Insoluble solids at high concentrations repress yeast’s response against stress and increase intracellular ROS levels

Lignocellulosic ethanol production requires high substrate concentrations for its cost-competitiveness. This implies the presence of high concentrations of insoluble solids (IS) at the initial stages of the process, which may limit the fermentation performance of the corresponding microorganism. The presence of 40–60% IS (w/w) resulted in lower glucose consumption rates and reduced ethanol volumetric productivities of Saccharomyces cerevisiae F12. Yeast cells exposed to IS exhibited a wrinkled cell surface and a reduced mean cell size due to cavity formation. In addition, the intracellular levels of reactive oxygen species (ROS) increased up to 40%. These ROS levels increased up to 70% when both lignocellulose-derived inhibitors and IS were simultaneously present. The general stress response mechanisms (e.g. DDR2, TPS1 or ZWF1 genes, trehalose and glycogen biosynthesis, and DNA repair mechanisms) were found repressed, and ROS formation could not be counteracted by the induction of the genes involved in repairing the oxidative damage such as glutathione, thioredoxin and methionine scavenging systems (e.g. CTA1, GRX4, MXR1, and TSA1; and the repression of cell cycle progression, CLN3). Overall, these results clearly show the role of IS as an important microbial stress factor that affect yeast cells at physical, physiological, and molecular levels.

. Upregulated and downregulated genes with molecular function unknown Gene (Systematic name) Brief description Log2-fold change Upregulated genes: ECM1 Pre-ribosomal factor involved in 60S ribosomal protein subunit export 0.9 SEO1 Putative permease 1.5

YBL028C
Protein of unknown function that may interact with ribosomes 0.7

PAU9
Protein of unknown function 1.9

MIC10
Conserved component of the MICOS complex 0.8

YCR051W
Putative protein of unknown function; non-essential gene 1.0

YDL012C
Tail-anchored plasma membrane protein with a conserved CYSTM module; possibly involved in response to stress 0.7

KNH1
Protein with similarity to Kre9p; required for propionic acid resistance 1.1

RAD59
Protein involved DNA double-strand break repair; repairs breaks in DNA during vegetative growth via recombination and single-strand annealing 1.1

YDL121C
Putative protein of unknown function; non-essential gene 0.8

YDR034C-A
Putative protein of unknown function 1.9

BCP1
Essential protein involved in nuclear export of Mss4p, which plays a role in actin cytoskeleton organization and vesicular transport 1.2

IRC22
Protein of unknown function; non-essential gene 0.7

GTT3
Protein of unknown function may be involved in glutathione metabolism 0.6

SCC4
Subunit of cohesin loading factor (Scc2p-Scc4p); involved in establishing sister chromatid cohesion during double-strand break repair via phosphorylated histone H2AX

MTC4
Cytoplasmic protein of unknown function -1.7

YBR285W
Putative protein of unknown function -1.2

FUS1
Membrane protein localized to the shmoo tip that regulates the termination of mating projection growth; has a role in cortical protein anchoring -1.0

GID7
Subunit of GID Complex that binds directly to central component Vid30p -0.9

HSP30
Negative regulator of the H(+)-ATPase Pma1p involved in the response to various stresses such as heat, osmotic stress, and DNA damage; also involved in negative regulation of ATPase activity -2.0

USO1
Essential protein involved in vesicle-mediated ER to Golgi transport; binds membranes and functions during vesicle docking to the Golgi; required for assembly of the ER-to-Golgi SNARE complex -2.5

PEX19
Chaperone and import receptor for newly-synthesized class I PMPs; binds peroxisomal membrane proteins (PMPs) in the cytoplasm and delivers them to the peroxisome for subsequent insertion into the peroxisomal membrane -1.2

BUG1
Cis-golgi localized protein involved in ER to Golgi transport; forms a complex with the mammalian GRASP65 homolog, Grh1p -1.2

MSS2
Peripherally bound inner membrane protein of the mitochondrial matrix; involved in membrane insertion of C-terminus of Cox2p -1.9

IWR1
RNA polymerase II transport factor, conserved from yeast to humans; also has a role in transporting RNA polymerase III into the nucleus -1.1

SAS10
Subunit of U3-containing Small Subunit (SSU) processome complex; involved in production of 18S rRNA and assembly of small ribosomal subunit; disrupts silencing when overproduced -1.5

MSH5
Protein of the MutS family; forms a dimer with Msh4p that facilitates crossovers between homologs during meiosis -1.1

RTN2
Reticulon protein; involved in nuclear pore assembly and maintenance of tubular ER morphology; promotes membrane curvature; regulates the ER asymmetry-induced inheritance block during ER stress; role in ER-derived peroxisomal biogenesis -1.5

FMP45
Integral membrane protein localized to mitochondria; required for sporulation and maintaining sphingolipid content -1.6

ADY3
Protein required for spore wall formation; mediates assembly of the LEP complex, formation of the ring-like structure via interaction with spindle pole body components and prospore membrane maturation -1.6

GAL3
Transcriptional regulator; involved in activation of the GAL genes in response to galactose -1.1

RAD61
Subunit of a complex that inhibits sister chromatid cohesion; also negatively regulates chromosome condensation -1.5

PST1
Cell wall protein that contains a putative GPI-attachment site; secreted by regenerating protoplasts; upregulated by cell wall damage via disruption of FKS1

LAM5
Putative sterol transfer protein with a role in intracellular sterol transport, by similarity with Lam6p -0.9

COS4
Endosomal protein involved in turnover of plasma membrane proteins; predicted to have a role in the multivesicular body sorting pathway -0.9

YFL065C
Putative protein of unknown function; induced by treatment with 8-methoxypsoralen and UVA irradiation -1.4

FAR7
Protein involved in recovery from pheromone-induced cell cycle arrest -1.4

YFR016C
Protein of unknown function -1.5

ERJ5
Type I membrane protein with a J domain; required to preserve the folding capacity of the endoplasmic reticulum; loss of the nonessential ERJ5 gene leads to a constitutively induced unfolded protein response -0.9

RIM8
Protein involved in proteolytic activation of Rim101p; part of response to alkaline pH -1.0

YGL081W
Putative protein of unknown function; non-essential gene; interacts genetically with CHS5, a gene involved in chitin biosynthesis -1.1

RRT6
Putative protein of unknown function; non-essential gene identified in a screen for mutants with increased levels of rDNA transcription -1.1

NUT1
Component of the RNA polymerase II mediator complex; mediator is required for transcriptional activation and also has a role in basal transcription -1.0

VID30
Central component of GID Complex, involved in FBPase degradation; required for association of Vid vesicles and actin patches in vacuole import and degradation pathway; shifts the balance of nitrogen metabolism toward glutamate production -1.2

YGL262W
Putative protein of unknown function -1.1

STF2
Protein involved in resistance to desiccation stress; exhibits antioxidant properties, and its overexpression prevents ROS accumulation and apoptosis -1.1 SEC9 t-SNARE protein required for secretory vesicle-plasma membrane fusion -1.7

MTL1
Putative plasma membrane sensor; involved in cell integrity signaling and stress response during glucose starvation and oxidative stress -0.9

MCO32
Putative protein of unknown function -1.6

YGR066C
Putative protein of unknown function -1.8

YGR130C
Component of the eisosome with unknown function -1.6

ECL1
Protein of unknown function; overexpression increases oxygen consumption and respiratory activity while deletion results in reduced oxygen consumption under conditions of caloric restriction -0.9

YHR022C
Putative protein of unknown function; non-essential gene -1.4

RTC3
Protein of unknown function involved in RNA metabolism -1.2

YHR140W
Putative integral membrane protein of unknown function -1.2

KEL1
Protein required for proper cell fusion and cell morphology -1.3

PEX18
Peroxin; peroxisomal protein involved in the import of proteins into the peroxisomal matrix -1.3

VID28
GID Complex subunit, serves as adaptor for regulatory subunit Vid24p; vacuolar import and degradation -0.9

YIL055C
Putative protein of unknown function -1.8

YRB2
Protein of unknown function; involved in nuclear processes of the Ran-GTPase cycle; involved in nuclear protein export; contains Ran Binding Domain and FxFG repeats -1.2

SPO22
Meiosis-specific protein essential for chromosome synapsis; involved in synaptonemal complex assembly and protein sumoylation -1.1

MGA2
ER membrane protein involved in regulation of OLE1 transcription; inactive ER form dimerizes and one subunit is then activated by ubiquitin/proteasome-dependent processing followed by nuclear targeting -0.9

TPH3
Putative protein of unknown function -1.1

PET130
Protein required for respiratory growth -1.4

CHM7
Yeast homolog of human CHMP7, localizes to the endoplasmic reticulum -1.8

NCA3
Protein involved in mitochondrion organization -1.3

MTC1
Protein of unknown function that may interact with ribosomes -0.7

YJL132W
Putative protein of unknown function -1.0

YJL144W
Cytoplasmic hydrophilin essential in desiccation-rehydration process; expression induced by osmotic stress, starvation and during stationary phase -1.2

FMP33
Putative protein of unknown function -1.2

JJJ2
Protein of unknown function -1.6

RCY1
F-box protein involved in recycling endocytosed proteins; involved in recycling plasma membrane proteins internalized by endocytosis -1.0

LSO1
Protein with a potential role in response to iron deprivation -1.0

MHO1
Protein of unknown function -1.1

MNN14
Putative protein of unknown function; non-essential gene -0.9

BIR1
Subunit of chromosomal passenger complex (CPC); required for chromosome bi-orientation and for spindle assembly checkpoint activation upon reduced sister kinetochore tension; relative distribution to shortened microtubules increases upon DNA replication stress -1.5

SGM1
Protein of unknown function; required for wild-type growth rate on galactose and mannose -2.3

YJR149W
Putative protein of unknown function -1.5

YJR154W
Putative protein of unknown function -1.1

DID4
Class E Vps protein of the ESCRT-III complex; required for sorting of integral membrane proteins into lumenal vesicles of multivesicular bodies, and for delivery of newly synthesized vacuolar enzymes to the vacuole, involved in endocytosis -0.6

VPS24
One of four subunits of the ESCRT-III complex; forms an endosomal sorting complex required for transport III (ESCRT-III) subcomplex with Did4p; involved in the sorting of transmembrane proteins into the multivesicular body (MVB) pathway -0.9

BLI1
Subunit of the BLOC-1 complex involved in endosomal maturation -1.1

STB6
Protein that binds Sin3p in a two-hybrid assay -1.4

RRP14
Essential protein, constituent of 66S pre-ribosomal particles; interacts with proteins involved in ribosomal biogenesis and cell polarity -2.3

YJU2
Essential protein required for pre-mRNA splicing -1.3

SBA1
Co-chaperone that binds and regulates Hsp90 family chaperones; involved in protein folding, positive regulation of telomere maintenance via telomerase, negative regulation of DNA binding, and regulation of telomerase activity -0.4

RCI50
Putative protein of unknown function; not required for growth of cells lacking the mitochondrial genome; SWAT-GFP and mCherry fusion proteins localize to the mitochondria -1.1

COY1
Golgi membrane protein with similarity to mammalian CASP; genetic interactions with GOS1 (encoding a Golgi snare protein) suggest a role in Golgi function -1.4

YKR011C
Protein of unknown function; protein abundance increases in response to DNA replication stress -1.0

YKR015C
Putative protein of unknown function -1.1

MIC60
Component of the MICOS complex; involved in mitochondrial cristae formation and the import of proteins into the mitochondrial intermembrane space -0.7

YKR023W
Putative protein of unknown function; is detected in highly purified mitochondria in high-throughput studies -1.6

YKR041W
Protein of unknown function; localizes to the mitotic spindle; its overexpression affects endocytic protein trafficking -1.3

THI73
Putative plasma membrane permease; proposed to be involved in carboxylic acid uptake and repressed by thiamine -1.9

SNF7
One of four subunits of the ESCRT-III complex; involved in ubiquitin-dependent protein catabolism via the multivesicular body sorting pathway, late endosome to vacuole transport, intralumenal vesicle formation, ATP export, and the cellular response to anoxia -1.0

OSW2
Protein of unknown function reputedly involved in spore wall assembly -1.0

GIS3
Protein of unknown function -1.3

AVL9
Conserved protein involved in exocytic transport from the Golgi -1.5

YLR149C
Protein of unknown function; non-essential gene; null mutation results in a decrease in plasma membrane electron transport; protein abundance increases in response to DNA replication stress -1.1

YLR179C
Protein of unknown function with similarity to Tfs1p -0.7

YLR225C
Putative protein of unknown function; non-essential gene -1.0

CMG1
Putative protein of unknown function -2.0

IMH1
Protein involved in vesicular transport -1.5

YML003W
Putative protein of unknown function -1.2

ITT1
Protein that modulates the efficiency of translation termination -1.1

COG8
Component of the conserved oligomeric Golgi complex; involved in vesicle-mediated transport within the Golgi and CVT pathway -1.0

DDR2
Multi-stress response protein; expression is activated by a variety of xenobiotic agents and environmental or physiological stresses -1.0

IZH4
Membrane protein involved in zinc ion homeostasis; possible role in sterol metabolism; protein increases in abundance and relocalizes from nucleus to ER upon DNA replication stress

PEP12
Target membrane receptor (t-SNARE); for vesicular intermediates traveling between the Golgi apparatus and the vacuole; controls entry of biosynthetic, endocytic, and retrograde traffic into the prevacuolar compartment; syntaxin -0.9 TMC1 AN1-type zinc finger protein, effector of proteotoxic stress response -0.6

SLD7
Protein with a role in chromosomal DNA replication -1.5

YOR097C
Putative protein of unknown function; non-essential gene -1.1

SIA1
Protein of unassigned function; involved in activation of the Pma1p plasma membrane H+-ATPase by glucose -1.1

SLK19
Kinetochore-associated protein; equired for chromosome segregation and kinetochore clustering; required for normal segregation of chromosomes in meiosis and mitosis -2.2

FIT2
Mannoprotein that is incorporated into the cell wall; involved in the retention of siderophore-iron in the cell wall -0.6

FIT3
Mannoprotein that is incorporated into the cell wall; involved in the retention of siderophore-iron in the cell wall -1.0

YOR389W
Putative protein of unknown function; expression regulated by copper levels -1.5

ATG29
Autophagy-specific protein; involved in macroautophagy, late nucleophagy, mitochondrion degradation and piecemeal microautophagy of nucleus; required for recruiting other ATG proteins to the pre-autophagosomal structure (PAS) -1.3

YPL191C
Protein of unknown function -1.2

APL5
Delta adaptin-like subunit of the clathrin associated protein complex -1.1

YPL247C
Putative protein of unknown function -1.1

YPL277C
Putative protein of unknown function; gene expression regulated by copper levels -1.5

YPL278C
Putative protein of unknown function; gene expression regulated by copper levels -1.1

THP3
Protein that may have a role in transcription elongation; possibly involved in splicing based on pre-mRNA accumulation defect for many intron-containing genes -1.1