ABSTRACT
Multi-omics datasets are becoming of key importance to drive discovery in fundamental research as much as generating knowledge for applied biotechnology. However, the construction of such large datasets is usually time-consuming and expensive. Automation might enable to overcome these issues by streamlining workflows from sample generation to data analysis. Here, we describe the construction of a complex workflow for the generation of high-throughput microbial multi-omics datasets. The workflow comprises a custom-built platform for automated cultivation and sampling of microbes, sample preparation protocols, analytical methods for sample analysis and automated scripts for raw data processing. We demonstrate possibilities and limitations of such workflow in generating data for three biotechnologically relevant model organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida.
Subject(s)
Multiomics , WorkflowABSTRACT
Exploring the dynamic behavior of cellular metabolism requires a standard laboratory method that guarantees rapid sampling and extraction of the cellular content. We propose a versatile sampling technique applicable to cells with different cell wall and cell membrane properties. The technique is based on irreversible electroporation with simultaneous quenching and extraction by using a microfluidic device. By application of electric pulses in the millisecond range, permanent lethal pores are formed in the cell membrane of Escherichia coli and Saccharomyces cerevisiae, facilitating the release of the cellular contents; here demonstrated by the measurement of glucose-6-phosphate and the activity of the enzyme glucose-6-phosphate dehydrogenase. The successful application of this device was demonstrated by pulsed electric field treatment in a flow-through configuration of the microfluidic chip in combination with sampling, inactivation, and extraction of the intracellular content in a few seconds. Minimum electric field strengths of 10 kV/cm for E. coli and 7.5 kV/cm for yeast S. cerevisiae were required for successful cell lysis. The results are discussed in the context of applications in industrial biotechnology, where metabolomics analyses are important.
ABSTRACT
Viscosity is an inherent characteristic of fluids and is therefore an important parameter in many different processes. Current methods to measure viscosity involve direct contact with the liquid sample, which is often undesirable. Here we present a simple, precise and robust contact-free method to determine viscosity, using a single drive motor, inexpensive components and disposable sample vessels. The measurement principle involves the detection of viscosity-dependent angular positions in a rotating liquid relative to the direction of centrifugal acceleration in an orbitally shaken vessel. The signal can be detected using different optical methods, as shown here using fluorescence and transmitted light. The sensitivity of the system can be adjusted over a wide range by varying the sample volume or the shaking diameter, and multiple samples can be analysed in parallel. This novel viscometer is also applicable to characterize non-Newtonian shear rate-dependent fluids.
ABSTRACT
The production of poly-γ-glutamic acid (γ-PGA), a biopolymer consisting of D- and L-glutamic acid monomers, currently relies on L-glutamate, or citrate as carbon substrates. Here we aimed at using plant biomass-derived substrates such as xylose. γ-PGA producing microorganisms including Bacillus subtilis natively metabolize xylose via the isomerase pathway. The Weimberg pathway, a xylose utilization pathway first described for Caulobacter crescentus, offers a carbon-efficient alternative converting xylose to 2-oxoglutarate without carbon loss. We engineered a recombinant B. subtilis strain that was able to grow on xylose with a growth rate of 0.43 h-1 using a recombinant Weimberg pathway. Although ion-pair reversed-phase LC/MS/MS metabolome analysis revealed lower concentrations of γ-PGA precursors such as 2-oxoglutarate, the γ-PGA titer was increased 6-fold compared to the native xylose isomerase strain. Further metabolome analysis indicates a metabolic bottleneck in the phosphoenolpyruvate-pyruvate-oxaloacetate node causing bi-phasic (diauxic) growth of the recombinant Weimberg strain. Flux balance analysis (FBA) of the γ-PGA producing B. subtilis indicated that a maximal theoretical γ-PGA yield is achieved on D-xylose/ D-glucose mixtures. The results of the B. subtilis strain harboring the Weimberg pathway on such D-xylose/ D-glucose mixtures demonstrate indeed resource efficient, high yield γ-PGA production from biomass-derived substrates.
ABSTRACT
Salt-enhanced cultivation as a morphology engineering tool for the filamentous actinomycete Actinomadura namibiensis was evaluated in 500-mL shaking flasks (working volume 100 mL) with the aim of increasing the concentration of the pharmaceutically interesting peptide labyrinthopeptin A1. Among the inorganic salts added to a complex production medium, the addition of (NH4)2SO4 led to the highest amount of labyrinthopeptin A1 production. By using 50 mM (NH4)2SO4, the labyrinthopeptin A1 concentration increased up to sevenfold compared to the non-supplemented control, resulting in 325 mg L-1 labyrinthopeptin A1 after 10 days of cultivation. The performance of other ammonium- and sulfate-containing salts (e.g., NH4Cl, K2SO4) was much lower than the performance of (NH4)2SO4. A positive correlation between the uptake of glycerol as one of the main carbon sources and nongrowth-associated labyrinthopeptin productivity was found. The change in the cell morphology of A. namibiensis in conjunction with increased osmolality by the addition of 50 mM (NH4)2SO4, was quantified by image analysis. A. namibiensis always developed a heterogeneous morphology with pellets and loose mycelia present simultaneously. In contrast to the non-supplemented control, the morphology of (NH4)2SO4-supplemented cultures was characterized by smaller and circular pellets that were more stable against disintegration in the stationary production phase.
