Exploiting Single Domain Antibodies as Regulatory Parts to Modulate Monoterpenoid Production in E. coli

Abstract

Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at the transcriptional and translational level have been developed, relatively few exist at the protein level. Single domain antibodies (sdAb) such as camelid heavy chain variable fragments (V HH ) possess binding characteristics which could be exploited for their development and use as novel parts for regulating metabolic pathways at the protein level in microbial cell factories. Here, a platform for the use of V HH as tools in Escherichia coli is developed and subsequently used to modulate linalool production in E. coli. The coproduction of a Design of Experiments (DoE) optimized pBbE8k His 6 -V HH CyDisCo system alongside a heterologous linalool production pathway facilitated the identification of anti-bLinS V HH that functioned as modulators of bLinS. This resulted in altered product profiles and significant variation in the titers of linalool, geraniol, nerolidol, and indole obtained. The ability to alter the production levels of high value terpenoids, such as linalool, in a tunable manner at the protein level could represent a significant step forward for the development of improved microbial cell factories. This study serves as a proof of principle indicating that V HH can be used to modulate enzyme activity in engineered pathways within E. coli. Given their almost limitless binding potential, we posit that single domain antibodies could emerge as powerful regulatory parts in synthetic biology applications.

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

“…VHH72 is predominantly expressed in the insoluble fraction until all genetic modifications are combined in autoDC REdox strain, allowing for soluble nanobody expression. Having established the benefits of autoDC REdox strain for soluble VHH expression, we compared its performance with previously reported SHuffle and CyDisCo strains 15,24,60,64 . Considering that protein sequences can influence expression, it is essential to evaluate consistency in platform strains to enable broader applicability.…”

Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80

“…A naïve llama VHH phage library was kindly provided by UCB Biopharma. Enrichment of the phage against PD-L1 was performed as described previously ( 61 ), with some modifications. Panning was performed with Nunc Maxisorp ELISA plates (Invitrogen), with 1 μM biotinylated PD-L1 immobilized on the plate using neutravidin (Thermo Fisher) and streptavidin (Thermo Fisher), for the first and second rounds of panning, respectively.…”

“…Individual colonies of phage from round 2 of the panning were subjected to monoclonal rescue, as previously described ( 61 ). An ELISA assay was used to assess binding to PD-L1 D1D2 and PD-L1 D1.…”

Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery

“…Monoclonal rescue was performed on individual phage colonies resulting from R2 of biopanning, as previously described (24). Subsequently, monoclonal ELISA assays were performed to confirm binding of VHHs to IL-13.…”