Abstract
BackgroundYeast 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.ResultsIn 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 (Δψm) 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.ConclusionsThe 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.
Highlights
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
This study investigated the relationships between transcriptomic profile and cellular physiological responses of yeast engineered for increased lipid production
The transcriptomic analysis showed that the lipid engineering strategy redirected the global metabolic pathway towards lipid production, such as upregulated pathways for the production of precursors including acetyl-CoA, malonylCoA, and acyl-CoA
Summary
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. 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. Investigation of the basis of productivity loss and physiological impacts to cells subject to lipid pathway engineering may reveal mechanisms that could be addressed through further genetic modification or culture conditions, as examples. Two powerful approaches for revealing the responses of cells to metabolic engineering include transcriptome and physiological parameter analysis. While there have been few prior reports of transcriptomic analyses of yeast engineered for enhanced lipid production [8, 9], analysis to date has revealed likely oxidative stress impacts on cell growth and additional demand for NADPH in yeast engineered for high production of fatty acid esters. Regardless of the technology used, transcriptomics has been broadly applied to great effect in the study of cell metabolism [10]
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