Dataset Information

Flow-cytometry-based physiological characterisation and transcriptome analyses reveal a mechanism for reduced cell viability in yeast engineered for increased lipid content.

ABSTRACT:

Background

Yeast has been the focus of development of cell biofactories for the production of lipids and interest in the field has been driven by the need for sustainably sourced lipids for use in a broad range of industrial applications. Previously, we reported a metabolic engineering strategy for enhanced lipid production in yeast which delivered high per-cell lipid but with low cell growth and compromised physiology. To investigate the relationship between lipid engineering and cellular physiological responses and to identify further metabolic engineering targets, we analysed transcriptomes and measured cell physiology parameters in engineered strains.

Results

In the engineering strategy, the central carbon pathway was reprogrammed to provide more precursors for lipid production and lipid accumulation and sequestration steps were enhanced through the expression of heterologous genes. Genes coding for enzymes within the pentose phosphate, beta-oxidation pathways, ATP and NADPH biosynthesis had lower transcript levels in engineered cells. Meanwhile, flow-cytometry analysis of fluorescent-dye stained cells showed the highest reactive oxygen species (ROS) levels and mitochondrial membrane potential (??m) in cells with the highest lipid content, supporting the known relationship between mitochondrial activity and ROS generation. High intracellular ROS and low membrane integrity were not ameliorated by application of antioxidants.

Conclusions

The limited intracellular energy supplies and the unbalanced redox environment could be regarded as targets for further lipid engineering, similarly for native lipid accumulation genes that were upregulated. Thus, lipid pathway engineering has an important effect on the central carbon pathway, directing these towards lipid production and sacrificing the precursors, energy and cofactor supply to satisfy homeostatic metabolic requirements.

SUBMITTER: Peng H

PROVIDER: S-EPMC6477733 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Flow-cytometry-based physiological characterisation and transcriptome analyses reveal a mechanism for reduced cell viability in yeast engineered for increased lipid content.

Peng Huadong H He Lizhong L Haritos Victoria S VS

Biotechnology for biofuels 20190423

<h4>Background</h4>Yeast has been the focus of development of cell biofactories for the production of lipids and interest in the field has been driven by the need for sustainably sourced lipids for use in a broad range of industrial applications. Previously, we reported a metabolic engineering strategy for enhanced lipid production in yeast which delivered high per-cell lipid but with low cell growth and compromised physiology. To investigate the relationship between lipid engineering and cellul ...[more]

PMID: 31044011

Similar Datasets

Project description:Bacteria modify their morphology in response to various factors including growth stage, nutrient availability, predation, motility and long-term survival strategies. Morphological changes may also be associated with specific physiological phenotypes such as the formation of dormant or persister cells in a "viable but non-culturable" (VBNC) state which frequently display different shapes and size compared to their active counterparts. Such dormancy phenotypes can display various degrees of tolerance to antibiotics and therefore a detailed understanding of these phenotypes is crucial for combatting chronic infections and associated diseases. Cell shape and size are therefore more than simple phenotypic characteristics; they are important physiological properties for understanding bacterial life-strategies and pathologies. However, quantitative studies on the changes to cell morphologies during bacterial growth, persister cell formation and the VBNC state are few and severely constrained by current limitations in the most used investigative techniques of flow cytometry (FC) and light or electron microscopy. In this study, we applied high-throughput Imaging Flow Cytometry (IFC) to characterise and quantify, at single-cell level and over time, the phenotypic heterogeneity and morphological changes in cultured populations of four bacterial species, Bacillus subtilis, Lactiplantibacillus plantarum, Pediococcus acidilactici and Escherichia coli. Morphologies in relation to growth stage and stress responses, cell integrity and metabolic activity were analysed. Additionally, we were able to identify and morphologically classify dormant cell phenotypes such as VBNC cells and monitor the resuscitation of persister cells in Escherichia coli following antibiotic treatment. We therefore demonstrate that IFC, with its high-throughput data collection and image capture capabilities, provides a platform by which a detailed understanding of changes in bacterial phenotypes and their physiological implications may be accurately monitored and quantified, leading to a better understanding of the role of phenotypic heterogeneity in the dynamic microbiome.

Project description:Background and aimsFlow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility.MethodsThe method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species.Key resultsData quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %.ConclusionsThe high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.

Project description:Over the past years, gut microbiota became a major field of interest with increasing reports suggesting its association with a large number of human diseases. In this context, there is a major interest to develop analysis tools allowing simple and cost-effective population pattern analysis of these complex ecosystems to follow changes over time. Whereas sequence-based metagenomics profiling is widely used for microbial ecosystems characterization, it still requires time and specific expertise for analysis. Flow cytometry overcomes these disadvantages, providing key information on communities within hours. In addition, it can potentially be used to select, isolate and cultivate specific bacteria of interest. In this study, we evaluated the culturability of strictly anaerobic bacteria that were stained with a classical Live/Dead staining, and then sorted using flow cytometry under anaerobic conditions. This sorting of "viable" fraction demonstrated that 10-80% of identified "viable" cells of pure cultures of strictly anaerobic bacteria were culturable. In addition, we tested the use of a combination of labeled vancomycin and Wheat Germ Agglutinin (WGA) lectin to discriminate Gram-positive from Gram-negative bacteria in complex ecosystems. After validation on both aerobic/anaerobic facultative and strictly anaerobic bacteria, the staining methods were applied on complex ecosystems, revealing differences between culture conditions and demonstrating that minor pH variations have strong impacts on microbial community structure, which was confirmed by 16S rRNA gene sequencing. This combination of staining methods makes it possible to follow-up evolutions of complex microbial communities, supporting its future use as a rapid analysis tool in various applications. The flow cytometry staining method that was developed has the potential to facilitate the analysis of complex ecosystems by highlighting changes in bacterial communities' dynamics. It is assumed to be applicable as an efficient and fast approach to improve the control of processes linked to a wide range of ecosystems or known communities of bacterial species in both research and industrial contexts.

Dataset Information

Flow-cytometry-based physiological characterisation and transcriptome analyses reveal a mechanism for reduced cell viability in yeast engineered for increased lipid content.

Background

Results

Conclusions

Publications

Flow-cytometry-based physiological characterisation and transcriptome analyses reveal a mechanism for reduced cell viability in yeast engineered for increased lipid content.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets