Data in support of substrate flexibility of a mutated acyltransferase domain and implications for polyketide biosynthesis.

Enzyme-directed mutasynthesis is an emerging strategy for the targeted derivatization of natural products. Here, data on the synthesis of malonic acid derivatives for feeding studies in Saccharopolyspora erythraea , the mutagenesis of DEBS and bioanalytical data on the experimental investigation of studies on the biosynthetic pathway towards erythromycin are presented.

Substrate Flexibility of a Mutated Acyltransferase Domain and Implications for Polyketide Biosynthesis.

Polyketides are natural products frequently used for the treatment of various diseases, but their structural complexity hinders efficient derivatization. In this context, we recently introduced enzyme-directed mutasynthesis to incorporate non-native extender units into the biosynthesis of erythromycin. Modeling and mutagenesis studies led to the discovery of a variant of an acyltransferase domain in the erythromycin polyketide synthase capable of accepting a propargylated substrate. Here, we extend molecular rationalization of enzyme-substrate interactions through modeling, to investigate the incorporation of substrates with different degrees of saturation of the malonic acid side chain. This allowed the engineered biosynthesis of new erythromycin derivatives and the introduction of additional mutations into the AT domain for a further shift of the enzyme's substrate scope. Our approach yields non-native polyketide structures with functional groups that will simplify future derivatization approaches, and provides a blueprint for the engineering of AT domains to achieve efficient polyketide synthase diversification.

Heterologous fermentation of a diterpene from Alternaria brassisicola.

A variety of different applications render terpenes and terpenoids attractive research targets. A promising but so far insufficiently explored family of terpenoids are the fusicoccanes that comprise a characteristic 5-8-5 fused tricyclic ring system. Besides herbicidal effects, these compounds also show apoptotic and anti-tumour effects on mammalian cells. The access to fusicoccanes from natural sources is scarce. Recently, we introduced a metabolically engineered Saccharomyces cerevisiae strain to enable the heterologous fermentation of the shared fusicoccane-diterpenoid precursor, fusicocca-2,10(14)-diene. Here, we show experiments towards the identification of bottlenecks in this process. The suppression of biosynthetic by-products via medium optimisation was found to be an important aspect. In addition, the fermentation process seems to be improved under oxygen limitation conditions. Under fed-batch conditions, the fermentation yield was reproducibly increased to approximately 20 mg/L. Furthermore, the impact of the properties of the terpene synthase on the fermentation yield is discussed, and the preliminary studies on the engineering of this key enzyme are presented.

A morphometric screen identifies specific roles for microtubule-regulating genes in neuronal development of P19 stem cells.

The first morphological change after neuronal differentiation is the microtubule-dependent initiation of thin cell protrusions called neurites. Here we performed a siRNA-based morphometric screen in P19 stem cells to evaluate the role of 408 microtubule-regulating genes during this early neuromorphogenesis step. This screen uncovered several novel regulatory factors, including specific complex subunits of the microtubule motor dynein involved in neurite initiation and a novel role for the microtubule end-binding protein EB2 in attenuation of neurite outgrowth. Epistasis analysis suggests that competition between EB1 and EB2 regulates neurite length, which links its expression to neurite outgrowth. We propose a model that explains how microtubule regulators can mediate cellular morphogenesis during the early steps of neuronal development by controlling microtubule stabilization and organizing dynein-generated forces.

Exploration of biosynthetic access to the shared precursor of the fusicoccane diterpenoid family.

Herein preparative scale access to the shared precursor of the fusicoccane family of diterpenoids through biosynthesis in three different microbial hosts is reported. A method to purify the metabolite in high purity and on a preparative scale was explored.

Quantitative analyses of cryptochrome-mBMAL1 interactions: mechanistic insights into the transcriptional regulation of the mammalian circadian clock.

The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537(mBMAL1) acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1.

Structural and functional analyses of PAS domain interactions of the clock proteins Drosophila PERIOD and mouse PERIOD2.

PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.