What is PBAT?

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.

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Challenges

Firstly, the natural degradation rate of PBAT is extremely slow. After three months of natural degradation, it can only be degraded by about 2.3%. Secondly, the PBAT decomposition rates of existing PBAT-degrading enzymes in room temperature are generally low, restricting its utilization in the context of agricultural practices. Additionally, PBAT hydrolysis products can be toxic, adversely affecting microbial metabolism and efficient substrate assimilation.

Solution

We identified three distinct enzyme candidates for targeted evolutionary engineering, with the objective of developing highly efficient PBAT hydrolases that exhibit optimal catalytic activity under room temperature conditions. Furthermore, we also aim to obtain an engineering Pseudomonas putida KT2440 strain capable of catabolizing of PBAT-derived degradation products.

Development of a secretion pathway in Bacillus subtilis

We first established an efficient secretion system within Bacillus subtilis. The signal peptide AprE was fused to the N-terminus of the enzymes to enable them to be effectively secreted out of the cells via the Sec pathway. The successfully constructed protein exocytosis pathway allows for intuitive hydrolysis zones screening of the mutation libraries.

Development of PBAT hydrolases

We selected IsPETase, BsLipA, and Lipase1028 as the starting points for directed evolution and established three mutation libraries using error-prone PCR and site-directed mutagenesis. Hydrolysis zone screening method was employed to endow them with the ability to degrade PBAT.

Development of an engineered Pseudomonas putida KT2440 strain

The evolved PBAT hydrolases were transformed into Pseudomonas putida KT2440-tph, which had undergone adaptive laboratory evolution to acquire the ability to utilize terephthalic acid (TPA) and 1,4-butanediol (BDO) as carbon sources. This allowed the construction of a KT2440 strain with high PBAT degradation capacity and the ability to grow using its degradation products.