Microbial production of aromatic chemicals is an attractive method for obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid, 4-amino-3-hydroxybenzoic acid (4,3-AHBA), is expected to be a precursor of highly functional polybenzoxazole polymers; however, methods for its microbial production have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) into the metabolic pathway of an industrially relevant bacterium, Corynebacterium glutamicum. Six different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains, which were then screened for the production of 4,3-AHBA by culturing with glucose as a carbon source. The highest concentration of 4,3-AHBA was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). A combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBHM106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) 4,3-AHBA and 0.059 g/L (0.43 mM) precursor 4-ABA in fed-batch culture using a nutrient-rich medium. The culture of this strain in the chemically defined CGXII medium yielded 9.8 C-mol% of 4,3-AHBA from glucose, corresponding to 12.8% of the theoretical maximum yield (76.8 C-mol%) calculated using a genome-scale metabolic model of C. glutamicum. Identification of PHBH mutants that could efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to a better understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials.

中文翻译：

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4-羟基苯甲酸3-羟化酶突变体能够在谷氨酸棒杆菌中从葡萄糖生产4-氨基-3-羟基苯甲酸

芳香族化学品的微生物生产是从生物质资源中获取高性能材料的一种有吸引力的方法。非蛋白氨基酸 4-氨基-3-羟基苯甲酸 (4,3-AHBA) 有望成为高功能聚苯并恶唑聚合物的前体；然而，其微生物生产方法尚未见报道。在这项研究中，我们尝试通过将 4-氨基苯甲酸 (4-ABA) 的 3-羟基化引入工业相关细菌谷氨酸棒杆菌的代谢途径中，从葡萄糖生产 4,3-AHBA。六种不同的 4-羟基苯甲酸 3-羟化酶 (PHBH) 在谷氨酸棒杆菌菌株中异源表达，然后通过以葡萄糖作为碳源培养来筛选生产 4,3-AHBA 的菌株。在弧形柄杆菌 (CvPHBH) 表达 PHBH 的菌株中检测到最高浓度的 4,3-AHBA。活性位点定点诱变和漆酶介导的比色测定随机诱变相结合，使我们能够获得在深孔板培养条件下增强 4,3-AHBA 生产力的 CvPHBH 突变体。表达 CvPHBHM106A/T294S 并具有增强的 4-ABA 生物合成途径的重组谷氨酸棒杆菌菌株在补料分批中产生 13.5 g/L (88 mM) 4,3-AHBA 和 0.059 g/L (0.43 mM) 前体 4-ABA使用营养丰富的培养基进行培养。该菌株在化学成分确定的 CGXII 培养基中培养，从葡萄糖中产生了 9.8 C-mol% 的 4,3-AHBA，相当于使用基因组规模代谢模型计算的理论最大产量 (76.8 C-mol%) 的 12.8%谷氨酸棒杆菌。能够有效催化谷氨酸棒杆菌中 4-ABA 3-羟基化的 PHBH 突变体的鉴定使我们能够构建能够使用葡萄糖作为碳源以克级生产 4,3-AHBA 的人工生物合成途径。这些发现将有助于更好地理解酶催化芳香族化合物的区域选择性羟基化，以及高性能材料的生物质衍生前体的多样化。