Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters.

微生物对苯二甲酸 (TPA) 分解代谢途径在少数已知能转化这种异生芳香化合物的细菌中是保守的。然而，迄今为止，很少有报道表明该途径已在异源宿主中成功表达以有效利用 TPA 作为唯一碳源。在这项工作中，我们旨在通过来自天然 TPA 利用细菌的分解代谢和转运蛋白基因的异源表达，在Acinetobacter baylyi ADP1 中设计 TPA 转化。具体来说，我们获得了 ADP1 衍生菌株，这些菌株能够在 TPA 作为唯一碳源上生长，使用染色体插入和来自Comamonas的tph分解代谢操纵子的靶向扩增sp. E6. 然后使用适应性实验室进化来改善该基质上的生长。保留了tph基因的多个拷贝的进化菌株的 TPA 消耗率为~0.2 g/L/h（或 ~1 g TPA/g 细胞/h），类似于Comamonas sp。E6 并且几乎是约氏红球菌RHA1（另一种利用 TPA 的天然菌株）的2 倍。为了评估进化的 ADP1 菌株中的 TPA 转运，我们设计了一个由转录因子 TphR 和荧光报告基因组成的 TPA 生物传感器。与全基因组测序相结合，TPA 生物传感器显示 TPA 的转运不受来自Comamonas的异源蛋白质的介导sp. E6. 相反，内源性 ADP1 粘蛋白转运蛋白 MucK 是主要促进剂超家族的成员，负责几种进化菌株中的 TPA 转运，其中发现 MucK 变体可增强 TPA 吸收。此外，IclR 型转录调节因子 DcaS 被鉴定为mucK表达的阻遏物。总的来说，这项工作展示了一种通过基因扩增、适应性实验室进化和筛选方法组合鉴定的天然蛋白质的意外功能。该研究还提供了一种用于 ADP1 的 TPA 生物传感器，并确定了用于代谢工程应用的转运蛋白变体，重点是聚酯的塑料升级。