Overview
Cercosporin is a photo-activated reactive oxygen species (ROS) that may be the key to combating algae blooms that are directly leading to the demise of seagrass in Florida, a primary food source for Florida's manatees. However, the cost of cercosporin application carries a hefty price tag, currently rendering it unfeasible as a solution. Florida State University’s Sea Clear iGEM team sought out a solution to this problem using synthetic biology to adapt an existing biosynthetic pathway in yeast. By using synthetic biology to fine-tune enzyme production within cercosporin's biosynthetic pathway, our team wishes to make cost effective large scale cercosporin manufacturing a reality.
These are the four cornerstones of any engineering success cycle. Throughout the course of this endeavor, the team rotated through these stages in an attempt to solve the plight of the manatees with biology.
Design
Our design was inspired by the potential of synthetic biology to maximimze cercosporin yield in ways that its native fungal host and chemical synthesis cannot. This is evidenced by the increases in fungal metabolite yields in yeast attained by modifying gene regulation, swapping protein domains, fusing enzymes and co-culturing with bacteria (10,11,12,13) . Given the complexity of the molecule and its efficient biological synthesis due to natural selection, our team sought to leverage this existing efficiency to further adapt production.
While biosynthesis of cercosporin in organisms such as Cercospora beticola has been made extremely efficient by natural selection, its regulation has not been fine-tuned for maximum yield: Many of the genes required for biosynthesis are regulated by factors that have not been fully elucidated, and the ones we know of such as sunlight, temperature, pH, and choice of media (1) make mass production challenging. To eliminate these restrictions, our idea was to isolate the necessary genes in C. beticola from their yield limiting regulatory networks and artificially control expression in a more tractable organism – Saccharomyces cerevisiae. With the competition timeframe and information gathered from literature in mind (enzymes required and speculated in biosynthesis, intermediate stability, and product detection protocols), we decided to transgenically express the first two enzymes (CTB1 and CTB3) in the biosynthesis pathway as a proof of concept for the yeast mediated production of cercosporin.
Biosynthetic Pathway of Cercosporin
To do this, we selected the first two enzymes in the pathway – CTB1 and CTB3 – from the genome of C. beticola and sequentially inserted them into shuttle vectors for cloning in E.coli and episomal expression in S. cerevisiae. Choices of plasmid copy number and promoter strength were modulated to encourage maximum production in our chassis, resulting in several combinatorial designs. Molecular cloning was done using NEB HiFi DNA assembly, allowing DNA fragments for coding sequences and regulatory elements to be interchanged by annealing purposefully designed overlaps. Assays were then implemented for enzyme and product detection, including HA epitope tagging on CTB1, 6xHis tagging on CTB3 and analytical chemistry assays for product identification and quantification inspired by existing literature (2,3,4).
Laboratory Workflow
Design Modularity
Given every possible variation in assembly, there were 24 vectors designed: 6 expressing CTB1 alone, and 18 co-expressing CTB1 and CTB3, with each vector utilizing different promoter and copy number variations.
Configurations For Enzyme Expression Tuning
Build
Bridging the gap between imagination and creation is much simpler said than done. Within the engineering cycle, our team constantly fluctuated between the build and design stages to ensure that we had the best chance of bringing our ideas to life. Failures within the build stage were taken as an opportunity to improve upon our own designs. We ensured that we were revising our workflow to optimize the effectiveness of each build iteration.
Objective 1: Linearizing Backbone
There were two versions of the linearized backbone created. The backbones originated from bacterial strains supplied by Dr. Tomko’s lab, based out of the FSU College of Medicine. The two strains were named P424 and P414, with P424 being a high copy version and P414 a low copy version. The first step to produce both linearized backbones was creating plasmid DNA from each of the bacterial strains. The plasmids were made using New England BioLab’s Monarch® Plasmid Miniprep Kit. From here, the first iteration of the build process commenced.
Phase 1A - Polymerase Chain Reaction (PCR)
Status: Troubleshooting
Polymerase chain reaction (PCR) was the first laboratory technique attempted to produce P414 and P424 backbones. Throughout the course of many trials, it was determined that PCR was not a viable option for obtaining either of the backbones. Electrophoresis technology was used to measure the size of experimental products. It was repeatedly found that the plasmid yielded by PCR was nowhere near the expected magnitude that was theorized within SnapGene.
Phase 1B - Restriction Enzyme Digest
Status: Completed
After deciding to shift away from PCR technology, the team focused on utilizing a unique restriction enzyme called PVUII to carry out a restriction enzyme digest. The enzyme allowed for the P414 and P424 plasmid DNA to be cleaved at specific sequences, essentially “cutting” out the desired segment to be used as the backbone. However, the enzyme chosen presented a unique challenge. An intrinsic property of PVUII is that it cannot be heat-inactivated. This proved to be a problem because remnants of the enzyme persisted within the plasmid, reeking havoc on a microscopic scale. To remedy this, an overnight digest was carried out to ensure the digestion was as complete as possible. Using electrophoresis, it was verified that the digest produced plasmids of the correct size.
Phase 2A - DNA Purification
Status: Completed
After a successful restriction enzyme digest, the plasmid DNA was purified using New England BioLab’s Monarch® PCR and DNA Cleanup Kit (5 μg). This step was intended to purify high quality DNA devoid of any residual restriction enzyme byproducts. This protocol was chosen during the initial workflow design because it is adaptable to both PCR and restriction enzyme digest products.
Phase 2B - Quality Check: Size Verification of Plasmids using Electrophoresis
Status: Completed
At this point, the team took a step back to analyze the plasmids at each stage of workflow being followed. This was done as a quality control check before proceeding to the next step in the pursuit of backbone isolation.
The results of this gel changed the trajectory of the build phase of the engineering cycle. Using the DNA ladder in the first lane of the gel as a reference, the sizes of the bands were approximated and analyzed for the presence of backbone. It was discovered that the plasmid created from purifying the restriction enzyme plasmid in Lane 4 contained significantly less backbone than expected. It was inferred that the cleanup step essentially “cleaned” too well, consequentially reducing the concentration of backbone present within the plasmid by a significant degree. At this point, it was determined that isolating the backbone should occur within the restriction enzyme stage and not the DNA purification stage as originally thought. This was found to be the case for both P424 and P414 subtypes.
Phase 3 - Isolating DNA Backbone using CloneWell™ II Electrophoresis
Status: Completed
Within the plasmids, the desired backbone exists alongside other DNA components. The team then had to cultivate the best method of isolating the backbone from its molecular neighbors. From here, it was decided that a unique type of agarose gel called CloneWell™ II would be used. This choice simplified isolation of the backbone because it allowed for the desired DNA to be isolated and extracted within the same medium. This allowed for the backbone to be extracted within the plasmid stage where it was the most abundant. Furthermore, the recovered sample does not need to be repurified using a DNA cleanup kit. This both aided in laboratory efficiency and avoided the problems caused by using New England BioLab’s Monarch® PCR and DNA Cleanup Kit (5 μg) that were discovered in the quality check. Within this phase, DNA backbone was successfully obtained and ready for use in DNA assemblies.
Objective 2: DNA Assembly
DNA assembly is the core of both synthetic biology and life itself. It is the process by which individual genetic components align and merge together to construct new DNA structures. This is no easy feat because this complex molecular process is subject to a plethora of factors unperceivable by the human eye.
Phase 4A - NEBuilder HiFi Assembly
Status: Troubleshooting
A series of assemblies were attempted using an adapted version of the protocol for HiFi DNA assembly authored by New England BioLabs. The volume of the reactions had to be scaled up to accommodate the magnitude of the DNA fragments making up each proposed design. To calculate the amount of each fragment in picomoles needed for optimal assembly, the NEBiocalculator was used.
The allowable range for DNA fragments established within the protocol is 0.2 to 0.5 picomoles. Each reaction fell at the upper limit of the established bounds. To preserve the 1-1 ratio between Master Mix and DNA fragments, more of the mix was added to the reaction volume.
The nature of a HiFi assembly is that efficiency decreases as the amount of fragments increases. As mentioned before, the assemblies were at the upper threshold of the allowable range. After a series of failures, it was decided that HiFi was not a feasible option for assembling the designs created by the team.
Phase 4B - Golden Gate Assembly
Status: In Progress
The Golden Gate Assembly is what the team hopes will be their golden ticket to achieving a successful assembly. Golden Gate can support the assembly of over fifty DNA fragments. The team hopes that this method would alleviate the challenges presented by HiFi. However, this decision was made very recently by the team. As of October 12, no assemblies have been able to be conducted. However, the team looks forward to sharing our proposal of this new venture at the 2022 iGEM Grand Jamboree.
Objective 3: DNA Transformation
Transformation is the process in which DNA is uptaken by a host cell and then seeded onto an antibiotic plate. For the scope of this project, bacterial cells were chosen as the vessel for transformation. However, this process can be done using other cells such as yeast or even plant cells. This is done because the plasmid was designed with a specific antibiotic resistance, in this case Ampicillin. As a result, only bacteria containing the desired product will grow. After obtaining a colony, it can be used to create a culture to make the designed plasmid.
Phase 5: High Efficiency Transformation using 10-beta Competent E. coli
Status: In Progress
After the completion of an assembly, a transformation will be carried out using 10-beta Competent E. Coli cells. This type of E. Coli was chosen because it has the capacity to handle assemblies with a high number of DNA fragments.
Numerous transformations were conducted using the assemblies produced by HiFi. Over the course of many attempts, no colonies were produced. This was the main reasoning as to why the team decided to no longer pursue HiFi as the assembly method. However, the team is optimistic that a transformation using Golden Gate will yield a successful set of colonies.
Objective 4: Test
The work done during the build stage was intended to culminate into establishing a pathway within yeast. Establishing a pathway is one matter, and ensuring its success is another. The success of the pathway is dependent on whether or not we are able to detect nortorolactone within the yeast. As mentioned before, nortorolactone is the first step in synthesizing cercosporin. Currently, an established methodology for detecting nortorolactone does not exist. However, the team is actively developing a dual-faceted detection protocol we hypothesize will be able to detect this chemical intermediate.
Phase 6: Establishing Yeast Pathway
Status: In Progress
The first step in creating a yeast pathway that this team would do is preparing competent yeast cells. Then, plasmid DNA created from the bacterial transformations will be transformed within yeast and subsequently plated.
Phase 7A: Detecting Nortorolactone in Yeast using UV-VIS-NIR 10-beta Competent E. coli
Status: In Progress
We would perform the characterization of the samples in a UV-VIS-NIR spectrometer. The spectra produced would show absorption peaks between 250 nm and 400 nm. We would need to select a relevant set of quartz curvatures to get reliable data specifically within the UV region. Depending on our sample volume, we would choose the length of the cuvette. We will need to use two sets of cuvettes, one for the sample being tested and the other for the solvent we choose. At this stage, we intend on using ethanol as the solvent.
Phase 7B: Detecting Nortorolactone in Yeast using HPLC
Status: In Progress
High-performance liquid chromatography (HPLC) is an analytical technique used in chemistry that has the capability of identifying components within a mixture. We are developing this protocol as an alternative method of detecting nortorolactone. It can also be used in conjunction with the UV-VIS-NIR protocol being developed in tandem.
Learn
Our team believes that whiplash from the endless loop that is the engineering success cycle is not a reminder of our own shortcomings, but a testament to the tenacious spirit embodied by each and every member of our team. Failure is an inescapable component of any scientific journey, and every setback is a stride towards success.
Design. Build. Test. Learn.
Learning is arguably the key component to this sometimes brutal cycle. The simple truth is that we were unable to adapt Cercosporin’s existing biosynthetic pathway into yeast at this time. However, we are optimistic that given more time and resources we will be able to actualize our initial goal. Our team has no doubt that the knowledge we cultivated over the course of this project will prove invaluable to both future innovators and the synthetic biology community as a whole.
Our team is proud to have spearheaded an avant-garde solution to a problem that hits very close to home-saving the Florida manatee.
What's Next? (Design Improvements)
the build process, TEST several limitations and potential workflow improvements were identified concerning plasmid elements and sequence specifications. For improvements to our overall solution inspired by dialgoue with stakeholders and experts, please see our human practices page.
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Kanamycin instead of Ampicillin Resistance for Colony Screening
Kanamycin is more stable than Ampicillin, resulting in a longer shelf life, both in powdered form and while plated on agar (5). For this reason we would prefer it over Ampicillin moving forward, as costs and workflow would be improved.
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Permanent Removal of Native GAL1 promoter in our Vectors
The inducible GAL1 promoter is naturally present in both of our plasmids - p414 and p424 - and is a choice of promoter we utilized; however, its basal presence is inconvenient and in the case of alternative promoters requires excision from the plasmid prior to any useful assembly. For this reason, we would preliminarily excise it in the future, starting with an unmodified multiple cloning site.
- Alternative Codon Usage to Maintain Uniqueness of Restriction Sites
Given the stepwise nature of our pathway assembly (both by choice and by nature of HiFi/Gibson cloning) and multitude of restriction sites, special care must be taken to a avoid accidentally cutting the plasmid and undoing earlier assembly portions. Our general cloning workflow is as follows:
- Linearization of plasmid using a unique resriction site, 2 close unique restriction sites, or a dual restriction site with both of its target sequences nearby
- Insertion of CTB1 where the cut occured
- Linearization of plasmid containing CTB1 using new/unused unique or dual cut restriction site(s)
- Insertion of CTB3 where the new cut occured
Continuing this further, if more enzymes were to be cloned into the same vector.
- Linearization of plasmid containing CTB1 and CTB3 using new/unused unique or dual cut restriction site(s)
- Insertion of new gene of choice where the newest cut occured
- Repeat assembly for as many genes as needed, utilizing unused cut sites each time (otherwise the assembly comes undone)
This sequential two step assembly can be repeated for as many genes as a plasmid can fit. The issue is that restriction sites to be utilized in later assemblies cannot be present within the sequences of genes inserted earlier; doing so would result in unintended cuts within the sequences of previously assembled genes and destruction of the plasmid. In order to avoid this catastrophe, enzymes should be selected from a multiple cloning site ahead of time, with special precaution taken to ensure that once a restriction enzyme is used, its cut sites will not be present in the sequences of future genes inserted. This can be done after initial codon optimization for yeast by screening future sequences for unwanted cut sites, followed by removal of those cut sites by alternative codon selection prior to gene fragment ordering.
Restriction Site Considerations
In our workflow for this project, we ignored the existing multiple cloning sites and started our assembly of CTB1 just upstream of them, reasoning that we could excise the problematic GAL1 promoter inherent to our plasmids and insert our first gene in a single step; However, the CTB1 sequence utilized was ordered without checking for problematic cut sites under the assumption that there would be many left to choose from, which was not the case given its large size. Becuase of this, we ended up having to use a suboptimal restriction enzyme for CTB3 insertion which could not be heat inactivated and hampered workflow, and had we wanted to insert even more genes into our vector there likely would not have been any more useable cut sites to pick from. To avoid these complications in further design iterations, it would be best to utilize restriction sites within the multiple cloning sites, and ensure that they remain single or dual cutters by screening future insertions for restriction sites ahead of time.
Further Improvements
For sustainable production of cercosporin in our chassis, there would ultimately need to be chromosomal integration of our vectors which could be done a number of ways (6,7,8). For maximum plasmid carrying capacity (especially in the context of episomal plasmid maintenance), self-cleaving peptides could minimize the number of regulatory elements that need to be cloned in to a vector by allowing multiple genes to be introduced in the same transcript (9). Modifications mentioned at the beginning of the page could also be implemented to further increase cercosporin yield.
References
(1) Daub, M. E., and Ehrenshaft, M. 2000. The photoactivated Cercospora toxin cercosporin: Contributions to plant disease and fundamental biology. Ann. Rev. Phytopath. 38:461-490.
(2) Newman AG, Townsend CA. Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae. J Am Chem Soc. 2016 Mar 30;138(12):4219-28. doi: 10.1021/jacs.6b00633. Epub 2016 Mar 16. PMID: 26938470; PMCID: PMC5129747.
(3) Adam G. Newman, Anna L. Vagstad, Philip A. Storm, and Craig A. Townsend. Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming. Journal of the American Chemical Society 2014 136 (20), 7348-7362 DOI: 10.1021/ja5007299
(4) de Jonge R, Ebert MK, Huitt-Roehl CR, Pal P, Suttle JC, Spanner RE, Neubauer JD, Jurick WM 2nd, Stott KA, Secor GA, Thomma BPHJ, Van de Peer Y, Townsend CA, Bolton MD. Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum. Proc Natl Acad Sci U S A. 2018 Jun 12;115(24):E5459-E5466. doi: 10.1073/pnas.1712798115. Epub 2018 May 29. Erratum in: Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):E8324. PMID: 29844193; PMCID: PMC6004482.
(5) Ryan KJ, Needham GM, Dunsmoor CL, Sherris JC. Stability of antibiotics and chemotherapeutics in agar plates. Appl Microbiol. 1970 Sep;20(3):447-51. doi: 10.1128/am.20.3.447-451.1970. PMID: 5485725; PMCID: PMC376956.
(6)Shao Z, Zhao H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 2009;37:e16
(7) Wingler LM, Cornish VW. Reiterative Recombination for the in vivo assembly of libraries of multigene pathways. Proc Natl Acad Sci U S A. 2011;108:15135–15140.
(8) Michael S. Siddiqui, Atri Choksi, Christina D. Smolke, A system for multilocus chromosomal integration and transformation-free selection marker rescue, FEMS Yeast Research, Volume 14, Issue 8, 1 December 2014, Pages 1171–1185, https://doi.org/10.1111/1567-1364.12210
(9) Lee JH, Won HJ, Oh E-S, Oh M-H and Jung JH (2020) Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants. Front. Plant Sci. 11:559365. doi: 10.3389/fpls.2020.559365
(10) Zhao, M., Zhao, Y., Yao, M. et al. Pathway engineering in yeast for synthesizing the complex polyketide bikaverin. Nat Commun 11, 6197 (2020). https://doi.org/10.1038/s41467-020-19984-3
(11) Gao J, Wenderoth M, Doppler M, Schuhmacher R, Marko D, Fischer R. Fungal Melanin Biosynthesis Pathway as Source for Fungal Toxins. mBio. 2022 Jun 28;13(3):e0021922. doi: 10.1128/mbio.00219-22. Epub 2022 Apr 27. PMID: 35475649; PMCID: PMC9239091.
(12) Scharf DH, Brakhage AA. Engineering fungal secondary metabolism: a roadmap to novel compounds. J Biotechnol. 2013 Jan 20;163(2):179-83. doi: 10.1016/j.jbiotec.2012.06.027. Epub 2012 Jul 20. PMID: 22820338.
(13) Zhou, T., Yu, S., Hu, Y. et al. Enhanced cercosporin production by co-culturing Cercospora sp. JNU001 with leaf-spot-disease-related endophytic bacteria. Microb Cell Fact 20, 100 (2021). https://doi.org/10.1186/s12934-021-01587-2