Water-soluble phenolic compounds produced from extractive ammonia pretreatment exerted binary inhibitory effects on yeast fermentation using synthetic hydrolysate

<div><p>Biochemical conversion of lignocellulosic biomass to liquid fuels requires pretreatment and enzymatic hydrolysis of the biomass to produce fermentable sugars. Degradation products produced during thermochemical pretreatment, however, inhibit the microbes with regard to both ethanol yield and cell growth. In this work, we used synthetic hydrolysates (SynH) to study the inhibition of yeast fermentation by water-soluble components (WSC) isolated from lignin streams obtained after extractive ammonia pretreatment (EA). We found that SynH with 20g/L WSC mimics real hydrolysate in cell growth, sugar consumption and ethanol production. However, a long lag phase was observed in the first 48 h of fermentation of SynH, which is not observed during fermentation with the crude extraction mixture. Ethyl acetate extraction was conducted to separate phenolic compounds from other water-soluble components. These phenolic compounds play a key inhibitory role during ethanol fermentation. The most abundant compounds were identified by Liquid Chromatography followed by Mass Spectrometry (LC-MS) and Gas Chromatography followed by Mass Spectrometry (GC-MS), including coumaroyl amide, feruloyl amide and coumaroyl glycerol. Chemical genomics profiling was employed to fingerprint the gene deletion response of yeast to different groups of inhibitors in WSC and AFEX-Pretreated Corn Stover Hydrolysate (ACSH). The sensitive/resistant genes cluster patterns for different fermentation media revealed their similarities and differences with regard to degradation compounds.</p></div

Correlation between chemical genomic profiles of SynH control media, ACSH and SynH+WSC.

<p>Chemical genomics is the study of chemical compound interactions with specific genes within an organism. This approach determined whether hydrolysate variability existed using a biological ‘‘sensor” (individual gene mutants) to create a genome-wide, biological ‘‘fingerprint” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194012#pone.0194012.ref014" target="_blank">14</a>]. In this study, we combined chemical genomics profiling with SynH, therefore determined both hydrolysate variability and gene fingerprints. This is a high-throughput method to test different compounds for their inhibitory effects, which can be widely applied in fermentation study and media development.</p

Fermentation performance of Y128 using different WSC fractions.

<p><b>Here,</b> (A) Glucose consumption; (B) Xylose consumption; (C) Ethanol production and (D) Cell growth OD₆₀₀. ACSH: AFEX corn stover hydrolysate; SynH-W: SynH with 20 g/L water phase extract after ethyl acetate-water partitioning; SynH-P: SynH with 20 g/L ethyl acetate phase extract after ethyl acetate-water partitioning; SynH-WSC: SynH with 20 g/L WSC; SynH-Control: SynH-base media with no inhibitors added. Both phenolic compounds and nutrient components were re-dissolved in SynH-base media at 20 g/L. Fermentations were conducted in Erlenmeyer flasks (50 mL at pH 4.8, 30 °C and 150 RPM with inoculum at 0.8 OD₆₀₀.</p

Chemical genomic profiling of WSC on using a yeast deletion strain library.

<p>(A). Chemical genomic profile of SynH+WSC, with the top sensitive deletion mutants (red) and top resistant mutants (green) highlighted (mean profile n = 3); (B). Gene clusters correlation between SynH, ACSH and SynH+WSC (n = 3). Mutants in <i>ERJ1</i> (involved in ER protein folding), <i>PDX1</i> (a subunit of the mitochondrial dehydrogenase complex), and <i>GOS1</i> (involved Golgi transport) were especially sensitive to WSC. The sensitive genes gave insight into the mechanism of toxicity, confirming that cell membranes were the likely target of WSC toxicity; and overexpression of the sensitive genes could be used to confer resistance. Comparing the chemical genetic profiles between different hydrolysates and fermentation media, we found that the profile of SynH + WSC exhibited a high correlation with that of ACSH (Fig 6B, R = 0.69), and showed greater similarity to ACSH compared to SynH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194012#pone.0194012.g007" target="_blank">Fig 7</a>). The strong correlation suggests that the degradation compounds in WSC can represent the real inhibitors in ACSH to a large extent.</p

Methodology and mass balance of water extraction and ethyl acetate extraction of crude lignin.

<p>Methodology and mass balance of water extraction and ethyl acetate extraction of crude lignin.</p

Water soluble aromatic compounds isolated from EA crude lignin stream using ethyl acetate.

<p>Here, (A) Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and (B) gas chromatography-mass spectrometry (GC-MS) chromatograms were used to identify phenolic compounds.</p

Fermentation performance of Y128 under varying concentrations of WSC.

<p>(A) Glucose consumption; (B), Xylose consumption; (C), Ethanol production and (D), Cell growth OD₆₀₀. Syn-4WSC: SynH with 40 g/L WSC added; SynH-3WSC: SynH with 30 g/L WSC added; SynH 2WSC: SynH with 20 g/L WSC added; SynH-1WSC: SynH with 10 g/L WSC added; SynH-Control: SynH-base media with no inhibitors added. Fermentation was conducted in Erlenmeyer flasks (50 mL) at pH 4.8, 30 °C and 150 RPM with inoculum at 2 (OD₆₀₀).</p

Array-Comparative Genome Hybridization (aCGH) caryoscopes of Chromosome 14 rearrangement seen in three independently-evolved F1 hybrids.

<p>Along the top is shown the gene map of a “zoomed-in” 10 Kb portion of Chromosome 14 (from coordinates 355,000 to 365,000) corresponding to the <i>MEP2</i> rearrangement region. The aCGH data are shown for 200-generation evolved clones isolated from each independent vessel: GSY2532 from Vessel A, GSY2533 from Vessel B, and GSY2535 from Vessel C. The aCGH data shown are for the entire chromosome 14, with data shown separately for the <i>S. cerevisiae</i> and the <i>S. uvarum</i> chromosomes. Bars along the chromosome represent red∶green log ratios, with length of the bar proportional to the value of the log ratio. Red bars indicate positive log ratios (i.e., the presence and/or amplification of the genomic region corresponding to that probe) and green bars indicate negative log ratios (i.e., the depletion or deletion of the genomic region corresponding to that probe). The scale to the left indicates how the height of the bars corresponds to log ratio. Black vertical bars in the <i>S. cerevisiae</i> chromosomes correspond to their centromeres (the location of the <i>S. uvarum</i> centromeres has not been determined but is thought to be similar to that of <i>S. cerevisiae</i>).</p

Recurrent Rearrangement during Adaptive Evolution in an Interspecific Yeast Hybrid Suggests a Model for Rapid Introgression

<div><p>Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created <i>de novo</i> interspecific yeast hybrids between two diverged but largely syntenic <i>Saccharomyces</i> species, <i>S. cerevisiae</i> and <i>S. uvarum</i>, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow ∼1-kb region of chromosome 14, and all producing an “interspecific fusion junction” within the <i>MEP2</i> gene coding sequence, such that the 5′ portion derives from <i>S. cerevisiae</i> and the 3′ portion derives from <i>S. uvarum</i>. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of <i>S. uvarum</i> into an otherwise intact <i>S. cerevisiae</i> chromosome 14, while the homeologous <i>S. uvarum</i> chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within <i>MEP2</i>, yielding a chimaeric chromosome; these events result in the presence in the cell of two <i>MEP2</i> fusion genes having identical breakpoints. Given that <i>MEP2</i> encodes for a high-affinity ammonium permease, that <i>MEP2</i> fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying <i>MEP2</i> fusion genes are each more fit than their common ancestor, the novel <i>MEP2</i> fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the <i>MEP2</i> rearrangement suggests a model for rapid introgression, a phenomenon seen in numerous eukaryotic phyla, that does not require repeated backcrossing to one of the parental species.</p> </div

Further analysis of <i>MEP2</i> gene fusion rearrangements.

<p>(A) Depth of coverage plots from whole genome sequence of three independently evolved F1 hybrids. All panels show read coverage data from whole genome sequencing for the 20 kb region surrounding the <i>MEP2</i> gene on chromosome 14 for both <i>S. cerevisiae</i> and <i>S. uvarum</i> (with chromosomal coordinates shown below), such that the start codon of <i>MEP2</i> is precisely aligned between the two species. GSY86 is the ancestral unevolved F1 hybrid and GSY2532, GSY2533 and GSY2535 are 200-generation evolved clones isolated from Vessels A, B, and C, respectively. The lower plots show ancestor-normalized log-ratio values for the evolved clones, with the start and stop codon boundaries of the <i>MEP2</i> gene shown as dotted lines and the gene itself shown as a black arrow. In GSY86 there was no coverage for a small section of the <i>S. uvarum</i> genome upstream of the MEP2 start codon (upper left plot); this region coincides with the junction of two contigs in the original <i>S. uvarum</i> assembly. Based on our Sanger sequencing of the region, the lack of coverage likely corresponds to a small misassembly in the sequence we used as the reference genome. To avoid a divide by zero error, no log ratio data were calculated for this region, yielding a small “gap” in the <i>S. uvarum</i> log ratio plots. (B) Structure of <i>MEP2</i> region rearrangement found in three independently evolved F1 hybrids by whole genome sequencing. Schematic representation of the genome configuration of the <i>MEP2</i> fusion region for the <i>S. cerevisiae</i> and <i>S. uvarum</i> chromosome 14 s as found in the three evolved clones; thin black line = <i>S. cerevisiae</i> genomic sequences, thick light blue line = <i>S. uvarum</i> genomic sequences; arrowed box = coding region of the <i>MEP2</i> gene. Observed copy numbers for the <i>S. cerevisiae</i> (Sc) and <i>S. uvarum</i> (Su) genomic sequences across the junction region are indicated above. (C) Locations of <i>MEP2</i> gene fusion junctions found by targeted sequencing in multiple clones from independent evolved populations. The entire Mep2 protein is shown to scale, with signal peptide shown as labeled light green box on left; the 11 transmembrane domains are shown as black horizontal bars below. Vertical bars show the location of all characterized junctions; the width of each bar is to scale for the region of shared identity between the two species found at the particular junction. Green vertical bars show junctions found in Vessel A, orange for Vessel B, and yellow for Vessel C (note that multiple clones from Vessels A and B were characterized, compared with only one clone from Vessel C). The half green-half orange bar represents a junction found in both Vessels A and B. Junction positions of the whole-genome-sequenced clones GSY2532, 2533 and 2535 are indicated.</p