Microfluidic Irreversible Electroporation-A Versatile Tool to Extract Intracellular Contents of Bacteria and Yeast.

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.

High-Yield Production of 4-Hydroxybenzoate From Glucose or Glycerol by an Engineered Pseudomonas taiwanensis VLB120.

Aromatic compounds such as 4-hydroxybenzoic acid are broadly applied in industry for a myriad of applications used in everyday life. However, their industrial production currently relies heavily on fossil resources and involves environmentally unfriendly production conditions, thus creating the need for more sustainable biotechnological alternatives. In this study, synthetic biology was applied to metabolically engineer Pseudomonas taiwanensis VLB120 to produce 4-hydroxybenzoate from glucose, xylose, or glycerol as sole carbon sources. Genes encoding a 4-hydroxybenzoate production pathway were integrated into the host genome and the flux toward the central precursor tyrosine was enhanced by overexpressing genes encoding key enzymes of the shikimate pathway. The flux toward tryptophan biosynthesis was decreased by introducing a P290S point mutation in the trpE gene, and degradation pathways for 4-hydroxybenzoate, 4-hydroxyphenylpyruvate and 3-dehydroshikimate were knocked out. The resulting production strains were tailored for the utilization of glucose and glycerol through the rational modification of central carbon metabolism. In batch cultivations with a completely mineral medium, the best strain produced 1.37 mM 4-hydroxybenzoate from xylose with a C-mol yield of 8% and 3.3 mM from glucose with a C-mol yield of 19.0%. Using glycerol as a sole carbon source, the C-mol yield increased to 29.6%. To our knowledge, this is the highest yield achieved by any species in a fully mineral medium. In all, the efficient conversion of bio-based substrates into 4-hydroxybenzoate by these deeply engineered P. taiwanensis strains brings the renewable production of aromatics one step closer.