Team:Evry/Description/Context
Project Description
Context
Plastic waste: a worldwide problem
Yearly, roughly 260 million tons of plastic are being produced worldwide. This represents 8,880 kilograms per second. That plastic is far too frequently thrown off in natural environments, such as forests or oceans, producing visual pollution but also affecting the life in all the ecosystems [1].
Conventional plastics take from 500 to 1000 years to degrade. However, effort put into research have allowed the discovery of a new type of material having similar properties as petroleum-based plastics with lower degradation time: bioplastics. These bioplastics come from renewable sources, are compostable and they do not produce toxic fumes if incinerated. Our question is: can we use synthetic biology to promote the usage of bioplastics?
Figure 1. Plastic pollution. On the left, image representing the huge amount of plastic produced worldwide. On the right, a coastal environment covered in plastic waste.
On the origins of synthetic biology: bacterial production of bioplastics
Historically, one of Synthetic Biology's starting pillars was to engineer bacteria to produce bioplastics. Back to 1984, Oliver Peoples and Sinskey's lab started sequencing a bacterial gene that turned out to code for polyhydroxybutyrate (PHB), a natural polyester. From that, they inferred that they could take bacteria's plastic production at the industrial scale.
With that in mind, they created a company, Metabolix. There, they focused on metabolic engineering, trying to improve the yield of plastic production, which they did [2].
Nowadays, there are several research groups working on the bioproduction of different materials. With the same idea in mind, we decided to focus on engineering a bacterium for producing a biological alternative to petroleum-based plastic, called Poly-Lactic Acid.
PLA bioproduction: state of the art
Back in 2010, Sang Yup Lee's research group managed to produce PLA by modifying the metabolism of Escherichia coli [3]. They reported the heterologous biosynthesis of the PLA homopolymer and its copolymer, poly(3-hydroxybutyrate- co-lactate) or P(3HB-co-LA).
They described two key reactions: the transformation of lactate into lactyl-CoA, as intermediary metabolite, and the polymerization of this lactyl-CoA. For these reactions, E. coli needed to have an engineered propionate CoA-transferase (PctCp), originating from Clostridium propionicum, and also an engineered polyhydroxyalkanoate (PHA) synthase (PhaC1Ps6-19), coming this time from Pseudomonas sp. MBEL 6-19.
With these implementations, E. coli was able to produce PLA and P(3HB-co-LA), but with a relatively low efficiency. Thus, they performed metabolic flux analysis and proceed to knock out several genes, as described on the figure below. An important gene that they tried to enhance, was ldhA, in order to have a high lactate fraction and thus improve the PLA production.
Their results showed that PLA and P(3HB-co-LA) were indeed being produced, as they were characterized by gas chromatography (GC). They could also seen the products in vesicles in the cytoplasm of the E. coli mutants, using transmission electron microscopy.
After the publication of this paper, the same research group kept on investigating PLA bioproduction and published some reviews [4,5]. However, when browsing recent papers that aim at reviewing PLA synthesis methods, most of them do not include biosynthesis [6]. Overall, only the enterprise Carbios seems to have succeeded in engineering bacteria for industrial PLA production [7]. Thus, we aim to push towards this research line, following the path of previous iGEM teams (Yale 2013; UChile-OpenBio 2015), as we believe that it could help the bioplastic industry and, in consequence, also to tackle down the worldwide problem of plastic waste.
*Note: Figures 2 and 3 are from the original paper cited in [3]. Both the publishers and the corresponding author gave us permission to use them.
Figure 2.* Metabolic engineering of E. coli for the production of PLA and P(3HB-co-LA) (from [3]). Overall metabolic network. Right top, in silico PLA production rate versus cell growth rate. Right bottom, in silico flux response analysis.
Figure 3.* PLA and P(3HB-co-LA) accumulated in recombinant E. coli cells (from [3]). Transmission electron micrographs of intracellular PLA homopolymer and P(3HB-co-LA) copolymer granules.
References
- Lytle, C.G. Plastic Pollution. Coastal Care. Retrieved from: http://plastic-pollution.org (9 October 2016).
- Peoples, O. & Sinskey, A.J. Polyhydroxybutyrate (PHB): A Model System for Biopolymer Engineering II. E.A. Dawes (cd.), Novel Biodegradable Microbial Polymers, NATO Adv Sci Inst Series (Holland:Kluwer Academic Publishers), 186, 191-202 (1990)
- Jung, Y.K., Kim, T.Y., Park S.J. & Lee S.Y. Metabolic Engineering of Escherichia coli for the Production of PLA and Copolymers in E. coli. Biotech and Bioeng 105:1, 161-171 (2010)
- Park, S.J., Lee, S.Y., Kim, T.W., Jung, Y.K., Yang, T.H. Biosynthesis of lactate-containing polyesters by metabolically engineered bacteria. Biotechnol J. Feb;7(2):199-212. (2012)
- Yang, J.E., Choi, S.Y., Shin, J.H., Park, S.J., Lee, S.Y. Microbial production of lactate-containing polyesters.Microb Biotechnol. Nov;6(6):621-36. (2013)
- Pretula, J., Slomkowski, S., Penczek, S. Polylactides-Methods of synthesis and characterization. Adv Drug Deliv Rev. In Press, Corrected Proof. doi: 10.1016/j.addr.2016.05.002 (2016)
- Gameiro, D.N. Engineered Microorganisms now able to directly produce Bioplastics for 3D-Printing. Retrieved from: http://labiotech.eu/carbios-pla-metabolic-pathway-bioplastic (9 October 2016)
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