backbone, P1 Promoter, P2 RBS, P3 merA , P4 RBS, P5 merB and P6 Transcription terminator. The plasmid backbone is the main structure of the construct and contains all the necessary elements for replication and maintenance within the host cell. The first phase of protein synthesis, transcription, is initiated by the promoter and it ends with a signal from the terminator. The promoter has an important role of regulating gene expression and in turn the terminator ensures the proper processing and releasing of mRNA. There are two RBS’s separately located before each gene of interest in charge of facilitating translation by allowing the binding of ribosomes to mRNAs. (Fig. 4).

The gene elements used in our model are supported by literature research and contacting professionals. We used the same principle and assembly system described by Thiel et al 2019. Apart from the genes of interest, all the gene fragments were chosen from the plasmid library compiled and validated at UTU. The final position of each fragment is determined by their cohesive ends. In our model we used RSF1010 -derived pDF as a plasmid backbone (Nagy et al 2021) and PA1lac0-1/Riboj as a promoter which was located upstream of both merA and merB.

PA1lac0-1/Riboj is a lac-based inducible promoter which means it is not continuously active but it can be turned on with the presence of a specific inducer. The promoter has a “leaky transcription” and is passively around 50% active even without an inducer. IPTG is an efficient non-toxic inducer that has been widely used in Synechocystis . In bacteria IPTG mimics a sugar molecule, lactose, binding and preventing Lacl repressor protein from inhibiting the lac-based promoter which initiates the transcription of downstream genes. With this promoter, the bioremediation process can be controlled and induced if needed with the addition of IPTG (Guerrero et al., 2012).

Unlike with E. coli, there are currently no valid in silico tools for selecting the most efficient RBS’s for our genes of interest in Synechocystis . Therefore, one phase of our experimental work includes testing the constructs with three different RBS’s for each position (P2 and P4): RBS S3, RBS S4 and RBS S5. With these RBS’s we found the most promising evidence of their compatibility and efficiency (Thiel et al 2018). The goal of this experimentation is to optimize the most efficient RBS compatible with this system.

For optimization, we decided to make different variations of constructs changing between the RBS’s and the gene homologs. We will also make one-gene constructs with a reporter protein like sYFP2 or EFE to see the function and mechanism of each gene separately. (Fig. 5).

Here you can learn more about the parts we used: Parts page

DNA assembly and validation methods for RBS optimization

After compiling all the necessary DNA fragments in the library, the constructs will be assembled using a Golden Gate based assembly system for RBS optimization. It is an advanced operon-based assembly system that uses type IIs restriction enzymes. With this particular method we have an efficient way of changing the RBS’s separately from the promoter for RBS optimization. We are able to make operons with two genes regulated by the same promoter in a one-pot reaction which allows a simultaneous assembly of multiple gene fragments. We also have a great opportunity to work with professionals who have directly participated in the development of this assembly system. (Fig. 6).

In Synechocystis , the growth and selection period takes around 10-30 days. To maximize efficiency, we decided to first transform the assembled constructs in the bacterial model species E. coli DH5α. It has a growth and selection period of only around 12 hours and in which we can more easily amplify the plasmids. The success of the assembly would be confirmed by amplifying the 6-fragment insert in the plasmid backbone with colony-PCR and separating them by size using agarose gel electrophoresis. We would then transform the modified plasmids into Synechocystis with antibiotic selection and verify the results again with the same colony PCR and electrophoresis used in E. coli before moving on to further testing. (Fig. 7).

Testing our modified Synechocystis strains

The final test for our modified cyanobacteria is using methylmercury and mercuric (II) chloride to confirm the function of the expressed MerA and MerB enzymes in vivo. By adding methylmercury to our modified cyanobacteria we could test the function of the whole system and by adding mercuric chloride we could measure the function of MerA separately. With the one-gene constructs we can also conduct separate experiments with only one gene in mind. The goal is to evaluate the capacity of the generated strains to detoxify organic mercury present in the external medium.

Our first approach is to contact and collaborate with agencies that have the ability to conduct mercury measurements in Finland. Depending on the measurement method employed, we may need to precipitate specific mercury forms to prevent interference with our analysis. One of the most promising institutions we have engaged with is Suomen ympäristökeskus (SYKE), a renowned Finnish research and expert institution. They perform mercury measurements using CV-ICP-MS (Continuous-Flow Inductively Coupled Plasma Mass Spectrometer) which would be able to give us quantitative results by measuring the concentration of mercury in our modified samples and a wild type control.

The second approach involves experimenting with mercury resistance in our modified strains. We were inspired by the experimental setup of team Minnesota 2014 (Tarnowski Jessica, 2014) which we modified to work with Synechocystis . The experiment in question is a qualitative assay utilizing filter disks containing a controlled amount of methylmercury or mercuric(II)chloride. We anticipate that the growth of our genetically modified Synechocystis strains around the disks will outperform that of the control plates based on the hypothesis that enhanced detoxification capabilities should lead to an improved overall growth and survival. (Fig. 8). This method requires careful consideration of the safety risks involved but might not be as time-consuming as working out a collaboration with an external source and it offers more chances for modifications.

One way we can also approach measuring the function of our constructs is to test the protein expression in the modified E. coli strains with SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). SDS-PAGE separates different proteins based on their size so we could analyze the expression of MerA and MerB enzymes compared to the control.