Benzoic acid is a building block of a multitude of well-known plant natural products, such as paclitaxel and cocaine. Its simple chemical structure contrasts with its complex biosynthesis. Hypericum species are rich in polyprenylated benzoic acid-derived xanthones, which have received attention due to their biological impact on human health. The upstream biosynthetic sequence leading to xanthones is still incomplete. To supply benzoic acid for xanthone biosynthesis, Hypericum calycinum cell cultures use the CoA-dependent non-β-oxidative pathway, which starts with peroxisomal cinnamate CoA-ligase (HcCNL). Here, we use the xanthone-producing cell cultures to identify the transcript for benzaldehyde dehydrogenase (HcBD), a pivotal player in the non-β-oxidative pathways. In addition to benzaldehyde, the enzyme efficiently catalyzes the oxidation of trans-cinnamaldehyde in vitro. The enzymatic activity is strictly dependent on the presence of NAD⁺ as co-factor. HcBD is localized to the cytosol upon ectopic expression of reporter fusion constructs. HcBD oxidizes benzaldehyde, which moves across the peroxisome membrane, to form benzoic acid. Increases in the HcCNL and HcBD transcript levels precede the elicitor-induced xanthone accumulation. The current work addresses a crucial step in the yet incompletely understood CoA-dependent non-β-oxidative route of benzoic acid biosynthesis. Addressing this step may offer a new biotechnological tool to enhance product formation in biofactories.

苯甲酸是许多著名的植物天然产物的构建基，例如紫杉醇和可卡因。其简单的化学结构与其复杂的生物合成形成鲜明对比。金丝桃属物种富含聚戊二酰化苯甲酸衍生的氧杂蒽，由于它们对人类健康的生物学影响，受到人们的关注。导致氧杂蒽酮的上游生物合成序列仍然不完整。为了提供苯甲酸用于黄酮的生物合成，贯叶连翘细胞培养物使用了依赖于CoA的非β-氧化途径，该途径始于过氧化物酶体肉桂酸CoA-连接酶（Hc CNL）。在这里，我们使用产生黄酮的细胞培养物来鉴定苯甲醛脱氢酶（HcBD），是非β-氧化途径的关键参与者。除苯甲醛外，该酶在体外还有效催化反式肉桂醛的氧化。酶活性严格取决于NAD ⁺作为辅助因子的存在。当报告融合构建体异位表达时，Hc BD定位于胞质溶胶。HC BD氧化苯甲醛，苯甲醛穿过过氧化物酶体膜移动，形成苯甲酸。Hc CNL和Hc BD转录物水平的增加在激发子诱导的蒽酮积累之前。当前的工作是在尚未完全理解CoA依赖性非β的关键步骤-苯甲酸生物合成的氧化途径。解决此步骤可能会提供一种新的生物技术工具，以增强生物工厂中产品的形成。