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

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 His₆-V_HHCyDisCo 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.