Biological α-olefins can be used as both biofuels and high value-added chemical precursors to lubricants, polymers, and detergents. The prototypic CYP152 peroxygenase family member OleTJE from Jeotgalicoccus sp. ATCC 8456 catalyzes a single-step decarboxylation of free fatty acids (FFAs) to form α-olefins using H2O2 as a cofactor, thus attracting much attention since its discovery. To improve the productivity of α-olefins, significant efforts on protein engineering, electron donor engineering, and metabolic engineering of OleTJE have been made. However, little success has been achieved in obtaining α-olefin high-producer microorganisms due to multiple reasons such as the tight regulation of FFA biosynthesis, the difficulty of manipulating multi-enzyme metabolic network, and the poor catalytic performance of OleTJE. In this study, a novel enzyme cascade was developed for one-pot production of α-olefins from low-cost triacylglycerols (TAGs) and natural oils without exogenous H2O2 addition. This artificial biocatalytic route consists of a lipase (CRL, AOL or Lip2) for TAG hydrolysis to produce glycerol and free fatty acids (FFAs), an alditol oxidase (AldO) for H2O2 generation upon glycerol oxidation, and the P450 fatty acid decarboxylase OleTJE for FFA decarboxylation using H2O2 generated in situ. The multi-enzyme system was systematically optimized leading to the production of α-olefins with the conversion rates ranging from 37.2 to 68.5%. Furthermore, a reaction using lyophilized CRL/OleTJE/AldO enzymes at an optimized ratio (5 U/6 μM/30 μM) gave a promising α-olefin yield of 0.53 g/L from 1500 μM (~1 g/L) coconut oil. The one-pot enzyme cascade was successfully established and applied to prepare high value-added α-olefins from low-cost and renewable TAGs/natural oils. This system is independent of exogenous addition of H2O2, thus not only circumventing the detrimental effect of H2O2 on the stability and activity of involved enzymes, but also lower the overall costs on the TAG-to-olefin transformation. It is anticipated that this biotransformation system will become industrially relevant in the future upon more engineering efforts based on this proof-of-concept work.

中文翻译：

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建立一个酶级联，用于从低成本甘油三酯和油中一锅法生产 α-烯烃，无需添加外源 H2O2

生物α-烯烃既可以用作生物燃料，也可以用作润滑剂、聚合物和清洁剂的高附加值化学前体。来自 Jeotgalicoccus sp. 的原型 CYP152 过氧合酶家族成员 OleTJE。ATCC 8456 以 H2O2 为辅因子，催化游离脂肪酸 (FFA) 一步脱羧形成 α-烯烃，因此自发现以来备受关注。为了提高α-烯烃的产率，在OleTJE的蛋白质工程、电子供体工程和代谢工程方面进行了重大努力。然而，由于FFA生物合成调控严格、多酶代谢网络难以操控、OleTJE催化性能差等多重原因，在获得α-烯烃高产微生物方面收效甚微。在这项研究中，开发了一种新的酶级联反应，用于从低成本三酰基甘油 (TAG) 和天然油中一锅法生产 α-烯烃，而无需添加外源 H2O2。这种人工生物催化途径包括用于 TAG 水解以产生甘油和游离脂肪酸 (FFA) 的脂肪酶 (CRL、AOL 或 Lip2)、用于在甘油氧化时产生 H2O2 的糖醇氧化酶 (AldO) 和用于生产的 P450 脂肪酸脱羧酶 OleTJE使用原位产生的 H2O2 进行 FFA 脱羧。系统优化了多酶系统，从而生产出转化率在 37.2% 至 68.5% 之间的 α-烯烃。此外，以优化比例 (5 U/6 μM/30 μM) 使用冻干 CRL/OleTJE/AldO 酶的反应从 1500 μM (~1 g/L) 椰子油中得到 0.53 g/L 的有希望的 α-烯烃产量. 一锅酶级联反应成功建立并应用于从低成本和可再生的TAGs/天然油中制备高附加值的α-烯烃。该系统不依赖外源添加 H2O2，因此不仅避免了 H2O2 对相关酶的稳定性和活性的不利影响，而且降低了 TAG 到烯烃转化的总体成本。预计这种生物转化系统将在未来在基于此概念验证工作的更多工程努力下变得具有工业相关性。同时也降低了 TAG 到烯烃转化的总体成本。预计这种生物转化系统将在未来在基于此概念验证工作的更多工程努力下变得具有工业相关性。同时也降低了 TAG 到烯烃转化的总体成本。预计这种生物转化系统将在未来在基于此概念验证工作的更多工程努力下变得具有工业相关性。