A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii.

A novel peptidyl-lys metalloendopeptidase (Tc-LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc-LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc-LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc-LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5-7.5 and ≥30% activity between pH values 8.5-10.0, indicating its broad applicability. The temperature maximum of Tc-LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc-LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc-LysN's activity up to ~100% and ~50%, respectively. Tc-LysN's thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)'s sequence coverage of 84% using Tc-LysN which was comparable to the sequence coverage of 90% by trypsin peptides. KEY POINTS: •A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity •Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants •Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting.

In vivo identification of Drosophila rhodopsin interaction partners by biotin proximity labeling.

Proteins exert their function through protein-protein interactions. In Drosophila, G protein-coupled receptors like rhodopsin (Rh1) interact with a G protein to activate visual signal transduction and with arrestins to terminate activation. Also, membrane proteins like Rh1 engage in protein-protein interactions during folding within the endoplasmic reticulum, during their vesicular transport and upon removal from the cell surface and degradation. Here, we expressed a Rh1-TurboID fusion protein (Rh1::TbID) in Drosophila photoreceptors to identify in vivo Rh1 interaction partners by biotin proximity labeling. We show that Rh1::TbID forms a functional rhodopsin that mediates biotinylation of arrestin 2 in conditions where arrestin 2 interacts with rhodopsin. We also observed biotinylation of Rh1::TbID and native Rh1 as well as of most visual signal transduction proteins. These findings indicate that the signaling components in the rhabdomere approach rhodopsin closely, within a range of ca. 10 nm. Furthermore, we have detected proteins engaged in the maturation of rhodopsin and elements responsible for the trafficking of membrane proteins, resembling potential interaction partners of Rh1. Among these are chaperons of the endoplasmic reticulum, proteins involved in Clathrin-mediated endocytosis as well as previously unnoticed contributors to rhodopsin transportation, such as Rab32, Vap33, or PIP82.

Proteomic differences in apple spur buds from high and non-cropping trees during floral initiation.

The cropping behavior of biennial apple (Malus ×domestica Borkh.) cultivars is irregular and often follows a biennial bearing pattern with 'On' years (high crop load and inhibited floral bud formation) followed by 'Off' years (little crop load and a promoted formation of floral buds). To study proteomic differences between floral and vegetative buds, trees of the strongly alternating cultivar 'Fuji' and the regular bearing cultivar 'Gala' were either completely thinned or not thinned at full bloom to establish two cropping treatments with no ('Off') or a high ('On') crop load, respectively. Student's t-Tests indicated significant differences of protein profiles in buds from 2-year old spurs from both treatments at each sampling date. Abundance patterns of protein clusters coincided with the onset of floral bud initiation and were most noticeable in buds from 'On' trees with a decreased abundance of key enzymes of the phenylpropanoid and flavonoid pathways and an increased abundance of histone deacetylase and ferritins. Furthermore, an increased abundance of proteins involved in histone and DNA methylation was found in the buds from 'Off' trees. This study presents the first large-scale, label-free proteomic profiling of floral and vegetative apple buds during the period of floral bud initiation. SIGNIFICANCE: Although several studies exist that address the complex developmental processes associated with the formation of floral buds in apple (Malus ×domestica Borkh.) at transcriptomic level, no data is available for explaining the difference between floral and vegetative buds or biennial and regular bearing cultivars on a proteomic level. This study presents the first large-scale, label-free proteomic profiling of floral and vegetative apple buds from the two cultivars 'Fuji' and 'Royal Gala' during the period of floral bud initiation and renders possible the development of suitable biomarkers for biennial bearing in apple.

Modeling the time course of ComX: towards molecular process control for Bacillus wild-type cultivations.

Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer's demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus. One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10 T. First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.