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Inspiration

As our world population rises, more materials are needed to produce the various daily necessities and goods each person uses in their lives. Lactams, a kind of chemical commodity, are used as a raw material in the production of many goods prevalent in our lives, e.g. pharmaceuticals and nylon1. The production of lactams is expected to increase due to rising demand for its end-products, as seen in the expected global market size growth of nylon of USD 29.76 billion in 2020 to USD 46.31 billion in 20282. As the current production of lactams are all petrochemically based and require harsh conditions that harm the environment, it would not be a surprise to see increasing pollution caused by this industry as it expands to keep up with the demand1. Following the Sustainable Development Goals set by the United Nations, we wanted to look for a sustainable production method for lactams to build an industry that can support such demands in the long run3.


At the same time, global warming has also become a global concern, with Hong Kong also feeling the rising heat. The average annual mean temperature of Hong Kong SAR from 1885 to 2020 had an average increasing rate of 0.13ºC per decade, with a noticeable quickening during 1991 to 2020 at 0.24ºC per decade4. With carbon dioxide the main potent greenhouse gas driving rising atmospheric temperature, controlling atmospheric carbon dioxide is an obvious approach to mitigating global warming5. In 2019, the CO2 emission per capita in China and Hong Kong SAR was 7.10 tons and 5.59 tons respectively, both higher than world average of 4.72 tons6. Atmospheric CO2 concentrations have been climbing since the Industrial Revolution, hitting a new high of 419 ppm in 20217. Even if CO2 emissions were stable, atmospheric CO2 still poses a problem as CO2 can reside in the atmosphere for thousands of years, and so it is important for us to try to sequester carbon8,9.


After research for a chassis organism that can produce lactams biologically while also taking up carbon dioxide, we settled down with a cyanobacteria strain. Cyanobacteria have emerged as potential “microbial cell factories” due to their photosynthetic abilities, using solar energy and carbon dioxide to churn out biomaterials and chemicals after being engineered metabolically10. As lactams are highly demanded and are currently produced petrochemically, we see an opportunity of designing a complete biosynthesis pathway to produce them using CO2 as a sole carbon source. This way, human needs in the market are met at the same time while mitigating global warming. By modifying cyanobacteria to produce lactams, we hope to contribute our community and to the world by proving a bio alternative for industries.


Introduction and Background

The aim of this project is to modify Synechococcus elongatus UTEX 2973 so that it can synthesize valerolactam using carbon dioxide as a sole carbon source.

Our Product: Valerolactam

Valerolactam is a type of lactam, which are a family of cyclic amides. Lactams are cyclized forms of ω-amino acids, or non-natural straight chain amino acids, differing from each other by the number of carbon atoms in their rings. They can be used as monomers for polyamides, also known as nylons, and are also used for synthesis of drug components and antibiotics11,12,13. Their ω-amino acids can be applied in synthesis of chemicals for pharmaceutical use too. Valerolactam has five carbons in its cyclic ring and can be used as building blocks for nylon-5 and nylon-6,5, and for synthesis for chemicals used in drugs, e.g. piperidines1,13. In the traditional petrochemical synthesis of various sorts of lactams, intense energy for high temperatures and acidic conditions are used while large amounts of salt waste are generated, posing a threat to the environment14. Some chemo-biological processes have been proposed to synthesize lactams, but they still rely of petrobased feedstocks and involve harsh conditions14. Therefore, since biological systems usually require mild conditions and bypass toxic chemicals, a complete biosynthesis of lactams could benefit the industry.1

Previous studies have focused on synthesizing valerolactam in heterotrophic bacteria, e.g. in Escherichia coli and in Corynebacterium glutamicum.1 With two enzymes, L-Lysine mono- oxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) from the aminovalerate pathway in Pseudomonas putida KT2440, the ω-amino acid precursor of valerolactam, 5-aminovalerate (5-AVA), is converted from lysine.20 Only a few enzymes have been reported to cyclise ω-amino acids to lactams. Unfortunately, they share the common difficulty of not being efficient enough, some requiring high reaction temperatures, others requiring high activation energy1. In our project, we will model a cyclase and use a photosynthetic organism to produce valerolactam by engineering it for ectopic expression of DavB, DavA and the cyclase.

Our Host Organism

We have chosen a cyanobacteria as our host organism since direct photosynthetic production of valerolactam avoids extra costs used for fermentation of sugar by heterotrophic bacterial hosts8. Our chosen cyanobacteria strain Synechococcus elongatus UTEX 2973 is a novel strain with a record doubling time of 1.9 hours and exhibiting high light tolerance15. Compared to its close relative Synechococcus elongatus PCC 7942, its growth is more rapid and robust15. Available genetic tools for engineering S.elongatus UTEX 2973 have been characterized and optimized, laying a foundation for genetic manipulation of this strain for sustainable bioproduction of biomaterials or chemical commodities16. With a higher photosynthetic rate and carbon fixation rate, this strain has the potential to serve various biotechnology applications and mitigate increasing atmospheric carbon dioxide concentrations.17

The INTEGRATE System

Vo et al. (2020) proposed the insertion of transposable elements by guide RNA–assisted targeting (INTEGRATE) system. The INTEGRATE system is a CRISPR-transposon system, which involves a protein complex guided by a crRNA inserting a donor DNA into a designated site in an organism’s genome21. The donor DNA is the name for the piece of DNA being inserted into the genome. In this system, the crRNA complementary to a site in an organism’s genome guides the TniQ-Cascade protein complex to bind to the genome, and transposases excise the DNA and insert that donor DNA into the genome of the organism. The system is highly efficient and accurate, and we will use it to engineer S.elongatus UTEX 2973 so it can express DavB, DavA and a cyclase to synthesize valerolactam.

Methodology

The biosynthesis pathway of valerolactam in S.elongatus UTEX 2973 starts with carbon dioxide as its carbon source uses light energy. L-lysine is produced through the cyanobacterium’s L-lysine biosynthetic pathway, its synthesis regulated by an enzyme called aspartate kinase (AK)18. AK participates in L-lysine biosynthesis by converting L-aspartate to L-aspartyl-phosphate and it is subject to negative feedback by L-lysine, its activity decreased when there are high levels of L-lysine18. A feedback insensitive AK variant AK T359I for increased lysine yields in cyanobacteria has been characterized by Qi et al. and has been tested in S.elongatus PCC 7002 by Korosh et al., which we codon optimized for expression in S.elongatus UTEX 2973.19 The L-lysine is then converted to 5-aminovaleramide by L-Lysine mono- oxygenase (DavB), then to 5-aminovalerate (5-AVA) by 5-aminovaleramide amidohydrolase (DavA)20.


As current reported cyclases lack efficiency, we used computational modelling to improve a chosen cyclase CF3BD. We chose to model this cyclase due to its reaction temperature suitable for cyclization of 5-AVA to valerolactam in S.elongatus UTEX 2973 and its lack of need of other energy sources or activation factors1. Using Rosetta modelling, point mutations were performed on the cyclase and its stability was analyzed.


To engineer S.elongatus UTEX 2973, we used the Insertion of Transposable Elements by Guide RNA–Assisted Targeting (INTEGRATE) system developed by Vo et al.. The INTEGRATE system is a CRISPR-transposon system, which involves a protein complex guided by a crRNA inserting a piece of DNA, which is the gene(s) of interest called the Donor DNA in this system, into a designated site in an organism’s genome21. DavB, DavA, AK T359I and CF3BD capped with promoters and terminators characterized in the genetic toolbox by Li et al. will be inserted into the neutral sites in the genome of S.elongatus UTEX 297316,22.


As the plasmid pSPIN designed by Vo et al. (2020) holding the INTEGRATE system is designed for use in E.coli, we needed a plasmid for replication in S.elongatus UTEX 2973. We modified the pMC_0_7+8_panS_KanResLVL2 shuttle vector based on Golden Gate Assembly designed by iGEM Team Marburg 2019 to contain a multiple cloning site(MCS) and a Cre-lox system. The MCS enables combability for cloning with more restriction enzymes, including the ones necessary for us to subclone the INTEGRATE system into the newly modified shuttle vector, and two loxP sites facing the same direction flanking ColE1 allows deletion of this bacterial origin of replication by Cre recombinases. This way, after subcloning DavB, DavA, AK T359I and CF3BD with their corresponding promoters and terminators into the INTEGRATE system as the Donor DNA, the INTEGRATE system can then be subcloned into the new shuttle vector. This shuttle vector will then be amplified in E.coli DH5α so that a high plasmid concentration can be achieved for its transformation into S.elongatus UTEX 2973. Before transformation by electroporation of the shuttle vector into S.elongatus UTEX 2973, ColE1 will be removed through the Cre-lox system to reduce the metabolic burden of replicating the unnecessary bacterial origin of replication in the shuttle vector.


After engineering of S.elongatus UTEX 2973, it is expected to produce valerolactam. To obtain the valerolactam, the S.elongatus UTEX 2973 cells can be lysed and the valerolactam can be precipitated through solvent solvent extraction. However, this method may not be preferred in a large scale implementation of the production, and thus the extraction method of the valerolactam needs to be evaluated and revised in the second phase of our project.

Impact of COVID-19 on Our Project

Throughout our entire project, our biggest obstacle to overcome were the limitations brought by the COVID-19 social distancing restrictions. During the initial planning stage of our project in January 2021, the number of infected cases rose rapidly, and quarantine measures were tightened. For the safety of all citizens and students, the Educational Bureau announced that all face-to-face classes were suspended, thus making it hard for us to develop our project outline as all discussions had to be moved online, which lowered our communication efficiency.


Besides from running into issues during planning, we also encountered a series of problems during the implementation process. As we’ve chosen the cyanobacteria strain Synechococcus elongatus UTEX 2973 for our project, we hoped to gather more information on how to cultivate and grow the strain, thus we reached out to companies who are experienced with algae. Unfortunately, due to the economic recession caused by COVID-19, the companies were either disbanded or shut down. Since we weren’t able to gain relevant experience through interviewing such companies, we had to spend a longer period to figure out the best way to cultivate our strain. Also, since our target end-users were nylon manufactures, we planned to interview them as soon as possible so that we could get their advice. But due to social distancing limitations, our plan was postponed till July. Moreover, due to the strict COVID-19 restrictions, all lab sessions were canceled as students were not allowed to stay behind, such restrictions had a huge impact on our project as we weren’t able to perform the in vitro parts. We were forced to shift our focus to in vitro parts of our project until the labs were resumed. Luckily, the severity of the pandemic lowered around May and limited lab sessions were resumed.


Despite gaining access to labs, the time given was limited as we were not allowed to stay behind for a long time. The time left was far less than enough to complete what we’ve planned to achieve this year. Thus, after careful discussion and negotiations, we reached a consensus and agreed that we have no other way but to split our project into half and do a 2- year project under the inconvenience brought by COVID-19.


References:
  1. Gordillo Sierra, A. R., & Alper, H. S. (2020). Progress in the metabolic engineering of bio-based lactams and their ω-amino acids precursors. Biotechnology Advances, 43, 107587. https://doi.org/10.1016/j.biotechadv.2020.107587
  2. Ltd, R. A. M. (2021b, May). Global Nylon Market Size, Share & Trends Analysis Report by Product (Nylon 6, Nylon 66), by Application (Automobile, Electrical & Electronics, Engineering Plastics, Textiles), by Region, and Segment Forecasts, 2021–2028. Research and Markets Ltd 2021. https://www.researchandmarkets.com/reports/4375423/global-nylon-market-size-share-and-trends?utm_source=CI&utm_medium=PressRelease&utm_code=lk7nj3&utm_campaign=1597968+-+Global+Nylon+Market+Size%2c+Share+%26+Trends+Analysis+2021-2028+%7c+Nylon+6+Accounts+for+Highest+Revenue+Share&utm_exec=elco286prd
  3. Sustainable consumption and production | Department of Economic and Social Affairs. (n.d.-b). United Nations. Retrieved October 12, 2021, from https://sdgs.un.org/topics/sustainable-consumption-and-production
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