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  • Background
  • Biodegradation of PBAT meets challenges
  • Our goal
  • Reference
    • Background

    Poly (butylene adipate-co-terephthalate) (PBAT), a polymer synthesized from the monomers terephthalic acid (TPA), adipic acid (AA), and 1,4-butanediol (BDO), has garnered significant attention as a biocompatible and degradable polymeric material, exhibiting considerable potential for widespread application in the realm of agricultural film technology (Figure 1a)[1-3]. However, owever, the consequences of the growing and hard-to-reverse issue of polyethylene films on our planet are challenging, affecting both the physical environment and living organisms (Figure 1b and c)[4-8].

    Figure 1. (a) PBAT has a wide range of applications in various fields. (b) The use of PBAT agricultural film in agriculture. PBAT agricultural film can effectively reduce weeds and pests, regulate soil temperature, improve water retention capacity, reduce soil erosion, and increase crop yields by 20% to 60%. (c) Abandoned PBAT agricultural film causes large-scale plastic pollution.

    Biodegradation of PBAT meets challenges

    As an eco-friendly and sustainable strategy to reduce and recycle plastic waste, biodegradable catalysts using enzymes or microorganisms meet some challenges. Firstly, the degradation of PBAT yields a mixture of TPA and AA with distinct physical and chemical properties (Figure 2). Microorganisms typically encounter carbon catabolite repression when confronted with this varied substrate mix, impeding their ability to utilize multiple substrates simultaneously. Additionally, PBAT hydrolysis products can be toxic, adversely affecting microbial metabolism and efficient substrate assimilation, particularly at high concentrations or with crude hydrolysates[9-11].

    Figure 2. Chemical structures of PBAT and PET and respective ester hydrolysis products.

    The hydrolysis products of PBAT contain terephthalic acid (TPA), adipic acid (AA) and 1,4-butanediol (BDO)[3] . Biodegradation of PET also produces TPA, suggesting that certain enzymes that degrade PET have the potential to degrade PBAT.

    Existing PBAT-degrading enzymes and microorganisms typically require high temperatures (50-65℃) for effective composting (Table 1) [12]. Jia et al. identified a serine hydrolase enzyme from Thermobifida fusca FXJ-1 capable of degrading PBAT at 55℃, while Wallace et al. utilized proteomics to isolate an esterase from Pseudomonas pseudoalcaligenes effective at 65℃[13]. Nevertheless, the PBAT decomposition rates of existing PBAT-degrading enzymes in room temperature are generally low, restricting its utilization in the context of agricultural practices[14]. Moreover, Wang et al. reported that the degradation rate of PBAT films in conventional agricultural soil was only 2.3% over a three-month period[15].

    Table 1. Enzymes with PBAT degradation developed in recent years
    Enzyme Classification Source Activity (mol/mol)* PBAT Condition Ref. Genbank
    PfL1 Lipase Pelosinus Fermentans ~130 Film
    Milled    		 50°C 72h [16] EIW29778.1
    PpEst Esterase Pseudomonas pseudoalcaligenes ~8
    ~70    		 Film
    Milled    		 65°C 72h [17] W6R2Y2
    Cbotu_EstA Esterase Clostridium Botulinum <30 Not specified 50°C 72h [17] CAL82416.1
    Cbotu_EstB Esterase Clostridium botulinum ~12 Not specified 37°C 72h [17] CAL83600.1
    TfCut Cutinase Thermobifida fusca 5198 Film 70°C 48h [3] CBY05530
    IsPETase Cutinase Ideonella sakaiensis 4868 Film 30°C 48h [3] A0A0K8P6T7
    PbPL Cutinase Polyangium brachysporum - Film 30°C 48h - -
    BurPL Cutinase Burkholderiales bacterium 3208 Film 35°C 48h [3] -
    Ple628 Hydrolases Marine microbial consortium 121 Film 30°C 144h [18] OK558824
    Ple629 Hydrolases Marine microbial consortium 1704 Film 30°C 72h [18] OK558825
    HiC Cutinase Humicola insolens ~10,000 Film 50°C 72h [17] A0A075B5G4
    LCC Cutinase Leaf-branch compost 4636 Film 70°C 24h [3] G9BY57.1
    ICCG Cutinase Leaf-branch compost 5275 Film 75°C 24h [3] USU85609.1
    TcCut Cutinase Thermobifida cellulolysitica 5361 Film 65°C 48h [3] ADV92526.1

    *The activity is the quantitation of products containing TPA and BTa (mol) per mol enzymes. - refers to not find.

    Our goal

    In this work, we identified three distinct enzyme candidates for targeted evolutionary engineering, with the objective of developing highly efficient PBAT-degrading enzymes that exhibit optimal catalytic activity under room temperature conditions. These enzyme candidates include IsPETase, a cutinase derived from the PET-degrading bacterium Ideonella sakaiensis; BsLipA, a lipase from Bacillus subtilis; and Lipase1028, which has been newly isolated in our laboratory. IsPETase is capable of degrading PET in the glassy state at moderate temperatures (30-37°C)[19-22]. BsLipA exhibits good hydrolytic activity on ester bonds formed by medium-chain fatty acids[23, 24]. Lipase1028 demonstrates significant degradation capabilities for polyurethanes (PU). Based on the structural similarities between the substrates of these three enzymes and PBAT, we propose that all three possess considerable evolutionary potential[3, 25].

    Furthermore, we also aim to obtain an engineering single strain capable of catabolizing of PBAT-derived degradation products. Pseudomonas putida has been recognized as a potential host organism for a diverse range of biotechnological applications, including the metabolic processing of plastics [26, 27]. Wing-Jin Li et al. reported that the wild-type Pseudomonas putida KT2440 could degrade TPA but at a very slow rate, requiring over 50 hours to degrade 20 mM substrate[28]. In this study, we transformed the gene cluster tph derived from Pseudomonas stutzeri TPA3 into KT2440 to enable its degradation of TPA[29, 30]. Then, we performed adaptive laboratory evolution and metabolic engineering to isolate KT2440 variants capable of utilizing 1,4-butanediol as the sole carbon source. Ultimately, we will transform the evolved PBAT degradation enzyme genes which are obtained from directed evolution, into the engineered KT2440 strain, aiming to create a single strain capable of efficiently degrading PBAT and utilizing its degradation products, thus facilitating sustainable degradation processes eventually.

    Reference

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