Maximizing Photosynthesis-Driven Baeyer–Villiger Oxidation Efficiency in Recombinant Synechocystis sp. PCC6803

A. Tüllinghoff, Magdalena B. Uhl, Friederike E H Nintzel, A. Schmid, B. Bühler, J. Toepel
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引用次数: 11

Abstract

Photosynthesis-driven whole-cell biocatalysis has great potential to contribute to a sustainable bio-economy since phototrophic cells use light as the only energy source. It has yet to be shown that phototrophic microorganisms, such as cyanobacteria, can combine the supply of high heterologous enzyme levels with allocation of sufficient reduction equivalents to enable efficient light-driven redox biocatalysis. Here, we demonstrated that the heterologous expression of an NADPH-dependent Baeyer–Villiger monooxygenase (BVMO) gene from Acidovorax sp. CHX100 turns Synechocystis sp. PCC6803 into an efficient oxyfunctionalization biocatalyst, deriving electrons and O2 from photosynthetic water oxidation. Several expression systems were systematically tested, and a PnrsB-(Ni2+)–controlled expression based on a replicative plasmid yielded the highest intracellular enzyme concentration and activities of up to 60.9 ± 1.0 U gCDW −1. Detailed analysis of reaction parameters, side reactions, and biocatalyst durability revealed—on the one hand—a high in vivo BVMO activity in the range of 6 ± 2 U mgBVMO −1 and—on the other hand—an impairment of biocatalyst performance by product toxicity and by-product inhibition. Scale-up of the reaction to 2-L fed-batch photo-bioreactors resulted in the stabilization of the bioconversion over several hours with a maximal specific activity of 30.0 ± 0.3 U g CDW −1, a maximal volumetric productivity of 0.21 ± 0.1 gL−1 h−1, and the formation of 1.3 ± 0.1 gL−1 of ε-caprolactone. Process simulations based on determined kinetic data revealed that photosynthesis-driven cyclohexanone oxidation on a 2-L scale under high-light conditions was kinetically controlled and not subject to a limitation by photosynthesis.
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重组胞囊藻PCC6803光合作用驱动Baeyer-Villiger氧化效率的最大化
光合作用驱动的全细胞生物催化具有巨大的潜力,有助于可持续的生物经济,因为光营养细胞使用光作为唯一的能量来源。目前还没有研究表明,光养微生物,如蓝藻,可以将高异源酶水平的供应与足够的还原等效物的分配相结合,以实现高效的光驱动氧化还原生物催化。在这里,我们证明了来自Acidovorax sp. CHX100的nadph依赖性Baeyer-Villiger单加氧酶(BVMO)基因的异源表达将Synechocystis sp. PCC6803转化为有效的氧官能化生物催化剂,从光合作用的水氧化中获得电子和O2。系统测试了几种表达系统,基于复制质粒的PnrsB-(Ni2+)控制表达产生了最高的细胞内酶浓度和活性,高达60.9±1.0 U gCDW−1。对反应参数、副反应和生物催化剂耐久性的详细分析表明,一方面,BVMO在体内的活性在6±2 μ mgBVMO−1范围内,另一方面,由于产物毒性和副产物抑制作用,生物催化剂的性能受到损害。将反应扩大到2 l进料间歇式光生物反应器后,生物转化在数小时内稳定下来,最大比活性为30.0±0.3 U g CDW−1,最大体积生产力为0.21±0.1 gL−1 h−1,ε-己内酯的生成为1.3±0.1 gL−1。基于确定的动力学数据的过程模拟表明,在强光条件下,光合作用驱动的2-L环己酮氧化是动力学控制的,不受光合作用的限制。
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