Werner Allison Z, Avina Young-Saeng C, Johnsen Josefin, Bratti Felicia, Alt Hannah M, Mohamed Elsayed T, Clare Rita, Mand Thomas D, Guss Adam M, Feist Adam M, Beckham Gregg T
Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA; BOTTLE Consortium, Golden, CO, USA.
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
Metab Eng. 2025 Mar;88:196-205. doi: 10.1016/j.ymben.2024.12.006. Epub 2024 Dec 17.
Poly (ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.
聚对苯二甲酸乙二酯（PET）是最常见的塑料之一，可通过生物和化学催化途径解聚为其组成单体对苯二甲酸（TPA）和乙二醇（EG）。TPA和EG可重新合成PET用于闭环回收，或通过微生物转化为高价值产品用于开环回收。在此，我们通过采用适应性实验室进化（ALE）来改善TPA分解代谢，扩展了我们之前对恶臭假单胞菌KT2440进行工程改造并应用于PET转化的工作。三株对EG分解代谢进行了不同程度代谢工程改造的恶臭假单胞菌菌株，在TPA、TPA和EG混合物以及作为对照的葡萄糖上进行了自动化ALE实验。在大约350代的时间里，ALE分别使TPA和TPA - EG混合物上的生长速率提高了4.1倍和3.5倍。在39个独立的ALE实验的中点和终点收集进化菌株，在表现最佳的菌株中，TPA和TPA - EG上的生长速率分别提高了0.15 h⁻¹和0.20 h⁻¹。全基因组重测序鉴定出多个趋同突变，包括全局调节因子gacS、gacA和turA的功能丧失突变，以及影响异源表达的tphAB分解代谢基因的大的重复和基因间缺失事件。对这些靶点的逆向工程证实了因果关系，与基础菌株相比，一株缺失了所有三个调节因子且有tphAB和tpaK的第二个拷贝的菌株显示出更好的TPA利用能力。总之，一个涉及异源途径工程、ALE、全基因组测序和基因组编辑的迭代菌株工程过程确定了五种改善恶臭假单胞菌在TPA上生长的基因干预措施，旨在开发用于PET升级回收的增强型全细胞生物催化剂。
