Light-Dependent and Aeration-Independent Gram-Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803
Light-Dependent and Aeration-Independent Gram-Scale Hydroxylation of Cyclohexane to Cyclohexanol by CYP450 Harboring Synechocystis sp. PCC 6803
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DOI：
10.1002/biot.201800724
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发表时间：
2019-08-01
影响因子：
4.7
通讯作者：
Schmid, Andreas
中科院分区：
文献类型：
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作者：
Hoschek, Anna;Toepel, Joerg;Schmid, Andreas
含加氧酶的蓝藻是一种很有前途的全细胞生物催化剂。光合水氧化，从而提供所需的共底物，即活化还原当量和O-2，可持续。开发了显示出前所未有的高光合作用驱动的氧官能化活性的重组集胞藻PCC 6803菌株，并评估了其技术适用性。细胞功能性合成异源细胞色素P450单加氧酶，使环己烷羟基化。发现生物催化剂特异性反应速率是光依赖性的，在150 μ mol（光子）m（-2）s（-1）的光强度下达到26.3 +/- 0.6 U g（CDW）（-1）（U = μ mol min（-1）和细胞干重[CDW]）。通过双液相系统的原位底物供应将初始比活性增加到39.2 +/- 0.7 U g（CDW）（-1），并通过防止细胞变性来稳定生物转化。与单一水相系统相比，这导致比产物产率增加十倍，为4.5g（环己醇）g（CDW）（-1）。随后，将生物转化从摇瓶扩大到3L搅拌罐光生物反应器装置。原位O-2生成通过光合水氧化允许一个nonaerated过程操作，从而规避基板蒸发作为最关键的因素限制的过程的性能和稳定性。这项研究首次证实了蓝藻的技术适用性曝气独立的光驱动的氧化功能化反应，涉及高毒性和挥发性底物。
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 O-2, 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 g(CDW)(-1) (U = mu mol min(-1) and cell dry weight [CDW]) at a light intensity of 150 mu mol(photons) 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 g(CDW)(-1) and stabilizes the biotransformation by preventing cell toxification. This results in a tenfold increased specific product yield of 4.5 g(cyclohexanol) g(CDW)(-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 O-2 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.