In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.

A deeper investigation of the chemistry that occurs on the newly discovered epigenetic DNA bases 5-hydroxymethyl-(hmdC), 5-formyl-(fdC), and 5-carboxy-deoxycytidine (cadC) requires chemical tool compounds, which are able to dissect the different potential reaction pathways in cells. Here we report that the 2'-(R)-fluorinated derivatives F-hmdC, F-fdC, and F-cadC, which are resistant to removal by base excision repair, are good substrates for DNA polymerases and TET enzymes. This result shows that the fluorinated compounds are ideal tool substances to investigate potential C-C-bond cleaving reactions in the context of active demethylation.

We report the development of a new heterobase that is held together through reversible bonding. The so-formed cross-link adds strong stabilization to the DNA duplex. Despite this, the cross-link opens and closes through reversible imine bonding. Moreover, even enzymatic incorporation of the cross-link is possible. The new principle can be used to stabilize DNA duplexes and nanostructures that otherwise require high salt concentrations, which may hinder biological applications.

We have recently characterized the role of the riboflavinbinding protein (RfBP) dodecin from Halobacterium salinarum as clearing free riboflavin from the cytoplasm with riboflavin protein dissociation constants in the low nanomolar range, [1] and as providing riboflavin as the direct precursor for FMN and FAD biosynthesis. To prevent cellular damage, dodecin seals riboflavin in deeply buried binding cavities and neutralizes riboflavin reactivity by extensively quenching photoactivated states. [2] Both properties are achieved by a remarkable binding mode. Binding to dodecin, riboflavin aligns into a sandwich of aromatic systems in which extensive stacking compensates for minimal hydrogen bonding ( Figure 1). In the key step of the relaxation process of the light-activated riboflavin, an electron of tryptophan W36 is transferred to the excited flavin, generating a charge-separated intermediate state that subsequently recombines to the ground state. Recently, we were able to assign time constants to the individual processes in the photocycle of dodecin: 1) charge separation faster than the resolution of the experiment (< 0.2 ps, t 1 ); 2) electron back-transfer with a time constant of 0.9 ps (t 2 ); (3) a relaxation process with 6 ps parallel to (2) with an intermediate absorbing at 500 nm (t 3 ); and (4) proton transfer from the surrounding water coupled with the electron-transfer/back-transfer cycle (Scheme 1). [3] Based on high-resolution X-ray structural data and a concise functional characterization, establishing a system of extraordinarily well-defined structure-function relationships, we considered dodecin as excellently suited for modulating biological electron-transfer reactions by rational protein design, thereby studying the protein in a manipulative manner.This approach should be achieved by exchanging the native W36 with analogues of varying ionization potential leading to W36-riboflavin pairs of modulated redox potential difference. Given the computed value of 7.42 eV for the indole unit of tryptophan at the DFT/B3LYP/6-31G* level of theory in the gas phase, [4] we chose the derivatives 4-aminotryptophan (4NH 2 -W), 4-fluorotrypthophan (4F-W), and 4azatryptophan (4Aza-W) for shifting the ionization potential to 6.68, 7.49, and 7.96 eV, respectively (see Supporting Information). The C-4 position of the flavin-holding W36 was chosen for derivatization, as X-ray structural analysis indicated an empty bulge in the binding pocket allowing noninvasive modification. To prepare noncanonical W36-modified dodecin analogues, we applied the supplementationbased incorporation method (SPI) [5] together with the established procedures for dodecin purification and folding Figure 1. Sandwich-like incorporation of flavins. In dodecin, dimers of riboflavin (Rf) are bound between tryptophans (W36) and aligned antiparallelly by glutamines (Q55) at the interface of two protomers related by C 2 symmetry.