32 research outputs found
Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum
ART: A machine learning Automated Recommendation Tool for synthetic biology
Biology has changed radically in the last two decades, transitioning from a descriptive science into a design science. Synthetic biology allows us to bioengineer cells to synthesize novel valuable molecules such as renewable biofuels or anticancer drugs. However, traditional synthetic biology approaches involve ad-hoc engineering practices, which lead to long development times. Here, we present the Automated Recommendation Tool (ART), a tool that leverages machine learning and probabilistic modeling techniques to guide synthetic biology in a systematic fashion, without the need for a full mechanistic understanding of the biological system. Using sampling-based optimization, ART provides a set of recommended strains to be built in the next engineering cycle, alongside probabilistic predictions of their production levels. We demonstrate the capabilities of ART on simulated data sets, as well as experimental data from real metabolic engineering projects producing renewable biofuels, hoppy flavored beer without hops, and fatty acids. Finally, we discuss the limitations of this approach, and the practical consequences of the underlying assumptions failing
Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000
Establishment of high throughput enzyme analytics on an automated liquid handling platform to characterize the central metabolism of Corynebacterium glutamicum
Beyond Growth Rate 0.6: What Drives Corynebacterium glutamicum to Higher Growth Rates in Defined Medium
In a former study we showed that Corynebacterium glutamicum grows much faster in defined CGXII glucose medium when growth was initiated in highly diluted environments [Grunberger et al. (2013b) Biotechnol Bioeng]. Here we studied the batch growth of C. glutamicum in CGXII at a comparable low starting biomass concentration of OD approximate to 0.005 in more detail. During bioreactor cultivations a biphasic growth behavior with changing growth rates was observed. Initially the culture grew with mu = 0.61 +/- 0.02 h(-1) before the growth rate dropped to mu = 0.46 +/- 0.02 h(-1). We were able to confirm the elevated growth rate for C. glutamicum in CGXII and showed for the first time a growth rate beyond 0.6 in lab-scale bioreactor cultivations on defined medium. Advanced growth studies combining well-designed bioreactor and microfluidic single-cell cultivations (MSCC) with quantitative transcriptomics, metabolomics and integrative in silico analysis revealed protocatechuic acid as a hidden co-substrate for accelerated growth within CGXII. The presented approach proves the general applicability of MSCC to investigate and validate the effect of single medium components on microorganism growth during cultivation in liquid media, and therefore might be of interest for any kind of basic growth study. (C) 2013 Wiley Periodicals, Inc
14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban
The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17. β-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. β-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to β-adrenergic–stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism
14-3-3 binding creates a memory of kinase action by stabilizing the modified state of phospholamban
The cardiac membrane protein phospholamban (PLN) is targeted by protein kinase A (PKA) at Ser16 and by Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr17 β-Adrenergic stimulation and PKA-dependent phosphorylation of Ser16 acutely stimulate the sarcoplasmic reticulum calcium pump (SERCA) by relieving its inhibition by PLN. CaMKII-dependent phosphorylation may lead to longer-lasting SERCA stimulation and may sustain maladaptive Ca2+ handling. Here, we demonstrated that phosphorylation at either Ser16 or Thr17 converted PLN into a target for the phosphoadaptor protein 14-3-3 with different affinities. 14-3-3 proteins were localized within nanometers of PLN and endogenous 14-3-3 coimmunoprecipitated with pentameric PLN from cardiac membranes. Molecular dynamics simulations predicted different molecular contacts for peptides phosphorylated at Ser16 or Thr17 with the binding groove of 14-3-3, resulting in varied binding affinities. 14-3-3 binding protected either PLN phosphosite from dephosphorylation. β-Adrenergic stimulation of isolated adult cardiomyocytes resulted in the membrane recruitment of endogenous 14-3-3. The exogenous addition of 14-3-3 to β-adrenergic-stimulated cardiomyocytes led to prolonged SERCA activation, presumably because 14-3-3 protected PLN pentamers from dephosphorylation. Phosphorylation of Ser16 was disrupted by the cardiomyopathy-associated ∆Arg14 mutation, implying that phosphorylation of Thr17 by CaMKII may become crucial for 14-3-3 recruitment to ∆Arg14 PLN. Consistent with PLN acting as a dynamic hub in the control of Ca2+ handling, our results identify 14-3-3 binding to PLN as a contractility-augmenting mechanism