Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

We previously described the design of triacetic acid lactone (TAL) biosensor “AraC-TAL1”, based on the AraC regulatory protein. While useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity, and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a “decoy” ligand during negative selection, resulting in isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the IPRO computational framework was employed, followed by two rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid

We previously described the design of triacetic acid lactone (TAL) biosensor ‘AraC-TAL1’, based on the AraC regulatory protein. Although useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a ‘decoy’ ligand during negative selection, resulting in the isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the iterative protein redesign and optimization computational framework was employed, followed by 2 rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

Sporormielones A–E, bioactive novel C–C coupled orsellinic acid derivative dimers, and their biosynthetic origin

Sporormielones A–E, novel C–C coupled orsellinic acid derivative dimers containing tricyclic cores with a dimethylcyclopentenone unit, were isolated from Sporormiella sp., and their plausible biosynthetic mechanism was proposed.

Chemical Composition of Umbilicaria crustulosa and U. cylindrica

The chemical composition of ether, ethyl acetate and dichloromethane extracts of Umbilicaria crustulosa and U. cylindrica were determined for the first time by HPLC-UV, GC-FID and GC-MS. The HPLC-UV analysis was revealed the presence of methyl orsellinate, lecanoric acid, crustinic acid, gyrophoric acid and atranorin in U. crustulosa extracts while in U. cylindrica extracts were detected following compounds: salazinic acid, norstictic acid, atraric acid, gyrophoric acid and atranorin. GC-FID and GC-MS analysis were enabled the identification of orsellinic acid (40.4%) and orcinol (48.1%) as main components of U. crustulosa extracts and atraric acid (8.5%) and orcinol (10.3%) in U. cylindrica extracts.