Institute for Research Initiatives, Division for Research Strategy, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan.
Graduate School of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan.
Appl Microbiol Biotechnol. 2022 Dec;106(23):7867-7878. doi: 10.1007/s00253-022-12244-y. Epub 2022 Oct 27.
Poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis produces hydrolytic enzymes that convert PET, via mono(2-hydroxyethyl) terephthalate (MHET), into the monomeric compounds, terephthalic acid (TPA), and ethylene glycol (EG). Understanding PET metabolism is critical if this bacterium is to be engineered for bioremediation and biorecycling. TPA uptake and catabolism in I. sakaiensis have previously been studied, but EG metabolism remains largely unexplored despite its importance. First, we identified two alcohol dehydrogenases (IsPedE and IsPedH) and one aldehyde dehydrogenase (IsPedI) in I. sakaiensis as the homologs of EG metabolic enzymes in Pseudomonas putida KT2440. IsPedE and IsPedH exhibited EG dehydrogenase activities with Ca and a rare earth element (REE) Pr, respectively. We further found an upregulated dehydrogenase gene when the bacterium was grown on EG, whose gene product (IsXoxF) displays a minor EG dehydrogenase activity with Pr. IsPedE displayed a similar level of activity toward various alcohols. In contrast, IsPedH was more active toward small alcohols, whereas IsXoxF was the opposite. Structural analysis with homology models revealed that IsXoxF had a larger catalytic pocket than IsPedE and IsPedH, which could accommodate relatively bulkier substrates. Pr regulated the protein expression of IsPedE negatively; IsPedH and IsXoxF were positively regulated. Taken together, these results indicated that the combination of IsPedH and IsXoxF complements the function of IsPedE in the presence of REEs. IsPedI exhibited dehydrogenase activity toward various aldehydes with the highest activity toward glycolaldehyde. This study demonstrated a unique alcohol oxidation pathway of I. sakaiensis, which could be efficient in EG utilization. KEY POINTS: • IsPedH and IsXoxF complement IsPedE function in the presence of REEs. • IsPedI displayed the highest dehydrogenase activity toward glycolaldehyde. • Unique alcohol oxidation pathway of I. sakaiensis identified for EG utilization.
聚对苯二甲酸乙二醇酯（PET）降解菌 Ideonella sakaiensis 产生的水解酶可将 PET 通过单（2-羟乙基） terephthalate（MHET）转化为单体化合物对苯二甲酸（TPA）和乙二醇（EG）。如果要对该细菌进行生物修复和生物回收利用的工程改造，了解 PET 代谢至关重要。先前已经研究了 I. sakaiensis 中的 TPA 摄取和分解代谢，但 EG 代谢仍在很大程度上未被探索，尽管它很重要。首先，我们确定了 Ideonella sakaiensis 中的两种醇脱氢酶（IsPedE 和 IsPedH）和一种醛脱氢酶（IsPedI）是 Pseudomonas putida KT2440 中 EG 代谢酶的同源物。IsPedE 和 IsPedH 分别具有 Ca 和稀土元素（REE）Pr 的 EG 脱氢酶活性。当细菌在 EG 上生长时，我们进一步发现了一个上调的脱氢酶基因，其基因产物（IsXoxF）显示出与 Pr 一起具有较小的 EG 脱氢酶活性。与各种醇相比，IsPedE 显示出相似的活性。相反，IsPedH 对较小的醇更活跃，而 IsXoxF 则相反。同源建模的结构分析表明，与 IsPedE 和 IsPedH 相比，IsXoxF 具有更大的催化口袋，可容纳相对较大的底物。Pr 负调控 IsPedE 的蛋白表达；IsPedH 和 IsXoxF 呈正调控。总的来说，这些结果表明，REE 存在时，IsPedH 和 IsXoxF 的组合可补充 IsPedE 的功能。IsPedI 对各种醛显示出脱氢酶活性，对乙二醇醛的活性最高。这项研究表明了 Ideonella sakaiensis 的独特醇氧化途径，该途径在 EG 利用中可能很有效。关键点：•REE 存在时，IsPedH 和 IsXoxF 补充 IsPedE 的功能。•IsPedI 对乙二醇醛显示出最高的脱氢酶活性。•鉴定了 Ideonella sakaiensis 用于 EG 利用的独特醇氧化途径。
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