ACYL‐LIPID THIOESTERASES (ALT) are a type of plant acyl–acyl carrier protein thioesterase that generate a wide range of medium‐chain fatty acids and methylketone (MK) precursors when expressed heterologously in Escherichia coli. While this makes ALT‐type thioesterases attractive as metabolic engineering targets to increase production of high‐value medium‐chain fatty acids and MKs in plant systems, the behavior of ALT enzymes in planta was not well understood before this study. To profile the substrate specificities of ALT‐type thioesterases in different plant tissue types, AtALT1‐4 from Arabidopsis thaliana, which have widely varied chain length and oxidation state preferences in E. coli, were overexpressed in Arabidopsis seeds, Camelina sativa seeds, and Nicotiana benthamiana leaves. Seed‐specific overexpression of ALT enzymes led to medium‐chain fatty acid accumulation in Arabidopsis and Camelina seed triacylglycerols, and transient overexpression in N. benthamiana demonstrated that the substrate preferences of ALT‐type thioesterases in planta generally agree with those previously determined in E. coli. AtALT1 and AtALT4 overexpression in leaves and seeds resulted in the accumulation of 12–14 carbon‐length fatty acids and 6–8 carbon‐length fatty acids, respectively. While it was difficult to completely profile the products of ALT‐type thioesterases that generate MK precursors (i.e. β‐keto fatty acids), our results nonetheless demonstrate that ALT enzymes are catalytically diverse in planta. The knowledge gained from this study is a significant step towards being able to use ALT‐type thioesterases as metabolic engineering tools to modify the fatty acid profiles of oilseed crops, other plants, and microorganisms.

中文翻译：

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通过单热狗折叠酰基脂质硫酯酶的过度表达在种子和叶子中产生 C6-C14 中链脂肪酸

酰基脂质硫酯酶 (ALT) 是一种植物酰基-酰基载体蛋白硫酯酶，当在大肠杆菌中异源表达时，可产生多种中链脂肪酸和甲基酮 (MK) 前体。虽然这使得 ALT 型硫酯酶作为代谢工程目标具有吸引力，以增加植物系统中高价值中链脂肪酸和 MK 的产量，但在本研究之前，植物中 ALT 酶的行为尚不清楚。为了分析 ALT 型硫酯酶在不同植物组织类型中的底物特异性，来自拟南芥的AtALT1-4，在大肠杆菌中具有广泛不同的链长和氧化态偏好, 在拟南芥种子、亚麻荠种子和本氏烟草叶子中过度表达。ALT 酶的种子特异性过表达导致拟南芥和亚麻荠种子三酰甘油中链脂肪酸的积累，本氏烟草中的瞬时过表达表明植物中 ALT 型硫酯酶的底物偏好与先前在大肠杆菌中确定的一致。大肠杆菌。AtALT1和AtALT4叶子和种子中的过度表达分别导致 12-14 个碳长脂肪酸和 6-8 个碳长脂肪酸的积累。虽然很难完全描述产生 MK 前体（即β-酮脂肪酸）的 ALT 型硫酯酶的产物，但我们的结果仍然表明 ALT 酶在植物中具有催化多样性。从这项研究中获得的知识是朝着能够使用 ALT 型硫酯酶作为代谢工程工具来改变油料作物、​​其他植物和微生物的脂肪酸谱的重要一步。