Optogenetic control of Cdc48 for dynamic metabolic engineering in yeast.

Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid ß-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.

Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum-associated degradation pathway.

Protein quality mechanisms are fundamental for proteostasis of eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) is a well-studied pathway that ensures quality control of secretory and endoplasmic reticulum (ER)-resident proteins. Different branches of ERAD are involved in degradation of malfolded secretory proteins, depending on the localization of the misfolded part, the ER lumen (ERAD-L), the ER membrane (ERAD-M), and the cytosol (ERAD-C). Here we report that modification of several ER transmembrane proteins with the photosensitive degron (psd) module resulted in light-dependent degradation of the membrane proteins via the ERAD-C pathway. We found dependency on the ubiquitylation machinery including the ubiquitin-activating enzyme Uba1, the ubiquitin--conjugating enzymes Ubc6 and Ubc7, and the ubiquitin-protein ligase Doa10. Moreover, we found involvement of the Cdc48 AAA-ATPase complex members Ufd1 and Npl4, as well as the proteasome, in degradation of Sec62-myc-psd. Thus, our work shows that ERAD-C substrates can be systematically generated via synthetic degron constructs, which facilitates future investigations of the ERAD-C pathway.

Nitrogen stress induction on Levisticum officinale hairy roots grown in darkness and under photoperiod conditions: effect on growth and volatile components.

Six-year-old Levisticum officinale (lovage) hairy root cultures were used to study the effect of eight different NH(4) (+):NO(3) (-) ratios on their growth and volatile components. All cultures were kept at 24 degrees C on orbital shakers at 80 rpm, in darkness or in a 16 h light/8 h dark photoperiod. Growth was evaluated by dry and fresh weight determination. The volatiles were isolated by distillation-extraction and analysed by GC and GC-MS. Greater growth was attained in darkness with 10:90 (control, SH medium), 50:50 and 25:75 NH(4) (+):NO(3) (-) ratios, and also with SH control medium under the photoperiod condition, with a 10, 14, 12.5 and 12.5 fold increase of biomass in terms of dry weight, respectively, at the end of 42 days of growth. UPGMA cluster analysis of the mixtures of volatiles isolated from the hairy roots grown with different NH(4) (+):NO(3) (-) ratios confirmed their chemical variability. Although no particular grouping was detected in relation to the NH(4) (+):NO(3) (-) ratios or light conditions studied, most of the mixtures of volatiles isolated from the hairy roots were either dominated by n-octanal, (Z)-falcarinol or both components in about the same relative amounts.