Herold, R. A. | bioRxiv Science

bioRxiv2026

De novo Design of a Vanadium Haloperoxidase

Organohalogens are an essential class of compounds for both synthetic chemistry and natural systems, yet the design of halogenases to catalyze carbon-halogen (C-X) bond formation has remained elusive. Vanadium-dependent haloperoxidases (V-HPOs) are attractive targets for such an effort, but they pose an unusual design problem: the vanadate (VO43-) cofactor is an anionic oxometalate that must be held by a precisely arranged network of hydrogen-bonding and electrostatic interactions rather than by the strong coordinate-covalent bonds through which proteins bind cationic transition metals. We addressed this challenge by utilizing a machine-learning-guided workflow to design VAN1, a highly stable 20 kDa protein that binds VO43- and brominates activated aromatic substrates, including L-tyrosine. X-ray crystal structures of metal-bound VAN1 closely matched the computational model and confirmed the designed active site, and structure-guided mutagenesis improved VO43- affinity 15-fold and bromination activity 2.6-fold, in quantitative agreement with the resulting change in cofactor occupancy. VAN1 remains folded in neat organic solvents and is substantially more active in aqueous/organic mixtures than in water, representing an important advantage for synthetic applications. As the first example of a de novo enzyme that catalyzes C-X bond formation, VAN1 establishes a new starting point for V-HPO engineering and mechanistic studies.

biochemistry↗

bioRxiv2020

Engineered Terpenoid Production in Synechocystis sp. PCC 6803 Under Different Growth Conditions

Terpenoids are the largest class of natural products and have applications in a wide variety of industries. Many terpenoids can be chemically synthesized or extracted from plants, but this is often uneconomical or unsustainable. An alternative production method relies on the heterologous expression of terpene synthase enzymes in cyanobacteria, producing the desired compounds directly from carbon dioxide. In this work, a patchoulol synthase enzyme from Pogostemon cablin (patchouli) was expressed in the cyanobacterium Synechocystis sp. PCC 6803 under four different growth conditions. Final yields of patchoulol from each growth condition were as follows: 249 g L-1, photoautotrophic growth; 6.5 g L-1, mixotrophic growth; 27.6 g L-1, bicarbonate low light; 31.7 g L-1, bicarbonate high light. By comparing patchoulol production across growth conditions, we identified a significant positive correlation between the production of photopigments (chlorophyll and carotenoids) and the production of patchoulol. Importantly, this relationship was found to be stronger than the correlation between cell density and patchoulol production across growth conditions, which was not statistically significant. The relationship between photopigments and patchoulol should be generalizable to the production of other terpenoids that rely on expression of the endogenous methylerythritol phosphate (MEP) pathway in cyanobacteria. Based on the results of this work, we propose a strategy for maximizing terpenoid production in cyanobacteria by optimizing growth conditions for photopigment production, resulting in increased flux through the MEP pathway. This strategy has the advantage of facile photopigment quantification using simple spectroscopic methods, and optimized growth conditions can be utilized in partnership with conventional terpenoid production strategies to further improve yields.

bioengineering↗