DataSheet2_Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.xlsx

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.</p

DataSheet1_Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.docx

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.</p

MOESM10 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 10: Table S9. Assignments of plasmid genes to expression clusters identified by inter-center expression meta-analysis (see Additional file 1: Figure S3)

MOESM5 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 5: Table S4. Plasmid gene RNA-Seq counts

MOESM1 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 1: Figure S1. Completion of plasmid sequences by primer walking with a list of the primers used for each plasmid (A). PCR amplification of ZM4 chromosome region containing a 2.4-kb fragment near ZMO0133 locus that is absent in previously reported ZM4 genome sequence (B). A schematic is shown detailing the location of primers used to PCR, and PCR products on agarose gel are also shown. Figure S2. Customized rRNA depletion kit was developed with Life Technologies for Z. mobilis mRNA enrichment, and RNA-Seq result of the percentage of rRNA, tRNA, and mRNA in Z. mobilis total RNA was calculated (A). qRT-PCR measurement of rRNA content before and after rRNA depletion of total RNA using the customized kit (B). rRNA reduction is reported as the fold change in the target rRNA in total RNA relative to depleted RNA. Measurements were collected in WT (Z. mobilis strain 33C derived from Z. mobilis 8b) and MT (a mutant strain of 33C) grown in either rich media with 5% glucose (RMG) or rich media with 5% xylose (RMX) and collected in two biological replicates. Error is reported as standard deviation. Residual rRNA contamination and rRNA depletion efficiency of samples described in (B) was detected by RNA-Seq (C). Error is reported as standard deviation. An example of pairwise replicate correlation of RNA-Seq pseudo read counts (i.e. log2 transformed following addition of La Place constant of 1) for two biological replicates after rRNA depletion (D). Figure S3. Heatmap of RNA-Seq data from 6% and 9% ACSH, anaerobic (AN) and aerobic (AE) conditions. Coloring by condition (left color bar) corresponds to the one used for the Fig. 4. Blue, NREL, fermentor with biomass hydrolysates; black, NREL, flasks with rich RMG medium; light grey, GLBRC, 6% ACSH; Orange, GLBRC, 9% ACSH; light green, Univ. Athens, anaerobic; dark green, Univ. Athens (UA), anaerobic; dark green, UA, aerobic. Top index bar shows expression clusters (see Additional file 6: Table S5 for gene-cluster assignments). Right annotation bar shows generalized factor that is applicable to experimental designs across all the 3 research centers: “Early” and “Late” are growth stages

MOESM6 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 6: Table S5. Comparison of RNA-Seq from 6% and 9% ACSH

MOESM9 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 9: Table S8. Proteomic data of ZM4 in anaerobic and aerobic fermentation in rich and minimal media

MOESM7 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 7: Table S6. Comparison of RNA-Seq from anaerobic and aerobic conditions

MOESM8 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 8: Table S7. Proteomic data of ZM4 in ethanol shock and sodium acetate stress conditions

MOESM4 of Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032

Additional file 4: Table S3. RNA-Seq metadata