The three devices are:

Device 1: J23101 + I13504, in pSB1C3

Device 2: J23106 + I13504, in pSB1C3

Device 3: J23117 + I13504, in pSB1C3

(1) Increasing P(3HB) Yield

Increasing the yield of P(3HB) from a cell culture would decrease the time needed to complete the plastic production and extraction process by making smaller cultures more efficient. We have used the phaCAB operon from Ralstonia eutropha H16 that encodes the three genes required for P(3HB) production: PhaA, PhaB, and PhaC. Previous iGEM teams (Tokyo Tech 2012 and Imperial 2013) were able to successfully produce P(3HB) in vivo using BioBricks that encode these three genes. The pathway for P(3HB) production is as follows. First, 3-ketothiolase, encoded by PhaA, combines two molecules of acetyl-CoA to form acetoacetyl-CoA. This is then reduced by acetoacetyl-CoA reductase, encoded by PhaB, to form (R)-3-hydroxybutyl-CoA. This is then polymerized by PHA synthase, encoded by PhaC, forming poly-3-hydroxybuterate.

To increase the yield of P(3HB), we looked for a way to increase the amount of acetyl-coA, the precursor molecule to P(3HB) formation. Acetyl-CoA is composed of an acetyl group bound to coenzyme A. Coenzyme A consists of a beta-mercaptoethylamine group linked to pantothenic acid. On the Tokyo Tech 2012 iGEM team’s wiki, their results showed that adding pantothenic acid into their culture media led to increased yields of P(3HB). Therefore, we decided to increase the production of coenzyme A, which would then allow the cells to synthesize greater amounts of P(3HB).

Coenzyme A biosynthesis is a five-step process that is primarily regulated by the first enzyme in the pathway, pantothenate kinase (encoded by the gene PanK, which is also called CoaA) [4]. Pantothenate kinase stringently controls amount of coenzyme A produced in E. coli because the pantothenate kinase produced by E. coli is significantly inhibited by coenzyme A and its thioesters (such as acetyl-coA). Therefore, since we wanted to increase coenzyme A synthesis, we had to find a way to get around this negative feedback inhibition. Fortunately, the pantothenate kinase made in Staphylococcus aureus does not experience feedback inhibition from coenzyme A or its thioesters [5]. Indeed, this allows S. aureus to accumulate high levels of coenzyme A. We ordered the S. aureus pantothenate kinase sequence from IDT and inserted into the backbone PSB1C3 in front of the phaCAB operon designed by the Tokyo Tech iGEM team in 2012 with the hybrid promoter designed by the Imperial College iGEM team in 2013. The results section below shows that inserting the gene for pantothenate kinase allows for higher levels of P(3HB) production in E. coli.

(2) Facilitating P(3HB) Extraction

Since bacteria that naturally synthesize P(3HB) use the plastic as an energy storage mechanism, the plastic is produced inside the cell and stays there. Currently, the isolation of P(3HB) from the cells and culture media is one of the main bottlenecks in the P(3HB) production process – and the main source of its high cost of approximately $4-6 USD [6]. Traditional isolation methods include the use of large amounts of chemicals that can be hazardous to human health and the environment, such as chloroform and sodium hypochlorite, or other problems such as large volumes of waste water produced or impurities in the polymers [6]. Since one of our team’s goals has been to reduce up-mass of materials on space flights, we wanted to minimize the amounts of harmful (and heavy) chemicals that would need to be brought up as payload to extract the P(3HB).

We accomplished this by introducing a lysis device into the PSB1C3 plasmid that contained the phaCAB operon and the gene for pantothenate kinase. The lysis device was developed by the 2008 Berkeley iGEM team. The device contains T7 phage antiholin under a weak promoter, and T7 phage holin and endolysin inducible under (L)-arabinose. This lysis device was not reported to kill entire populations of E. coli, and we saw this as an advantage for a continuous plastic-production system. We would be able to cause part of the population to lyse and release its P(3HB) into the media. The rest of the population would continue growing and producing plastic. The plastic can be recovered from the media by causing the granules to aggregate, and then requiring lower amounts of solvents and less time to complete the purification process [7]. The results section below demonstrates the success of the lysis system and compares the efficiency of the recovery methods with and without the release of the P(3HB) into the media.

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References

[1] Anderson, A. J. and Dawes, E. A. Occurrence, Metabolism, Metabolic Role, and Industrial Uses of Bacterial Polyhydroxyalkanoates. Microbiological Reviews 54 (4), 1990. 450 – 472.

[2] Madison, L. L. and Huisman, G. W. Metabolic Engineering of Poly(3-Hydroxyalkanoates): From DNA to Plastic. Microbiol Mol Biol Rev 63 (1), 1999. 21 – 53.

[3] Kunasundari, B. and Sudesh, K. Isolation and recovery of microbial polyhydroxyalkanoates. eXPRESS Polymer Letters 5 (7), 2011. 620 – 634.

[4] Leonardi, R. et al. Coenzyme A: Back in action. Progress in Lipid Research 44, 2005. 125 – 153.

[5] Leonardi, R. et al. A Pantothenate Kinase from Staphylococcus aureus Refractory to Feedback Regulation by Coenzyme A. The Journal of Biological Chemistry 280, 2005. 3312 – 3322.

[6] Jacquel, N. et al. Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochemical Engineering Journal 39 (1), 2008. 15 – 27.

[7] Rahman, A. et al. Secretion of polyhydroxybuterate in Escherichia coli using a synthetic biological engineering approach. Journal of Biological Engineering 7 (24), 2013.