We have gained several mutants of PETase, PET hydrolysis activity of which are relatively improved compared to PETase Wild-type. Among them, it’s worth mentioning that I208V shows an activity increase of two-fold. However, all of our mutants up to now are single-site mutant, which have been merely changed only single one amino acid residue.
In our future work, we plan to design double-site or even multiple-site mutants according to our single-site mutants. We hope to combine two or several improved sites together into one protein, like I108V and S207T, to further extend the range of activity improvement. The hydrolysis activity of double-site mutants may not be linear addition of hydrolysis activity of the respective single mutants, but based on rational design rationales, the improvement is promising.
1. A Bigger Family
After our 3-bacterium consortium was established stably, the first thought came to our brains was whether we could expand this bacteria family. How to make this system work better and even perfectly is the first problem that we faced.
During our bacterial consortium experiment, actually, we tried many kinds of carbon sources, nitrogen sources and energy sources. Our bacteria could not live unless those sources were provided. From this point, our bacterial consortium is not an autotrophic system. Without laboratory environment, this system cannot naturally work. Due to abundant natural resources, each bacterium will not choose to work together to degrade PET unless they are forced to. In nature, the first choice for each creature is intake of energy and resources in the simplest way to maintain the reproduction and continuity of life.
Adding Cyanobacteria to our bacterial consortium is at the top of our list. Cyanobacteria are excellent organisms for biofuel production. Cyanobacterium Synechocystis sp. PCC6803 is a perfect lipid producer. However, Synechococcus elongatus PCC7942 has larger capacity of lipid production than Synechocystis sp. PCC6803 but accumulates most of the product in the cell because of the imbalance of the rates of lipid production and secretion. Initially, we intended to do something to increase lipid secretion by knocking the wzt gene(Akihiro Kato et al. 2016). However, Synechococcus elongatus PCC7942 wasn’t able to revive in two-week shaking cultivation. So we turned into Synechocystis sp.PCC6803.
The main lipid produced by PCC6803 is free fatty acid. The length of fatty acid is about 14-carbon to 16-carbon long. To our surprise, B.s 168, P.p KT2440 and R.j RHA1, according to KEGG, all have the pathways for fatty acid. It is reported that P.p KT2440 really like to live in a condition with lipid. Actually, the optimal carbon source for it to accumulate PHA is lipid. This provide a nice base for adding Synechocystis to our bacterial consortium.
2. Make it autotrophic
Adding Synechocystis sp.PCC6803 to our bacterial consortium can make this system become autotrophic. It will unlock the limit of energy source and help the whole system can concentrate on secreting PETase and MHETase and removing substrate inhibition.
In order to increase lipid secretion, we knocked out the wzt gene to make it easier for lipid pass across the cell membrane. To make PCC6803 controllable and avoid outbreak, we established a Nickel sensing and responding signal system. With this system, we can control the timing of phage lysis genes by adding Nickel ion. In this way, we can easily control the carbon source and cyanobacteria’s explosion, even if it would be used in nature one day.
Actually, there are still many works need to be done on this aspect. we constructed the expression vector successfully and transformed it into Synechocystis PCC6803 via electroporation. But the final results still remain unknown. We need more time to prove our view and let Synechocystis PCC6803 get into our big bacterial consortium family.
We have constructed the novel inclusion body based reporting system and cell lysis regulation system and introduce the TPA positive feedback regulation system to Saccharomyces cerevisiae. However, we have only tested the CpxR promoter and there are much more than only one promoter that can be induced by inclusion body or misfold protein. For instance, the degP promoter, ibpAB-fsxA promoter, etc. These promoters are also in the iGEM parts database (degP:BBa_K339008, ibpAB-fsxA:BBa_K339011). Which one has the best effect is still unknown yet and more experiments need to be done by later teams or people.
We use the RFP as the reporting protein because of it’s easy to detect, even by bare eyes if expression level is high enough. However, unless applying the damaging way like centrifugation or expensive device like 96-well Microplate Reader, the phenomenon is still not so clear. A more intuitive reporting strategy need to be developed.
We used the ddpX gene to automatically break the cells, however, it might be better to keep the bacterial alive and make the misfold protein recovered by itself. There have been some researches about this, and some particular enzymes in E.coli can help protein folding and inclusion body degrading in envelope such as degP, fkpA, PpiA, PpiD, DsbA, etc. We are looking forward to more researches on this field so that we can not only get TPA induced gene, but also genes induced by other substances via sequence design according to the inducing mechanism.
Tracy L. Raivio, Michael W. Laird, John C. Joly and Thomas J. Silhavy. Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress response. Molecular Microbiology (2000) 37(5), 1186-1197