Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm.
Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm.
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DOI：
10.1186/s12934-016-0579-3
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发表时间：
2016-10-21
影响因子：
6.4
通讯作者：
Goss RJ
中科院分区：
文献类型：
--
作者：
Tong X;Barberi TT;Botting CH;Sharma SV;Simmons MJ;Overton TW;Goss RJ
用于酶促生成精细化学品的单物种生物膜工程很有吸引力。我们最近展示了工程化大肠杆菌生物膜作为合成 5-卤色氨酸的平台的实用性。使用表达重组色氨酸合酶的大肠杆菌 PHL644 来生成生物膜。它的快速沉积和生物膜形成的促进是通过采用旋转下降方法来实现的。生物膜具有较大的三维表面积，非常适合生物催化。工程生物膜的催化寿命是惊人的，我们推测这可能很大程度上是由于生物膜的细胞外基质赋予重组酶的保护。 SILAC（细胞培养物中的稳定同位素标记氨基酸），特别是动态 SILAC，其中不同同位素标记氨基酸的脉冲在一段时间内给予细胞，已被用来追踪蛋白质的命运。为了在我们的旋涂生物膜中探索重组酶的长寿是否部分归因于其再生，我们将同位素标记的赖氨酸和苯丙氨酸脉冲引入到覆盖生物膜的培养基中，并在生物膜形成过程中跟踪它们的掺入。通过 SILAC 分析，我们揭示了旋涂生物膜内发生重组酶的持续且完全的再生。生物膜内惊人的催化寿命不仅仅源于生物膜及其相关细胞外基质对活性酶的简单保护。重组酶的补充可能对工程生物膜系统的催化寿命有显着贡献。在这里，我们提供了重组酶在工程生物膜中再生的第一个证据。动态 SILAC 证明了重组酶随着时间的推移不断补充，这表明工程大肠杆菌生物膜具有高度代谢活性，具有不可忽视的能量需求。重组酶的不断更新凸显了利用该生物膜系统作为动态平台的诱人可能性，可以以“即插即用”的方式将感兴趣的酶引入其中，并可能通过启动子切换进行控制，以生产一系列所需的精细化学品。本文的在线版本 (doi:10.1186/s12934-016-0579-3) 包含补充材料，可供授权用户使用。
Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm’s extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme’s longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. Here we provide the first evidence of a recombinant enzyme’s regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a “plug-and-play” fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals. The online version of this article (doi:10.1186/s12934-016-0579-3) contains supplementary material, which is available to authorized users.
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影响因子：
14.9
作者：
Benson, Dennis A.;Karsch-Mizrachi, Ilene;Sayers, Eric W.
通讯作者：
Sayers, Eric W.
影响因子：
4.1
作者：
Eijsink, VGH;Bjork, A;Vriend, G
通讯作者：
Vriend, G
影响因子：
3.8
作者：
Gross, Rainer;Buehler, Katja;Schmid, Andreas
通讯作者：
Schmid, Andreas
影响因子：
3.8
作者：
Gross, Rainer;Hauer, Bernhard;Schmid, Andreas
通讯作者：
Schmid, Andreas
影响因子：
4.4
作者：
Halan, Babu;Schmid, Andreas;Buehler, Katja
通讯作者：
Buehler, Katja