Jemma Pilcher discusses the advances made by a team from Imperial College London in designing a sustainable recycling platform for the biopolymer poly-3-hydroxybutyrate.
In today’s environmentally conscious society, the concern about conventional oil-derived polyolefin plastics is widespread. They are strong, mouldable materials, with imperviousness to water, enabling them to be used for numerous commercial applications. However, the large-scale production and disposal of plastics is having severe adverse effects on our environment – from drilling for the starting material, petroleum, to landfilling or releasing harmful gases during their incineration. Despite recycling systems being in place, 25Mt of petrochemical plastic waste is accumulating in the environment every year. To control this, a huge research effort is set on sourcing environmentally kinder alternatives to petrochemical plastics, such as naturally produced biopolymers.
Poly-3-hydroxybutyrate (P(3HB)) is a bio-polyester, which is naturally produced intracellularly as an emergency energy and carbon store by a number of bacterial and plant species. It is synthesised when the organism is in the presence of an excess carbon source, such as sugar, and can be microbially digested into CO2 and water. These qualities enable P(3HB) to be manufactured from renewable sources, such as plant biomass, and to completely biodegrade should it accidently be released in the environment.
When extracted and processed, P(3HB) has very similar properties to the petrochemical thermoplastic polypropylene. It can be injection moulded, extruded, thermoformed and electrospun into fibres, giving it the potential to replace some of the widely used petrochemical plastics. Recent studies have shown that the strength of P(3HB) can be improved by chemical modifications or blends with other polymers. Furthermore, biopolymers have additional desirable qualities, such as biocompatibility and biodegradability, which are not present in petrochemical plastics. As such, P(3HB) is an excellent material for agricultural applications, such as films for crop protection or capsules for the slow release of fertiliser. When P(3HB) decomposes, it breaks down into its monomeric unit, 3-hydroxybutyrate (3-HB). This ketone body is naturally present in mammalian blood, meaning that surgical sutures, pins and tissue scaffolds made out of P(3HB) can be inserted into the body without needing to be removed again. Moreover, engineered mammalian cells could be seeded onto a P(3HB) scaffold before being implanted. A recent study also demonstrated that P(3HB) can be used in 3D printing.
Despite having a range of qualities, this biopolymer is currently too expensive to produce on a commercial scale. The major contributors to its high production cost are the price of plant biomass feedstock and the low polymer yield in native P(3HB)-producing organisms. To make large-scale P(3HB) production commercially viable as well as investigate the use of cheaper and more abundant feedstock, research groups are genetically engineering model organisms, such as E. coli, to synthesise the biopolymer with greater yield than in the native organisms. As part of their iGEM project, the students from Imperial greatly increased P(3HB) production by improving a previously standardised and characterised DNA part, or a biobrick.
Producing the plastic
Ralstonia eutropha H16 is a species of bacteria that naturally produces P(3HB). The biopolymer is synthesised as a consequence of three enzymes, which are coded in a cluster of genes called the phaCAB operon – 3-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC). The operon had previously been cloned into E. coli and standardised, enabling continued improvements. E. coli is an appropriate organism for commercial-scale polymer synthesis, as its genetics, physiology and metabolism have been thoroughly studied and it can utilise a diverse range of carbon sources for its growth.
Alternative versions of the operon were constructed using computational model simulations to influence design choices, to increase the P(3HB) production in E. coli. The native operon is comprised of three genes alongside sequences known as ribosomal-binding sites, and preceded by another sequence called a promoter. The ribosomal-binding sites and promoters allow the cell’s expression machinery to recognise the operon and consequently synthesise the enzymes. By changing the ribosomal-binding sites and promoters in the operon, the interaction between these sequences and the expression machinery can be strengthened, which in turn increases the synthesis of the three enzymes and, therefore, P(3HB) yield. In addition to constructing a new constitutive operon with a stronger ribosomal-binding site and promoter, a version was also formulated with a dual ribosomal-binding site and promoter design, called the hybrid operon.
The three operons were characterised by comparing the weight of P(3HB) produced by E. coli carrying either the native, constitutive or hybrid constructs. Furthermore, bacterial cells containing P(3HB) could be visualised under an ultraviolet light when stained with a dye. Their results showed that P(3HB) production by the E. coli carrying the constitutive operon was around two-fold higher than those bearing the native operon. To their amazement, however, the hybrid operon displayed a larger improvement, as the cells engineered with this construct had a six-fold P(3HB) synthesis increase in comparison to the native operon. Deciphering the mechanism by which the dual ribosomal-binding site and promoter design works is the next step in this investigation, and may be applicable to a broader range of systems beyond the phaCAB operon.
Reaching the end
In conjunction with improving the production yield of the polymer, the Imperial team was very conscious of the end-of-life solution for P(3HB). Unlike petrochemical plastics, it cannot be recycled by melting down and reforming. In addition, P(3HB) may be a biodegradable polymer, but it can take months to years to decompose. Discarding it into the environment, therefore, would still result in waste accumulation, plus a loss of resources. To counteract this issue, the team designed and characterised the parts required to construct a synthetic biology, P(3HB) recycling platform. Again using engineered E. coli, this system degrades P(3HB) into 3-HB, then builds it back up again ready for purifying, processing and reuse.
First, the bacteria needed to be equipped with tools for degrading the biopolymer. The E. coli were engineered to express the degradation enzyme, P(3HB) depolymerase, encoded by the gene phaZ1. The enzyme was shown to work by applying the cell lysate of the E. coli, which had been engineered to express the phaZ1 gene, into wells within a set P(3HB) emulsion. The figure (overleaf, top) displays the result – after three days, there was a clear zone around the well where the phaZ1-engineered cell lysate was added, showing that the biopolymer had been degraded. This was compared to a control, whereby the lysate was applied from cells that had not been engineered to express phaZ1 and instead had an empty vector. Furthermore, the presence of 3-HB as a result of this breakdown process was confirmed using an adapted medical kit. The next step in the recycling platform is building up the polymer from 3-HB inside the cells, which occurs during a multi-phase pathway. Initially, 3-HB is converted to the central metabolite, acetoacetyl-CoA, by enzymes that are endogenously expressed in E. coli. Then, finally, the enzymes encoded by the phaCAB operon rebuild the polymer.
As such, the team successfully expressed and characterised the enzymes required to construct a P(3HB) recycling platform. A couple of the parts required to complete the system were particularly challenging to identify and could not be accomplished during the short duration of the project. In order to degrade P(3HB) outside of the cell, the depolymerase enzyme needs to be secreted. The team designed the phaZ1 construct with a sequence known as a secretion tag, which should enable the cell’s export machinery to expel the enzyme. Although the enzyme was synthesised, it was not exported from the cell. Further research needs to be conducted to identify an appropriate secretion tag. Another part of the platform that needs further investigation is a transport system that imports 3-HB into the cell, ready to convert into P(3HB).
In addition to increasing P(3HB) yield in E. coli, the Imperial team set out to identify a cheap and abundant feedstock for its production. The solution was to use mixed, non-recyclable waste, which would otherwise be incinerated or landfilled. This waste is largely composed of scraps of paper, wood and fibres, along with small pieces of plastic and metal, which remains after the valuable materials have been recovered. As proof of concept, the team demonstrated that their E. coli could grow and produce P(3HB) using this waste as a sole carbon source. For this concept to be commercially viable, the waste would need a pre-treatment to purify the sugar content to use as the feedstock.
Although this project is still in its infancy, it demonstrates the potential that synthetic biology has to address global environmental issues. Improving the yield, as well as designing a recycling system for the biopolymer, should enable sustainable P(3HB) production and commercial application in the future. This system, implemented on an industrial scale, would alleviate the necessity to drill for petroleum and reduce the accumulation of petrochemical plastics in the environment. Furthermore, using mixed, non-recyclable waste as a feedstock diverts these materials away from incinerators and landfill sites, which are detrimental to the environment.