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We're continuing our project from last year. We were inspired by Hong Kong‘s rich history as a thriving textile production centre in the global industry. However, this great history has a dark side. Chemical and physical hazards caused in textile processing are one of the major issues in pollution, especially for synthetic fabrics. Lactams, which can be used for making nylon, are also synthesized with petrochemical methods [1]. After research and discussion, the team agreed that a green pathway for lactams is a great idea to tackle the problem of environmental pollution. Not only are these cyclic amides a valuable commodity in the world of synthetic fabric production, they also can be used for the synthesis of drugs and household items [2] [3] [4]. Green pathways could reduce usage of harsh chemicals and production of harmful waste which are highly damaging to the environment.
We also wanted to include a solution to global warming in our project, which is a worldwide issue worsening by the day. Successful reports of producing chemicals through cyanobacteria are promising, and with its photoautotrophic nature, cyanobacteria was a perfect chassis for our project [5].
Therefore, we came up with the idea of using the cyanobacterial strain Synechococcus elongatus UTEX 2973 to synthesize 5AVA, then cyclizing it to valerolactam. This biosynthesis method can replace the industrial process of making valerolactam. Although only one part of the overall synthesis of nylon is replaced, it is still one step closer to a world made sustainable through synthetic biology.
As COVID-19 had a great impact on the time we could spend in the lab last year, we’re doing a two year project. This year, we focused on the steps of the pathway after the synthesis of 5AVA. Two proteins are involved: CF3HBD, the cyclase that cyclizes 5AVA to valerolactam, and NitR, the protein that binds to the valerolactam.
We based our protein-release method on a paper by Fujiwara & Doi, who developed the LoFT method of preparing cell extract [6]. It stands for “lysozyme treatment, osmotic shock, and freeze-thawing”. This allows us to obtain expressed CF3HBD and NitR from expression strains E.coli NiCo21 and E.coli T7 without relying on harsh chemicals to lyse the cells. At the same time, 5AVA is synthesized and released from engineered S.elongatus UTEX 2973 by cell lysis.
After 5AVA is released and cyclised by CF3HBD, we needed to separate the valerolactam from the rest of the solution. Yeom et al. gave us the idea to capture our cyclized valerolactam using proteins. NitR, a regulatory protein from the nitrile degradation operon of Alcaligenes faecalis, is activated by lactam compounds [7]. We plan to use NitR as a biosensor for valerolactam, and use the Spytag-Spycatcher system engineered by Zakeri et al. to anchor it onto magnetic beads so the binding of valerolactam can be upscaled [8]. Both are His-tagged for ease of purification; the Spytag is attached to NitR, while the Spycatcher is anchored to a magnetic bead. The Spytag and Spycatcher can form a strong isopeptide bond, holding the two parts together. After NitR captures the valerolactam, the whole complex can be separated out by magnetic force. The valerolactam is then released from NitR and the beads can be reused.
The valerolactam released from NitR can then be dissolved in an organic solvent, e.g. carbon tetrachloride, then be analyzed chemically by NMR. The enzymatic properties of NitR as well as CF3HBD can be analyzed by IR titration.
We hope that our project can help to alleviate the detrimental environmental impact of the production of this greatly important chemical commodity, lactams, locally and around the world.