Tong Xiaoxue, Barberi Tania Triscari, Botting Catherine H, Sharma Sunil V, Simmons Mark J H, Overton Tim W, Goss Rebecca J M
School of Chemistry, University of St. Andrews, St. Andrews, KY16 9ST, UK.
Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, KY16 9ST, UK.
Microb Cell Fact. 2016 Oct 21;15(1):180. doi: 10.1186/s12934-016-0579-3.
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.
构建用于酶促生成精细化学品的单物种生物膜具有吸引力。我们最近证明了工程化大肠杆菌生物膜作为合成5-卤代色氨酸平台的实用性。使用表达重组色氨酸合酶的大肠杆菌PHL644来生成生物膜。通过采用离心沉降法促进其快速沉积并引发生物膜形成。该生物膜具有很大的三维表面积，非常适合生物催化。工程化生物膜的催化寿命令人瞩目，我们推测这很可能主要是由于生物膜的细胞外基质对重组酶的保护作用。稳定同位素标记氨基酸在细胞培养中的应用（SILAC），特别是动态SILAC，即在一段时间内将不同同位素标记的氨基酸脉冲施加到细胞上，已被用于追踪蛋白质的命运。为了探究在我们的旋涂生物膜中，重组酶的寿命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覆盖生物膜的培养基中，并追踪它们在生物膜发育过程中的掺入情况。
通过SILAC分析，我们发现旋涂生物膜内发生了重组酶的持续且完全再生。生物膜内显著的催化寿命不仅仅是生物膜及其相关细胞外基质对活性酶的简单保护作用。重组酶的补充可能对工程化生物膜系统中观察到的催化寿命有显著贡献。
在此我们提供了工程化生物膜中重组酶再生的首个证据。动态SILAC证明重组酶随时间不断补充，这表明工程化大肠杆菌生物膜具有高代谢活性，存在相当可观的能量需求。重组酶的持续更新突出了利用该生物膜系统作为动态平台的诱人可能性，在该平台中，感兴趣的酶可以以“即插即用”的方式引入，并有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。
