CYP450窝藏集藻蓝藻的光依赖性和不依赖曝气的革兰氏级环己烷羟化反应生成环己醇。PCC 6803。,Biotechnology Journal

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CYP450窝藏集藻蓝藻的光依赖性和不依赖曝气的革兰氏级环己烷羟化反应生成环己醇。PCC 6803。
Biotechnology Journal ( IF 3.2 ) Pub Date : 2019-06-18 , DOI: 10.1002/biot.201800724
Anna Hoschek ₁ , Jörg Toepel ₁ , Adrian Hochkeppel ₁ , Rohan Karande ₁ , Bruno Bühler ₁ , Andreas Schmid ₁

Affiliation

含加氧酶的蓝细菌构成用于氧官能化反应的有前途的全细胞生物催化剂。光合水氧化从而可持续地输送所需的共底物，即被活化的还原当量和O2。重组集胞藻研制出显示出前所未有的高光合作用驱动的氧官能化活性的PCC 6803菌株，并对其技术适用性进行了评估。细胞在功能上合成异源细胞色素P450单加氧酶，可实现环己烷羟基化。发现生物催化剂特异性反应速率是光依赖性的，在150 µmol光子m-2 s-1的光强度下达到26.3±0.6 U gCDW -1（U =μmolmin-1和细胞干重[CDW]）。。通过两液相系统进行原位底物供应可将初始比活度提高到39。2±0.7 U gCDW -1并通过防止细胞毒性来稳定生物转化。与单水相系统相比，这导致4.5 g环己醇gCDW -1的比产品产率提高了十倍。随后，将生物转化物从摇瓶中缩放到3 L搅拌罐光生物反应器中。通过光合水氧化产生的原位O2允许进行非充气工艺操作，因此规避基材蒸发是限制工艺性能和稳定性的最关键因素。这项研究首次证明了蓝细菌在涉及高毒性和挥发性底物的不依赖曝气的光驱动氧官能化反应中的技术适用性。与单水相系统相比，这导致4.5 g环己醇gCDW -1的比产品产率提高了十倍。随后，将生物转化物从摇瓶中缩放到3 L搅拌罐光生物反应器中。通过光合水氧化产生的原位O2允许进行非充气工艺操作，因此规避基材蒸发是限制工艺性能和稳定性的最关键因素。这项研究首次证明了蓝细菌在涉及高毒性和挥发性底物的不依赖曝气的光驱动氧官能化反应中的技术适用性。与单水相系统相比，这导致4.5 g环己醇gCDW -1的比产品产率提高了十倍。随后，将生物转化物从摇瓶中缩放到3 L搅拌罐光生物反应器中。通过光合水氧化产生的原位O2允许进行非充气工艺操作，因此规避基材蒸发是限制工艺性能和稳定性的最关键因素。这项研究首次证明了蓝细菌在涉及高毒性和挥发性底物的不依赖曝气的光驱动氧官能化反应中的技术适用性。生物转化的规模从摇瓶扩展到3 L搅拌罐光生物反应器。通过光合水氧化产生的原位O2允许进行非充气工艺操作，因此规避基材蒸发是限制工艺性能和稳定性的最关键因素。这项研究首次证明了蓝细菌在涉及高毒性和挥发性底物的不依赖曝气的光驱动氧官能化反应中的技术适用性。生物转化的规模从摇瓶扩展到3 L搅拌罐光生物反应器。通过光合水氧化产生的原位O2允许进行非充气工艺操作，因此规避基材蒸发是限制工艺性能和稳定性的最关键因素。这项研究首次证明了蓝细菌在涉及高毒性和挥发性底物的不依赖曝气的光驱动氧官能化反应中的技术适用性。

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Light-Dependent and Aeration-Independent Gram-Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803.

Oxygenase-containing cyanobacteria constitute promising whole-cell biocatalysts for oxyfunctionalization reactions. Photosynthetic water oxidation thereby delivers the required cosubstrates, that is activated reduction equivalents and O2 , sustainably. A recombinant Synechocystis sp. PCC 6803 strain showing unprecedentedly high photosynthesis-driven oxyfunctionalization activities is developed, and its technical applicability is evaluated. The cells functionally synthesize a heterologous cytochrome P450 monooxygenase enabling cyclohexane hydroxylation. The biocatalyst-specific reaction rate is found to be light-dependent, reaching 26.3 ± 0.6 U gCDW -1 (U = μmol min-1 and cell dry weight [CDW]) at a light intensity of 150 µmolphotons m-2 s-1 . In situ substrate supply via a two-liquid phase system increases the initial specific activity to 39.2 ± 0.7 U gCDW -1 and stabilizes the biotransformation by preventing cell toxification. This results in a tenfold increased specific product yield of 4.5 gcyclohexanol gCDW -1 as compared to the single aqueous phase system. Subsequently, the biotransformation is scaled from a shake flask to a 3 L stirred-tank photobioreactor setup. In situ O2 generation via photosynthetic water oxidation allows a nonaerated process operation, thus circumventing substrate evaporation as the most critical factor limiting the process performance and stability. This study for the first time exemplifies the technical applicability of cyanobacteria for aeration-independent light-driven oxyfunctionalization reactions involving highly toxic and volatile substrates.

更新日期：2019-06-18

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