Abstract

We produced poly-(3-hydroxybutyrate) [P(3HB)] as a substrate to fold biOrigami. P(3HB) is a biopolymer with thermoplastic properties, meaning that it contracts in heat. Unlike polystyrene, P(3HB) is biodegradable. Building on previous iGEM teams’ work on P(3HB), we have contributed BioBricks that increase P(3HB) production and facilitate its extraction from Escherichia coli, and have processed the polymer into useful sheets. See our biobricks here:

See our BioBricks

Introduction

We are using recombinant E. coli to produce P(3HB) for folding biOrigami. Since plastics on earth have deleterious environmental impacts, we wanted to focus on producing one biodegradable plastic that could be synthesized for use on the moon or on Mars.

P(3HB) is the most common polyhydroxyalkanoate, which is a group of fully biodegradable polymers with properties similar to those of petroleum-based plastics. It can be produced in a variety of microorganisms, and its synthesis as a compound for energy storage in vivo has been extensively studied [1]. P(3HB) accumulates inside cells, and has been reported to occupy up to 90% (w/w) of the dry cell mass in certain species [2]. However, P(3HB) has yet to become integrated into the plastics market on Earth due to high costs of both fermentation and purification [3]. This summer, we focused on making the production, extraction, and use of P(3HB) more feasible on a lunar or Martian base.

(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.

Experiment Engineering E. coli to produce polystyrene

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Data and Results Optimizing the production of biological PHA

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Protocols

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References

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