Wada Ayumu, Prates Érica T, Hirano Ryo, Werner Allison Z, Kamimura Naofumi, Jacobson Daniel A, Beckham Gregg T, Masai Eiji
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Metab Eng. 2021 Mar;64:167-179. doi: 10.1016/j.ymben.2021.01.013. Epub 2021 Feb 4.
Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.
恶臭假单胞菌KT2440（以下简称KT2440）是一种经过充分研究的平台细菌，可用于从木质素解聚得到的芳香族化合物的异质混合物中生产具有工业价值的化学品。KT2440能够在与木质素相关的单体上生长，如阿魏酸（FA）、对香豆酸（4CA）、香草酸（VA）、4-羟基苯甲酸（4HBA）和原儿茶酸（PCA）。与其分解代谢相关的基因是已知的，但关于摄取系统的知识仍然有限。在这项工作中，我们研究了KT2440中与木质素相关单体的转运蛋白及其底物选择性。基于质子载体的抑制作用，我们重点关注了五个编码芳香酸/H同向转运体家族转运蛋白的基因，这些转运蛋白属于利用质子动力的主要易化子超家族。PP_1376（pcaK）和PP_3349（hcnK）的突变体分别在PCA/4HBA和FA/4CA上的生长显著降低，而五个基因突变体在VA上均未观察到变化。在pH 9.0时，hcnK突变体（FA/4CA）和vanK突变体（VA）对这些化合物的转化显著降低，这表明这些转运蛋白对于在高pH下摄取阴离子底物至关重要。在大肠杆菌中使用C标记底物的摄取试验和基于生物传感器的试验证实，PcaK、HcnK和VanK分别具有摄取PCA、FA/4CA和VA/PCA的能力。此外，对预测蛋白质结构的分析表明，这些转运蛋白底物结合位点的大小和亲水性质决定了它们的底物偏好。总体而言，这项研究表明，在生理pH下，PcaK和HcnK分别在摄取PCA/4HBA和FA/4CA中起主要作用，而VanK是一种VA/PCA转运蛋白。这些信息有助于设计菌株，将与木质素相关的单体高效转化为增值化学品。
