Metabolic and Biomolecular Engineering National Research Laboratory and Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Republic of Korea; BioProcess Engineering Research Center, KAIST, Daejeon, 34141, Republic of Korea.
Metabolic and Biomolecular Engineering National Research Laboratory and Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Republic of Korea; BioProcess Engineering Research Center, KAIST, Daejeon, 34141, Republic of Korea; BioInformatics Research Center, KAIST Institute for the BioCentury, and KAIST Institute for Artificial Intelligence, KAIST, Daejeon, 34141, Republic of Korea.
Metab Eng. 2023 Mar;76:75-86. doi: 10.1016/j.ymben.2023.01.007. Epub 2023 Jan 21.
Terephthalic acid (TPA) is an important commodity chemical used as a monomer of polyethylene terephthalate (PET). Since a large quantity of PET is routinely manufactured and consumed worldwide, the development of sustainable biomanufacturing processes for its monomers (i.e. TPA and ethylene glycol) has recently gained much attention. In a previous study, we reported the development of a metabolically engineered Escherichia coli strain producing 6.7 g/L of TPA from p-xylene (pX) with a productivity and molar conversion yield of 0.278 g/L/h and 96.7 mol%, respectively. Here, we report metabolic engineering of Pseudomonas putida KT2440, a microbial chassis particularly suitable for the synthesis of aromatic compounds, for improved biocatalytic conversion of pX to TPA. To develop a plasmid-free, antibiotic-free, and inducer-free biocatalytic process for cost-competitive TPA production, all heterologous genes required for the synthetic pX-to-TPA bioconversion pathway were integrated into the chromosome of P. putida KT2440 by RecET-based markerless recombineering and overexpressed under the control of constitutive promoters. Next, TPA production was enhanced by integrating multiple copies of the heterologous genes to the ribosomal RNA genes through iteration of recombineering-based random integration and subsequent screening of high-performance strains. Finally, fed-batch fermentation process was optimized to further improve the performance of the engineered P. putida strain. As a result, 38.25 ± 0.11 g/L of TPA was produced from pX with a molar conversion yield of 99.6 ± 0.6%, which is equivalent to conversion of 99.3 ± 0.8 g pX to 154.6 ± 0.5 g TPA. This superior pX-to-TPA biotransformation process based on the engineered P. putida strain will pave the way to the commercial biomanufacturing of TPA in an industrial scale.
对二甲苯（pX）生产对苯二甲酸（TPA）的代谢工程改造
对苯二甲酸（TPA）是一种重要的大宗化学品，用作聚对苯二甲酸乙二醇酯（PET）的单体。由于全世界常规生产和消费大量的 PET，因此其单体（即对苯二甲酸和乙二醇）的可持续生物制造工艺最近受到了广泛关注。在之前的一项研究中，我们报道了从对二甲苯（pX）生产 TPA 的代谢工程大肠杆菌菌株的开发，该菌株能够生产 6.7 g/L 的 TPA，生产力和摩尔转化率分别为 0.278 g/L/h 和 96.7 mol%。在这里，我们报告了适合芳香族化合物合成的微生物底盘假单胞菌 KT2440 的代谢工程改造，以提高 pX 到 TPA 的生物催化转化。为了开发具有成本竞争力的 TPA 生产无质粒、无抗生素和无诱导剂的生物催化工艺，所有用于合成 pX 到 TPA 生物转化途径的异源基因都通过基于 RecET 的无标记重组整合到假单胞菌 KT2440 的染色体中，并在组成型启动子的控制下过表达。接下来，通过基于重组的随机整合的迭代和随后的高性能菌株筛选，将异源基因的多个拷贝整合到核糖体 RNA 基因中，从而提高 TPA 的产量。最后，优化了分批补料发酵工艺，以进一步提高工程化假单胞菌菌株的性能。结果，从 pX 生产 38.25±0.11 g/L 的 TPA，摩尔转化率为 99.6±0.6%，相当于 99.3±0.8 g pX 转化为 154.6±0.5 g TPA。基于工程化假单胞菌菌株的这种优越的 pX 到 TPA 生物转化工艺将为工业规模的 TPA 商业生物制造铺平道路。
