Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase
Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase
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DOI：
10.1111/j.1742-4658.2008.06648.x
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发表时间：
2008-10-01
期刊：
影响因子：
5.4
通讯作者：
Schmid, Andreas
中科院分区：
文献类型：
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作者：
Blank, Lars M.;Ionidis, Georgios;Schmid, Andreas
全细胞氧化还原生物催化用于生产有价值的特定功能化产品的一个关键限制是底物/产品毒性，这一点可以通过使用耐溶剂微生物来克服。为了研究耐溶剂性和能量依赖性生物催化的相互关系，我们建立了一个在有毒的低logP(Ow)溶剂存在下的生物催化模型系统：耐溶剂重组恶臭假单胞菌DOT-T1E在水/辛醇两液相反应介质中催化苯乙烯的立体选择性环氧化反应。利用基于~(13)C示踪剂的代谢通量分析，我们研究了有毒溶剂存在和氧化还原生物催化作用下恶臭假单胞菌的中心碳和能量代谢，并量化了NAD(P)H的再生率，这都大大增加了耐溶剂恶臭假单胞菌的能量需求。根据需求驱动的概念，NAD(P)H的再生率通过两种机制提高到8倍：(A)增加葡萄糖摄取率而不分泌代谢副产物，(B)减少生物量的形成。然而，在辛醇存在下，苯乙烯环氧化反应的最大再生速率仅相当于NAD(P)H再生速率的1%，这一速率比使用无毒溶剂时的速率低三倍以上。这表明细胞维护需要高能量和氧化还原辅因子，这限制了辛醇存在下的氧化还原生物催化。可用于生物催化的NAD(P)H再生率的估计上限表明，在优化的条件下，辅因子的可用性不会限制氧化还原生物催化，例如在没有有毒溶剂的情况下，这说明了耐溶剂恶臭假单胞菌的高代谢能力。这项研究表明，耐溶剂性恶臭假单胞菌通过将其能量代谢提高到比无限生长时观察到的水平高一个数量级，具有显著的补偿高能量需求的能力。
A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only similar to 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.