Towards light activated enzyme clustering for metabolic flux redirection

Abstract

While there are many tools for metabolic manipulation at the transcriptional level, there are few such tools at the protein level. Recent developments present an opportunity to develop a tool for protein-level control. It has been shown that clustering of subsequent enzymes can divert flux in a metabolic pathway. The present work investigates how lightactivated clustering of Cryptochrome 2 (Cry2) can redirect flux at a metabolic branch point. The violacein pathway is used as a test system because the various products of the pathway are colored and as a result, flux redirection can be visually determined. VioE and VioC are two subsequent enzymes of the violacein pathway. If the product of VioE is channeled towards VioC, more deoxyviolacein (DV), a pink product, is produced. Otherwise, the product of VioE is spontaneously converted to prodeoxyviolacein (PDV), a green product. Different clustering tags to VioE and VioC are characterized and used in a visual screen for flux redirection towards DV. While light inducible flux redirection towards DV is currently under investigation, the system reveals interesting dynamics at an additional branchpoint. If the product of VioB is processed by VioE, PDV is produced. Otherwise, the product of VioB is spontaneously converted to chromopyrrolic acid (CPA). Strains with a clustering tag on VioE (VioB is not tagged) produce more PDV relative to CPA when grown in the light versus the dark. The mechanism for this flux redirection remains to be explored. Overall, these results suggest that flux redirection using light activated enzyme clustering offers a new protein-level tool for metabolic manipulation.