Ackermann Yannic S, Li Wing-Jin, Op de Hipt Leonie, Niehoff Paul-Joachim, Casey William, Polen Tino, Köbbing Sebastian, Ballerstedt Hendrik, Wynands Benedikt, O'Connor Kevin, Blank Lars M, Wierckx Nick
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany.
Institute of Applied Microbiology - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Aachen, Germany.
Metab Eng. 2021 Sep;67:29-40. doi: 10.1016/j.ymben.2021.05.001. Epub 2021 May 7.
Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates. The dcaAKIJP genes from Acinetobacter baylyi, encoding initial uptake and activation steps for dicarboxylates, were heterologously expressed. Genomic integration of these dca genes proved to be a key factor in efficient and reliable expression. In spite of this, adaptive laboratory evolution was needed to connect these initial steps to the native metabolism of P. putida, thereby enabling growth on adipate as sole carbon source. Genome sequencing of evolved strains revealed a central role of a paa gene cluster, which encodes parts of the phenylacetate metabolic degradation pathway with parallels to adipate metabolism. Fast growth required the additional disruption of the regulator-encoding psrA, which upregulates redundant β-oxidation genes. This knowledge enabled the rational reverse engineering of a strain that can not only use adipate, but also other medium-chain-length dicarboxylates like suberate and sebacate. The reverse engineered strain grows on adipate with a rate of 0.35 ± 0.01 h, reaching a final biomass yield of 0.27 ± 0.00 g g. In a nitrogen-limited medium this strain produced polyhydroxyalkanoates from adipate up to 25% of its CDW. This proves its applicability for the upcycling of mixtures of polymers made from fossile resources into biodegradable counterparts.
塑料的生物升级循环是一种新兴的替代方法，用于处理各种聚合物废物流，这些废物流因污染严重而无法采用传统回收技术进行处理。己二酸和其他中链长度的二羧酸盐是许多塑料的关键成分，包括聚酰胺、聚酯和聚氨酯。本研究赋予恶臭假单胞菌KT2440对这些二羧酸盐的高效代谢能力。来自拜氏不动杆菌的dcaAKIJP基因编码二羧酸盐的初始摄取和激活步骤，该基因被异源表达。这些dca基因的基因组整合被证明是高效可靠表达的关键因素。尽管如此，仍需要通过适应性实验室进化将这些初始步骤与恶臭假单胞菌的天然代谢联系起来，从而使其能够以己二酸作为唯一碳源生长。进化菌株的基因组测序揭示了一个paa基因簇的核心作用，该基因簇编码苯乙酸代谢降解途径的部分内容，与己二酸代谢相似。快速生长需要额外破坏编码调节因子的psrA，该调节因子会上调冗余的β-氧化基因。这些知识使得能够对一种菌株进行合理的逆向工程改造，该菌株不仅可以利用己二酸，还可以利用其他中链长度的二羧酸盐，如辛二酸和癸二酸。逆向工程改造后的菌株在己二酸上的生长速率为0.35±0.01 h，最终生物量产量达到0.27±0.00 g g。在氮限制培养基中，该菌株可将己二酸转化为聚羟基脂肪酸酯，产量高达其细胞干重的25%。这证明了其在将化石资源制成的聚合物混合物升级循环为可生物降解材料方面的适用性。
