Biotechnological polyphosphate as an opportunity to contribute to the circularization of the phosphate economy.

Polyphosphates, chains of polymerized phosphate subunits, are used as food additives for various applications such as conservation, water retention, and pH buffering. Currently, the value chain of phosphates is linear, based on mining fossil phosphate rock, which is anticipated to be depleted in a few hundred years. With no replacement available, a transition to a circular phosphate economy, to which biological systems can contribute, is required. Baker's yeast can hyperaccumulate phosphate from various phosphate-rich waste streams and form polyphosphates, which can be used directly or as polyphosphate-rich yeast extract with enhanced properties in the food industry. By maturing the technology to an industrial level and allowing upcycled waste streams for food applications, substantial contributions to a sustainable phosphate economy can be achieved.

Pseudomonas putida KT2440 endures temporary oxygen limitations.

The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen. Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrial-scale bioprocesses.

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.

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC.

BACKGROUND: Microbial production of chemicals from renewable carbon sources enables a sustainable route to many bioproducts. Sugar streams, such as those derived from biomass pretreated with ionic liquids (IL), provide efficiently derived and cost-competitive starting materials. A limitation to this approach is that residual ILs in the pretreated sugar source can be inhibitory to microbial growth and impair expression of the desired biosynthetic pathway. RESULTS: We utilized laboratory evolution to select Escherichia coli strains capable of robust growth in the presence of the IL, 1-ethyl-3-methyl-imidizolium acetate ([EMIM]OAc). Whole genome sequencing of the evolved strain identified a point mutation in an essential gene, cydC, which confers tolerance to two different classes of ILs at concentrations that are otherwise growth inhibitory. This mutation, cydC-D86G, fully restores the specific production of the bio-jet fuel candidate D-limonene, as well as the biogasoline and platform chemical isopentenol, in growth medium containing ILs. Similar amino acids at this position in cydC, such as cydC-D86V, also confer tolerance to [EMIM]OAc. We show that this [EMIM]OAc tolerance phenotype of cydC-D86G strains is independent of its wild-type function in activating the cytochrome bd-I respiratory complex. Using shotgun proteomics, we characterized the underlying differential cellular responses altered in this mutant. While wild-type E. coli cannot produce detectable amounts of either product in the presence of ILs at levels expected to be residual in sugars from pretreated biomass, the engineered cydC-D86G strains produce over 200 mg/L D-limonene and 350 mg/L isopentenol, which are among the highest reported titers in the presence of [EMIM]OAc. CONCLUSIONS: The optimized strains in this study produce high titers of two candidate biofuels and bioproducts under IL stress. Both sets of production strains surpass production titers from other IL tolerant mutants in the literature. Our application of laboratory evolution identified a gain of function mutation in an essential gene, which is unusual in comparison to other published IL tolerant mutants.

Quantitative measurements in single-cell analysis: towards scalability in microbial bioprocess development.

Single-cell analysis in microfluidic cultivation devices bears a great potential for the development and optimization of industrial bioprocesses. High parallelization allows running a large number of cultivation experiments simultaneously even under quick alteration of environmental conditions. For example, the impact of changes in media composition on cell growth during classical batch cultivation can be easily resolved. A missing link for the scalability of microfluidic experiments is, however, their complete characterization via conventional performance indicators such as product titer and productivity. While existing mass spectrometry technology is not yet sufficiently coupled with microfluidics, optical methods like enzymatic assays or fluorescence sensors are promising alternatives but require further improvement to generate quantitative measurements of extracellular metabolites.