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

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.

[1]  A. Trančíková,et al.  Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL. , 2004, FEMS yeast research.

[2]  G. Stephanopoulos,et al.  Engineering oxidative stress defense pathways to build a robust lipid production platform in Yarrowia lipolytica , 2017, Biotechnology and bioengineering.

[3]  Jens Nielsen,et al.  Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. , 2013, Metabolic engineering.

[4]  J. Cronan,et al.  Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli , 1999, Molecular microbiology.

[5]  Jens Nielsen,et al.  Metabolic engineering strategies for microbial synthesis of oleochemicals. , 2015, Metabolic Engineering.

[6]  G. Lidén,et al.  Anaerobic glycerol production by Saccharomyces cerevisiae strains under hyperosmotic stress , 2007, Applied Microbiology and Biotechnology.

[7]  E. Cadenas,et al.  Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. , 1977, Archives of biochemistry and biophysics.

[8]  Timothy S. Ham,et al.  Production of the antimalarial drug precursor artemisinic acid in engineered yeast , 2006, Nature.

[9]  Gemma Beltran,et al.  Melatonin Reduces Oxidative Stress Damage Induced by Hydrogen Peroxide in Saccharomyces cerevisiae , 2017, Front. Microbiol..

[10]  F. Bordes,et al.  Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica , 2013, Applied Microbiology and Biotechnology.

[11]  W. Liang,et al.  Comparative transcriptome analysis reveals multiple functions for Mhy1p in lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica. , 2018, Biochimica et biophysica acta. Molecular and cell biology of lipids.

[12]  L. Harvey,et al.  The Pichia pastoris transmembrane protein GT1 is a glycerol transporter and relieves the repression of glycerol on AOX1 expression. , 2016, FEMS yeast research.

[13]  I. Nookaew,et al.  A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae , 2012, Nucleic acids research.

[14]  Jens Nielsen,et al.  Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene. , 2019, Metabolic engineering.

[15]  J. Nielsen,et al.  Impact of forced fatty acid synthesis on metabolism and physiology of Saccharomyces cerevisiae. , 2018, FEMS yeast research.

[16]  J. Keasling,et al.  Alleviation of reactive oxygen species enhances PUFA accumulation in Schizochytrium sp. through regulating genes involved in lipid metabolism , 2018, Metabolic engineering communications.

[17]  V. Haritos,et al.  Enhanced Production of High-Value Cyclopropane Fatty Acid in Yeast Engineered for Increased Lipid Synthesis and Accumulation. , 2018, Biotechnology journal.

[18]  J. Förster,et al.  In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production. , 2006, Metabolic engineering.

[19]  Jens Nielsen,et al.  Physiological and transcriptional characterization of Saccharomyces cerevisiae engineered for production of fatty acid ethyl esters. , 2016, FEMS yeast research.

[20]  C. Henderson,et al.  Examining the Role of Membrane Lipid Composition in Determining the Ethanol Tolerance of Saccharomyces cerevisiae , 2014, Applied and Environmental Microbiology.

[21]  B. Nikoli,et al.  The Effects of Vitamin C on Oxidative DNA Damage and Mutagenesis , 2006 .

[22]  T. Chardot,et al.  Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast. , 2009, FEMS yeast research.

[23]  S. Sollott,et al.  Mitochondrial membrane potential. , 2017, Analytical biochemistry.

[24]  Jens Nielsen,et al.  Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production. , 2012, FEMS yeast research.

[25]  D. Bonatto,et al.  Antioxidant protection of resveratrol and catechin in Saccharomyces cerevisiae. , 2008, Journal of agricultural and food chemistry.

[26]  H. Alper,et al.  Engineering Yarrowia lipolytica for the production of cyclopropanated fatty acids , 2018, Journal of Industrial Microbiology & Biotechnology.

[27]  V. Siewers,et al.  Engineering microbial fatty acid metabolism for biofuels and biochemicals. , 2018, Current opinion in biotechnology.

[28]  Verena Siewers,et al.  Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters , 2014, Biotechnology and bioengineering.

[29]  H. Alper,et al.  Surveying the lipogenesis landscape in Yarrowia lipolytica through understanding the function of a Mga2p regulatory protein mutant. , 2015, Metabolic engineering.

[30]  J. Tzen,et al.  An in vitro system to examine the effective phospholipids and structural domain for protein targeting to seed oil bodies. , 2001, Plant & cell physiology.

[31]  V. Haritos,et al.  Metabolic engineering of lipid pathways in Saccharomyces cerevisiae and staged bioprocess for enhanced lipid production and cellular physiology , 2018, Journal of Industrial Microbiology & Biotechnology.

[32]  Thomas M. Wasylenko,et al.  The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. , 2015, Metabolic engineering.

[33]  David E. Levin,et al.  Cell Wall Integrity Signaling in Saccharomyces cerevisiae , 2005, Microbiology and Molecular Biology Reviews.

[34]  Michael Sauer,et al.  Biosynthesis of Vitamin C by Yeast Leads to Increased Stress Resistance , 2007, PloS one.

[35]  Peng Xu,et al.  Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism , 2017, Nature Biotechnology.

[36]  F. Schroeder,et al.  Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells. , 1978, Biochimica et biophysica acta.