On August 21, 2024, the research team led by Dai Zhuojun at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, published a study in Nature Chemical Biology titled "Degradable living plastics programmed by engineered spores." This project involved genetically editing microbes to produce spores with extreme environmental tolerance, enabling them to secrete plastic-degrading enzymes under specific conditions. Additionally, the spores were embedded in the plastic matrix through plastic processing methods (high temperature, high pressure, or organic solvents).
Screenshot of the article online
（Link:https://www.nature.com/articles/s41589-024-01713-2）
In daily use environments, the spores remain dormant while the plastic maintains its stable performance. Under specific conditions (such as surface erosion or composting), the spores in the plastic are activated and initiate the degradation process, leading to the complete breakdown of the plastic (Figure 1).
Figure 1. Overall Research Approach
Research Background
The invention of plastics has brought great convenience to our daily lives. However, the massive production of plastic waste and improper disposal methods have made plastic waste (white pollution) one of the most severe environmental issues today. In 2016, Yoshida et al. reported on the soil bacterium Ideonella sakaiensis, which was found growing in sediment contaminated with PET near a plastic recycling facility in Japan. This Gram-negative, aerobic, rod-shaped bacterium possesses an extraordinary ability to utilize PET as its primary carbon source by expressing two key enzymes: PETase and MHETase. In subsequent studies, a large body of work in synthetic biology has focused on the discovery, design, evolution, and modification of plastic-degrading enzymes, but there has been little innovation in the synthesis methods for degradable plastics.
In 2018 and 2021, Ting Xu's team at the University of California, Berkeley, with a background in polymer physics, published two articles in Science and Nature, respectively, advancing the development of degradable plastics from another perspective and dimension. In their research, the research team developed a polymer composed of four monomers (RHPs, random heteropolymers), each capable of interacting with chemical fragments on the surface of target proteins. These monomer subunits are linked together to mimic natural proteins, maximizing the flexibility of interactions between them and the protein surface. This rational design based on interaction allows proteins to fold correctly in cell-free synthesis and maintain the activity of water-soluble proteins in organic solvents. Building on this research, Ting Xu's team mixed plastic-degrading enzymes, RHPs, and plastic masterbatch (polycaprolactone, PCL) for processing, where RHPs protects the hydrolase's biological function in the harsh plastic processing environment. The plastic can be stably used in an anhydrous environment and rapidly degrade in aqueous environments or during composting (Nature, 2021).
Challenges remain in pre-embedding degradation enzymes into plastics to balance extreme conditions during processing with enzyme stability. Although Ting Xu's team proposed a definite solution through RHPs to regulate protein stability, the widespread application of this method still faces many challenges. First, the synthesis of RHPs is challenging, even for laboratories with general chemical synthesis backgrounds; second, the processing temperature of PCL (80-120 degrees Celsius) is almost the lowest among plastics, with common plastic processing temperatures mostly above 200 degrees Celsius, including PET (polyethylene terephthalate) at up to 300 degrees Celsius, presenting significant challenges to RHPs' protective capabilities in these systems.
Dormant Spores and Living Plastics
Throughout millions of years of natural evolution, many microorganisms have developed resistance to harsh environmental conditions. When extreme conditions arise, making survival and reproduction unsuitable, bacteria transform into spores. This transformation grants bacteria enhanced resilience. Spores can withstand extreme dryness, temperature, and pressure, precisely the conditions found in plastic processing environments. Consequently, the research team proposed engineering Bacillus subtilis through synthetic biology methods to incorporate genes for controlled secretion of plastic-degrading enzymes (lipase BC from C. oleovorans) and induce dormancy in Bacillus subtilis under manganese ion stress, forming spores. These engineered spores carry the edited genetic circuitry and exhibit tolerance to high temperatures, pressure, organic solvents, and dryness compared to bacteria. By directly mixing the engineered spore solution with PCL plastic masterbatch and using high-temperature melt extrusion or organic solvent methods, the team prepared a series of spore-containing plastics. In various physical property tests, they found no significant differences between these living plastics and regular PCL plastics in terms of yield strength, ultimate stress, maximum strain, and melting point. In daily use environments, spores remain dormant, allowing the plastic to maintain stable performance (Figure 2).
Figure 2. Macroscopic and Microscopic Images of Regular PCL Plastic and "Living" Plastic
Release of Spores and Initiation of Degradation Process
The first step in plastic degradation involves successfully releasing and reviving the spores inside the living plastics. Researchers initially explored two methods for spore release. One method employed the Lipase CA from Candida antarctica to erode the plastic surface. Lipase CA's hydrolytic action on PCL plastic acts like a "scissor" (Figure 3), macroscopically breaking down the external structure of PCL plastic. Under the influence of Lipase CA, the PCL surface is damaged, releasing the engineered spores embedded within the material to the external environment, where they begin to revive and grow, initiating the expression of Lipase BC. Lipase BC binds to the terminal ends of the PCL polymer chains, progressively degrading them completely (ultimate degradation molecular weight <500 g/mol). The results indicated that the living plastics could rapidly degrade within 6-7 days, whereas regular PCL plastics subjected only to surface erosion (by Lipase CA) still contained a significant amount of plastic fragments even after 21 days (Figure 4).
Figure 3. Schematic Diagram of the Degradation Mechanism of PCL Plastic by the Two Enzymes
Figure 4. Surface Structure and Molecular Weight Changes of PCL Plastic Before and After Degradation by Two Enzymes:
(a) Degradation effects on regular PCL plastic (left) and living functional plastic (right) by Lipase CA treatment; (b) Molecular weight change curve during the degradation process of living PCL; (c) Molecular weight change curve during the degradation process of regular PCL plastic by Lipase CA alone.
Another method of spore release involves composting. Without the need for any additional external agents, living plastics can be completely degraded within 25-30 days in a soil environment. In contrast, traditional PCL plastics require approximately 55 days to degrade to the point where they are no longer visible to the naked eye (Figure 5).
Figure 5. Degradation of "Living" Plastics under Soil Conditions:
(a) Degradation of living plastics in a soil environment; (b) Degradation of regular PCL plastics in a soil environment.
More Attempts
As mentioned earlier, the processing conditions for PCL in the plastics family are relatively "mild." The choice of the PCL system in this study was largely due to its efficient enzymatic degradation system foundation: Lipase BC, as a processive enzyme, can capture and completely degrade PCL chains. To verify the universality of the system, we continued to explore other plastic systems. We mixed spores carrying green fluorescent plasmids with PBS (polybutylene succinate), PBAT (polybutylene adipate-co-terephthalate), PLA (polylactic acid), PHA (polyhydroxyalkanoates), and even PET (polyethylene terephthalate), which has a processing temperature of up to 300 degrees Celsius. Afterwards, we released the spores through physical grinding. Interestingly, even the spores released from PET plastic could still revive and re-express green fluorescence. This also laid a solid foundation for creating living plastics based on other substrates (Figure 6).
Figure 6. Other Substrate "Living" Plastics:
(a) Different types of plastics and their processing temperatures; (b) Hot melt preparation of "living" plastics with various substrates; (c) Physical breaking of plastics to release and activate spores carrying green fluorescent protein; (d) Grinding and breaking of "living" plastics; (e) Successful release and expression of green fluorescent protein by engineered spores.
To verify the scalability of the system, the research team also conducted a small-scale industrial test using a single-screw extruder. The living PCL plastic obtained through the above methods still exhibited rapid and efficient degradation efficiency (Figure 7). Furthermore, researchers immersed the living plastic in Sprite for two months. Without any external intervention, the living plastic maintained a stable shape, indicating that it can be used like traditional plastic. The degradation process is initiated only when the plastic is damaged or discarded. This study provides a new perspective and method for the development of novel biodegradable plastics, potentially helping to address the severe issue of plastic pollution.
Figure 7. Single-screw extruder preparation of "living" plastic and its degradation performance test.
(a) Living functional plastics prepared by a single-screw extruder; (b) Degradation test of living functional materials prepared by a single-screw extruder
The corresponding author of the paper is Researcher Dai Zhuojun from the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. Tang Chenwang, a co-trained PhD student in Dai Zhuojun's group, is the first author of the paper. Wang Lin and Sun Jing made significant contributions to the experimental design, progress, and article revision. This research was supported by several projects, including the National Key R&D Program, the National Natural Science Foundation of China for Excellent Young Scholars and General Program, and the Guangdong Provincial Outstanding Youth Natural Science Foundation.
References:
[1] Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate)[J]. Science, 2016, 351: 1196-1199.
[2] Panganiban B, Qiao B, Jiang T, et al. Random heteropolymers preserve protein function in foreign environments[J]. Science, 2018, 359: 1239-1243.
[3] DelRe C, Jiang Y, Kang P, et al. Near-complete depolymerization of polyesters with nano-dispersed enzymes[J]. Nature, 2021, 592: 558–563.
