Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm

Microbial Cell Factories

Research

Catherine H. Botting¹  Mark J. H. Simmons²  Tim W. Overton²  Sunil V. Sharma³  Rebecca J. M. Goss³  Xiaoxue Tong³  Tania Triscari Barberi³ 

[1] Biomedical Sciences Research Complex, University of St. Andrews, KY16 9ST, St. Andrews, UK;School of Chemical Engineering, University of Birmingham, Edgbaston, B152TT, Birmingham, UK;School of Chemistry, University of St. Andrews, KY16 9ST, St. Andrews, UK;Biomedical Sciences Research Complex, University of St. Andrews, KY16 9ST, St. Andrews, UK;

关键词: E. coli;    Biotransformation;    Biocatalysis;    Tryptophan synthase;    Halotryptophan;    SILAC;    Proteomic analysis;   

DOI  :  10.1186/s12934-016-0579-3

 received in 2016-05-11, accepted in 2016-10-14,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundEngineering 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.ResultsThrough 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.ConclusionsHere 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.

【 授权许可】

CC BY   
© The Author(s) 2016