Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA.
Department of Microbiology and Immunology, BioProducts Institute, and the Life Sciences Institute, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Metab Eng. 2021 May;65:111-122. doi: 10.1016/j.ymben.2021.02.005. Epub 2021 Mar 16.
Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (k/K) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.
木质素是植物细胞壁的丰富成分，其增值利用对于建立木质纤维素生物经济至关重要。利用微生物生物催化剂进行生物趋同已成为一种很有吸引力的方法，可以将木质素解聚产物的复杂混合物转化为有价值的化合物。理想情况下，生物催化剂应该将源自三种典型木质素的芳香族化合物转化为：丁香基（S）、愈创木基（G）和对羟苯基（H）。恶臭假单胞菌 KT2440（以下简称 KT2440）已被开发为一种生物催化剂，部分原因是其天然的代谢能力，但目前已知它不能代谢 S 型木质素衍生化合物。在这里，我们证明了丁香酸盐，一种常见的 S 型木质素衍生化合物，只有在另一种能源存在或过表达 vanAB 时才被 KT2440 利用，因为丁香酸盐被发现被 VanAB 氧化脱甲基化为没食子酸，VanAB 是一种由两个部分组成的单加氧酶，然后通过外二醇裂解进一步代谢。出乎意料的是，VanAB 对丁香酸盐的特异性（k/K）与香草酸盐的特异性相差不到 25%，并且两种底物的 O-去甲基化与 O 消耗很好地偶联。然而，天然的 KT2440 没食子酸裂解双加氧酶 GalA 被 3-O-甲基没食子酸强烈失活。为了构建一种能够同时转化 S、G 和 H 型单体的生物催化剂，我们因此采用了来自假单胞菌 HR199 的 VanAB，它对 3MGA 的活性较低，以及 LigAB，一种能够裂解原儿茶酸和 3-O-甲基没食子酸的外二醇双加氧酶。该菌株将木质素单体混合物的 93%转化为 2-吡喃酮-4,6-二羧酸，这是一种很有前途的生物基化学品。总的来说，这项研究阐明了 KT2440 中一种用于代谢 S 型木质素衍生化合物的天然途径，并展示了这个强大底盘在木质素增值方面的潜力。
