Multienzyme Whole-Cell In Situ Biocatalysis for the Production of Flaviolin in Permeabilized Cells of Escherichia coli
Multienzyme Whole-Cell In Situ Biocatalysis for the Production of Flaviolin in Permeabilized Cells of Escherichia coli
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DOI：
10.1002/cctc.201100351
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发表时间：
2012-06-01
期刊：
影响因子：
4.5
通讯作者：
Heinzle, Elmar
中科院分区：
文献类型：
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作者：
Krauser, Steffen;Kiefer, Patrick;Heinzle, Elmar
多酶生物催化越来越多地被认为是合成更复杂的化合物，如次级代谢产物。在我们的策略中，我们使用定制的透化全细胞。细胞透化已经被用于原位酶活性测量，因为半个世纪前，证明甚至在真核生物中的区室特异性数据。[1-6]应用去污剂使细胞对低分子量化合物具有渗透性，同时将酶保留在细胞内。透化后细胞的显微结构基本保持不变。[1，3]将细胞中最初含有的所有低分子量化合物在培养基中稀释并通过洗涤除去。这允许改变为仍然包含在细胞中的所有酶的限定的替代环境。在活生物体中不可用的替代前体分子可能是适用的，甚至可能在对活细胞有毒的条件下。大肠杆菌由于其快速和稳健的生长、可用的基因组和遗传工具以及关于大量酶的详细生物化学信息（包括代谢网络活动的调节）而特别令人感兴趣。这允许在某些条件下预测和改变途径活性。然后可以以明确定义的方式添加辅因子和底物，通过设计限制透化细胞内可能的反应，因此可以指导转化选择性。在这里，我们提出了我们的结果，从多酶原位生物催化剂的生产与透化细胞的次级代谢产物黄拉醇的发展。大肠杆菌，其如实验部分中所述进行遗传修饰。此外，添加三氯生可抑制烯酰还原酶，从而抑制脂肪酸的生物合成。[7]黄醌（Flaviolin，2，5，7-trihydroxynaphthoquinone，4）是灰色链霉菌（Streptomycesgriseus）的一种红棕色聚酮类次级代谢产物，可在活的E.杆菌[8-10]中间体四羟基萘（THN，3）是实际的酶促产物。最终产物黄拉醇然后通过用分子氧氧化产生。选择Triton X-100作为透化剂，因为它可以在低浓度下用于透化过程。[1]通过丙酮酸脱氢酶活性测定，确定了最适浓度为0.25%（v/v）。在不同的细胞和表面活性剂浓度的透化的比较导致的结论是，所需的表面活性剂的量是成比例的总暴露的细胞膜面积。[1，3]显微镜观察显示细胞基本完整，如先前报道的。[3]透性化细胞的定制E。通过使用基质辅助激光解吸/电离飞行时间质谱（MALDITOF MS）和分光光度分析检测，大肠杆菌形成显著量的黄嘌呤，而不含腺苷5 '-三磷酸（ATP）的对照为阴性。透化细胞的生产率在第一阶段确定为0.025 μmol min-1 gwet细胞/ml，在第二阶段确定为0.015 μmol min-1 gwet细胞/ml（图1）。这种转变的原因至今尚未查明。除去催化剂细胞后，在308 ℃下孵育1天，用光度法检测最终浓度为0.93 mM的黄素/黄素二聚体。这相当于E浓度的一半。coliBL 21（DE 3）培养3d后达高峰。基于提供的ATP的化学计量产率为30%。ATP的损失可能是由导致ATP水解的“无效”循环引起的。乙酰辅酶A合成酶（acs）的过度表达和竞争途径的缺失导致了一个显着的...
Multienzyme biocatalysis is increasingly considered for the synthesis of more complex compounds such as secondary metabolites. In our strategy we use tailored whole cells that are permeabilized. Cell permeabilization has been used for insitu enzyme activity measurements since half a century ago, proving even compartment-specific data in eukaryotes.[1–6] Detergents applied makes cells permeable for low molecular mass compounds while retaining enzymes inside cells. The microscopic structure of the cells is largely maintained after permeabilization.[1, 3] All low molecular mass compounds originally contained in the cells are diluted in the medium and removed by washing. This allows the change to a defined alternative environment for all enzymes still contained in the cells. Alternative precursor molecules not available in living organisms might be applicable, possibly even at conditions that would be toxic for living cells. Escherichia coli is of special interest owing to its fast and robust growth, the available genome and genetic tools, and detailed biochemical information about a large number of enzymes, including regulation of metabolic network activities. This allows for the prediction and alteration of pathway activity under certain conditions. Cofactors and substrates can then be added in a well-defined way, limiting possible reactions inside the permeabilized cells by design and, thus, the conversion selectivity can be directed. Here we present our results from the development of a multienzyme in situ biocatalyst for the production of the secondary metabolite flaviolin with the permeabilized cells of E. coli, which were genetically modified as described in the Experimental Section. In addition, fatty acid biosynthesis was inhibited by the addition of triclosan that inhibits enoyl reductase.[7] Flaviolin (2, 5, 7-trihydroxynaphthoquinone, 4) is a red-brown polyketide-based secondary metabolite of Streptomyces griseus that can be produced heterologously in living E. coli.[8–10] The intermediate tetrahydroxynaphthalene (THN, 3) is the actual enzymatic product. The final product flaviolin is then produced by oxidation with molecular oxygen. Triton X-100 was chosen as the permeabilizing agent because it can be used in low concentration in the permeabilization process.[1] An optimal concentration of 0.25%(v/v) could be determined by pyruvate dehydrogenase activity. Comparison of permeabilization at various cell and surfactant concentrations led to the conclusion that the requested amount of the surfactant is proportional to the total exposed cell membrane area.[1, 3] Microscopic observations showed largely intact cells as was also previously reported.[3] Permeabilized cells of tailored E. coli formed significant amounts of flaviolin, detected by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) and spectrophotometric analysis, while a control without adenosine 5’-triphosphate (ATP) was negative. The productivity of permeabilized cells was determined as 0.025 μmol minÀ1 gwet cells À1 in the first phase and 0.015 μmol minÀ1 gwet cells À1 in the second phase (Figure 1). The reasons for this shift have not been identified so far. After removing the catalyst cells, a final concentration of 0.93 mM flaviolin/flaviolin dimer was detected photometrically for 1day incubation at 308C. This equals half the concentration of an E. coli BL21 (DE3) culture reached in 3 days. The stoichiometric yield based on the supplied ATP was 30%. The loss of ATP may be caused by the “futile” cycles that result in the hydrolysis of ATP. The overexpression of acetyl-CoA synthase (acs) and the deletion of competing pathways led to a significant …