OGDH and Bcl-xL loss causes synthetic lethality in glioblastoma.

Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the α-ketoglutarate dehydrogenase (OGDH) gene, which encodes an enzyme that is part of the tricarboxylic acid (TCA) cycle, as essential for GBM growth. Moreover, by combining transcriptome and metabolite screening analyses, we discovered that loss of function of OGDH by the clinically validated drug compound CPI-613 was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3 mimetic, ABT263) in patient-derived xenografts as well neurosphere GBM cultures. CPI-613-mediated energy deprivation drove an integrated stress response with an upregulation of the BH3-only domain protein, Noxa, in an ATF4-dependent manner, as demonstrated by genetic loss-of-function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with disruption of the TCA cycle might be a treatment strategy for GBM.

Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity.

Fusobacterium nucleatum (Fn) is a Gram-negative oral commensal, prevalent in various human diseases. It is unknown how this common commensal converts to a rampant pathogen. We report that Fn secretes an adhesin (FadA) with amyloid properties via a Fap2-like autotransporter to enhance its virulence. The extracellular FadA binds Congo Red, Thioflavin-T, and antibodies raised against human amyloid ß42. Fn produces amyloid-like FadA under stress and disease conditions, but not in healthy sites or tissues. It functions as a scaffold for biofilm formation, confers acid tolerance, and mediates Fn binding to host cells. Furthermore, amyloid-like FadA induces periodontal bone loss and promotes CRC progression in mice, with virulence attenuated by amyloid-binding compounds. The uncleaved signal peptide of FadA is required for the formation and stability of mature amyloid FadA fibrils. We propose a model in which hydrophobic signal peptides serve as "hooks" to crosslink neighboring FadA filaments to form a stable amyloid-like structure. Our study provides a potential mechanistic link between periodontal disease and CRC and suggests anti-amyloid therapies as possible interventions for Fn-mediated disease processes.

A short-chain dehydrogenase plays a key role in cellulosic D-lactic acid fermentability of Pediococcus acidilactici.

Phenolic aldehydes from lignocellulose pretreatment are strong inhibitors of cell growth and metabolism of cellulosic lactic acid bacteria. Their low solubility and recalcitrance highly reduce the removal efficiency of various detoxification methods. This study shows a simultaneous conversion of phenolic aldehydes and fermentation of D-lactic acid by Pediococcus acidilactici using corn stover feedstock. Vanillin was found to be the strongest phenolic aldehyde inhibitor to P. acidilactici. The overexpression of a short-chain dehydrogenase encoded by the gene CGS9114_RS09725 from Corynebacterium glutamicum was identified to play a key role in D-lactic acid fermentability of P. acidilactici. The engineered P. acidilactici with the genome integration of CGS9114_RS09725 showed the accelerated vanillin reduction and improved cellulosic D-lactic acid production. This study reveals that vanillin conversion is crucial for D-lactic acid fermentation, and the direct expression of a specific vanillin reduction gene in lactic acid bacterium efficiently improves cellulosic D-lactic acid production.

Mechanism of Tolerance to the Lignin-Derived Inhibitor p-Benzoquinone and Metabolic Modification of Biorefinery Fermentation Strains.

p-Benzoquinone (BQ) is a lignin-derived inhibitor of biorefinery fermentation strains produced during pretreatment of lignocellulose. Unlike the well-studied inhibitors furan aldehydes, weak acids, and phenolics, the inhibitory properties of BQ, the microbial tolerance mechanism, and the detoxification strategy for this inhibitor have not been clearly elucidated. Here, BQ was identified as a by-product generated during acid pretreatment of various lignocellulose feedstocks, including corn stover, wheat straw, rice straw, tobacco stem, sunflower stem, and corncob residue. BQ at 20 to 200 mg/liter severely inhibited the cell growth and fermentability of various bacteria and yeast strains used in biorefinery fermentations. The BQ tolerance of the strains was found to be closely related to their capacity to convert BQ to nontoxic hydroquinone (HQ). To identify the key genes responsible for BQ tolerance, transcription levels of 20 genes potentially involved in the degradation of BQ in Zymomonas mobilis were investigated using real-time quantitative PCR in BQ-treated cells. One oxidoreductase gene, one hydroxylase gene, three reductase genes, and three dehydrogenase genes were found to be responsible for the conversion of BQ to HQ. Overexpression of the five key genes in Z. mobilis (ZMO1696, ZMO1949, ZMO1576, ZMO1984, and ZMO1399) accelerated its cell growth and cellulosic ethanol production in BQ-containing medium and lignocellulose hydrolysates.IMPORTANCE This study advances our understanding of BQ inhibition behavior and the mechanism of microbial tolerance to this inhibitor and identifies the key genes responsible for BQ detoxification. The insights here into BQ toxicity and tolerance provide the basis for future synthetic biology to engineer industrial fermentation strains with enhanced BQ tolerance.

Expressing an oxidative dehydrogenase gene in ethanologenic strain Zymomonas mobilis promotes the cellulosic ethanol fermentability.

Phenolic aldehydes from lignocellulose pretreatment harshly inhibit the viability and metabolism of ethanol fermenting strains. Direct conversion of phenolic aldehydes is usually incomplete due to their low water solubility and recalcitrance to bioconversion. Here we consolidated phenolic aldehydes bioconversion and ethanol fermentation in a typical ethanologenic bacterium Zymomonas mobilis by constructing an intracellular oxidative pathway. The gene PP_2680 encoding NAD+-dependent aldehyde dehydrogenase from Pseudomonas putida KT2440 was expressed in Z. mobilis ZM4. The expression significantly improved both aldehyde inhibitor conversion and ethanol fermentability in corn stover hydrolysate. The purified PP_2680 aldehyde dehydrogenase showed strong in vitro oxidative capacity on phenolic aldehydes and its in vivo expression significantly up-regulated the key genes in the ED pathway and the oxidative phosphorylation. This study provided an important concept of simultaneous biodetoxification and fermentation in ethanologenic strains for the improvement of ethanol fermentability.

Heterozygous diploid structure of Amorphotheca resinae ZN1 contributes efficient biodetoxification on solid pretreated corn stover.

BACKGROUND: Fast, complete, and ultimate removal of inhibitory compounds derived from lignocellulose pretreatment is the prerequisite for efficient production of cellulosic ethanol and biochemicals. Biodetoxification is the most promising method for inhibitor removal by its unique advantages. The biodetoxification mechanisms of a unique diploid fungus responsible for highly efficient biodetoxification in solid-state culture was extensively investigated in the aspects of cellular structure, genome sequencing, transcriptome analysis, and practical biodetoxification. RESULTS: The inborn heterozygous diploid structure of A. resinae ZN1 uniquely contributed to the enhancement of inhibitor tolerance and conversion. The co-expression of gene pairs contributed to the enhancement of the degradation of lignocellulose-derived model inhibitors. The ultimate inhibitors degradation pathways and sugar conservation were elucidated by microbial degradation experimentation as well as the genomic and transcriptomic sequencing analysis. CONCLUSIONS: The finding of the heterozygous diploid structure in A. resinae ZN1 on biodetoxification took the first insight into the global overview of biodetoxification mechanism of lignocellulose-derived inhibitors. This study provided a unique and practical biodetoxification biocatalyst of inhibitor compounds for lignocellulose biorefinery processing, as well as the synthetic biology tools on biodetoxification of biorefinery fermenting strains.

Tolerance and transcriptional analysis of Corynebacterium glutamicum on biotransformation of toxic furaldehyde and benzaldehyde inhibitory compounds.

Furaldehydes and benzaldehydes are among the most toxic inhibitors from lignocellulose pretreatment on microbial growth and metabolism. The bioconversion of aldehyde inhibitors into less toxic alcohols or acids (biotransformation) is the prerequisite condition for efficient biorefinery fermentations. This study found that Corynebacterium glutamicum S9114 demonstrated excellent tolerance and biotransformation capacity to five typical aldehyde inhibitors including two furaldehydes: 2-furaldehyde (furfural), 5-(hydroxymethyl)-2-furaldehyde, and three benzaldehydes: 4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin), and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde). Transcription levels of 93 genes hypothesized to be responsible for five aldehydes biotransformation were examined by qRT-PCR. Multiple genes showed significantly up-regulated expression against furaldehydes or benzaldehydes. Overexpression of CGS9114_RS01115 in C. glutamicum resulted in the increased conversion of all five aldehyde inhibitors. The significant oxidoreductase genes responsible for each or multiple inhibitors biotransformation identified in this study will serve as a component of key gene device library for robust biorefinery fermentation strains development in the future biorefinery applications.

Converting lignin derived phenolic aldehydes into microbial lipid by Trichosporon cutaneum.

Lignin is one of the major components of lignocellulose biomass and chemically degrades into phenolic aldehydes including 4-hydroxybenzaldehyde, vanillin, and syringaldehyde. No lipid accumulation from the phenolic aldehydes by oleaginous microbes had been succeeded. Compared with vanillin and syringaldehyde, T. cutaneum ACCC 20271 have better tolerance to 4-hydroxybenzaldehyde. 4-Hydroxybenzaldehyde was found to be able as the substrate for lipid accumulation, while vanillin and syringaldehyde were only converted to less toxic phenolic alcohols and acids without observable lipid accumulation, perhaps due to the space shelling of methoxyl group(s) in the structures. A long term fed batch fermentation of 4-hydroxybenzaldehyde accumulated 0.85 g L-1 of lipid, equivalent to 0.039 g lipid per gram of 4-hydroxybenzaldehyde substrate, approximately 3.7 folds greater than the control without 4-hydroxybenzaldehyde addition. The fatty acid composition well met the need for biodiesel synthesis. The preliminary pathway from 4-hydroxybenzaldehyde to lipid was predicted. This study took the first experimental trial on utilizing phenolic aldehydes as the sole carbon sources for microbial lipid accumulation by T. cutaneum ACCC 20271.

Rich biotin content in lignocellulose biomass plays the key role in determining cellulosic glutamic acid accumulation by Corynebacterium glutamicum.

BACKGROUND: Lignocellulose is one of the most promising alternative feedstocks for glutamic acid production as commodity building block chemical, but the efforts by the dominant industrial fermentation strain Corynebacterium glutamicum failed for accumulating glutamic acid using lignocellulose feedstock. RESULTS: We identified the existence of surprisingly high biotin concentration in corn stover hydrolysate as the determining factor for the failure of glutamic acid accumulation by Corynebacterium glutamicum. Under excessive biotin content, induction by penicillin resulted in 41.7 ± 0.1 g/L of glutamic acid with the yield of 0.50 g glutamic acid/g glucose. Our further investigation revealed that corn stover contained 353 ± 16 µg of biotin per kg dry solids, approximately one order of magnitude greater than the biotin in corn grain. Most of the biotin remained stable during the biorefining chain and the rich biotin content in corn stover hydrolysate almost completely blocked the glutamic acid accumulation. This rich biotin existence was found to be a common phenomenon in the wide range of lignocellulose biomass and this may be the key reason why the previous studies failed in cellulosic glutamic acid fermentation from lignocellulose biomass. The extended recording of the complete members of all eight vitamin B compounds in lignocellulose biomass further reveals that the major vitamin B members were also under the high concentration levels even after harsh pretreatment. CONCLUSIONS: The high content of biotin in wide range of lignocellulose biomass feedstocks and the corresponding hydrolysates was discovered and it was found to be the key factor in determining the cellulosic glutamic acid accumulation. The highly reserved biotin and the high content of their other vitamin B compounds in biorefining process might act as the potential nutrients to biorefining fermentations. This study creates a new insight that lignocellulose biorefining not only generates inhibitors, but also keeps nutrients for cellulosic fermentations.

Improving cellulosic ethanol fermentability of Zymomonas mobilis by overexpression of sodium ion tolerance gene ZMO0119.

Inhibition of sodium ion (Na+) on Zymomonas mobilis represents an important obstacle for efficient cellulosic ethanol production. This study screened and overexpressed the genes responsible for transporting metal ions in Z. mobilis for increasing its Na+ tolerance. The ZMO0119 gene encoding Na+/H+ antiporter was identified to be highly effective for reducing intracellular Na+ concentration of Z. mobilis by improving the Na+ transport capacity. Overexpression of ZMO0119 gene in Z. mobilis significantly accelerated the cell growth, glucose consumption, and cellulosic ethanol production from the dry acid pretreated and biodetoxified corn stover feedstock. This study provided an important gene responsible for increasing the cellulosic ethanol fermentability by Z. mobilis.

Tolerance response and metabolism of acetic acid by biodetoxification fungus Amorphotheca resinae ZN1.

Removal of acetic acid from pretreated lignocellulose biomass is an important step for the consequent fermentation on production of cellulosic ethanol and biobased chemicals. This study elucidates the biological metabolism and tolerance response of acetic acid by a widely used biodetoxification fungus Amorphotheca resinae ZN1. Acetic acid is consumed as a prior substrate to glucose and xylose by A. resinae ZN1, and the consumption is highly accelerated by solid state culture. Acetic acid is metabolized through the tricarboxylic acid (TCA) cycle when glucose exists in the medium, while through the two cycles of both the TCA cycle and glyoxylate cycle when there is no sugar in the medium. The tolerance response of A. resinae ZN1 to acetic acid includes various biological processes such as activation of ions transport, increase in amino acids uptake and biosynthesis, as well as induction of ergosterol biosynthesis and ATP generation. The study provided important basis for the future biodetoxification strain modification for enhanced acetic acid removal.

Tolerance improvement of Corynebacterium glutamicum on lignocellulose derived inhibitors by adaptive evolution.

Robustness of fermenting strains to lignocellulose derived inhibitors is critical for efficient biofuel and biochemical productions. In this study, the industrial fermenting strain Corynebacterium glutamicum S9114 was evolved for improved inhibitor tolerance using long-term adaptive evolution by continuously transferring into the inhibitors containing corn stover hydrolysate every 24 h, and finally a stably evolved C. glutamicum was obtained after 128 days of serial transfers. The evolved strain exhibited the highly increased conversion rate to the typical lignocellulose derived inhibitors including furfural, 5-hydroxymethylfurfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, and acetic acid. Glucose consumption was obviously accelerated, and 22.4 g/L of glutamic acid was achieved in the corn stover hydrolysate, approximately 68.4% greater than that by the original strain. Whole genome re-sequencing revealed various mutations with the potential connection to the improved performance of the evolved strain. Transcriptional analysis further demonstrated that the glucose-PTS transport and the pentose phosphate pathway were significantly upregulated in the evolved strain, which very likely contributed to the accelerated glucose consumption, as well as sufficient NAD(P)H supply for aldehyde inhibitors reduction conversion and thus enhanced the inhibitor tolerance. This study provided important experimental evidences and valuable genetic information for robust strain construction and modification in lignocellulose biorefining processes.

Engineering Pediococcus acidilactici with xylose assimilation pathway for high titer cellulosic l-lactic acid fermentation.

Xylose-assimilating pathways were constructed in the parental Pediococcus acidilactici strain and evolutionarily adapted to yield a highly stable co-fermentation strain for l-lactic acid production. The phosphoketolase pathway (PK) was blocked for reduction of acetic acid generation by disrupting phosphoketolase (pkt) gene. The pentose phosphate pathway (PPP) was reconstructed for xylose assimilation by integrating four heterologous genes encoding transketolase (tkt), transaldolase (tal), xylose isomerase (xylA) and xylulokinase (xylB) into the P. acidilactici chromosome. The xylose-assimilating ability of the constructed strain was significantly improved by long term adaptive evolution. The engineered strain was applied to the simultaneous saccharification and co-fermentation (SSCF) under high solids loading of wheat straw. The l-lactic acid titer, productivity and xylose conversion reached the record high at 130.8±1.6g/L, 1.82±0.0g/L/h, and 94.9±0.0%, respectively. This study provided an important strain and process prototype for production of high titer cellulosic l-lactic acid.

Constructing xylose-assimilating pathways in Pediococcus acidilactici for high titer d-lactic acid fermentation from corn stover feedstock.

Xylose-assimilating pathway was constructed in a d-lactic acid producing Pediococcus acidilactici strain and evolutionary adapted to yield a co-fermentation strain P. acidilactici ZY15 with 97.3g/L of d-lactic acid and xylose conversion of 92.6% obtained in the high solids content simultaneous saccharification and co-fermentation (SSCF) of dry dilute acid pretreated and biodetoxified corn stover feedstock. The heterologous genes encoding xylose isomerase (xylA) and xylulokinase (xylB) were screened and integrated into the P. acidilactici chromosome. The metabolic flux to acetic acid in phosphoketolase pathway was re-directed to pentose phosphate pathway by substituting the endogenous phosphoketolase gene (pkt) with the heterologous transketolase (tkt) and transaldolase (tal) genes. The xylose-assimilating ability of the newly constructed P. acidilactici strain was significantly improved by adaptive evolution. This study provided an important strain and process prototype for high titer d-lactic acid production from lignocellulose feedstock with efficient xylose assimilation.

Enhancement of furan aldehydes conversion in Zymomonas mobilis by elevating dehydrogenase activity and cofactor regeneration.

BACKGROUND: Furfural and 5-hydroxymethylfurfural (HMF) are the two major furan aldehyde inhibitors generated from lignocellulose dilute acid pretreatment which significantly inhibit subsequent microbial cell growth and ethanol fermentation. Zymomonas mobilis is an important strain for cellulosic ethanol fermentation but can be severely inhibited by furfural and (or) HMF. Previous study showed that Z. mobilis contains its native oxidoreductases to catalyze the conversion of furfural and HMF, but the corresponding genes have not been identified. RESULTS: This study identified a NADPH-dependent alcohol dehydrogenase gene ZMO1771 from Z. mobilis ZM4, which is responsible for the efficient reduction of furfural and HMF. Over-expression of ZMO1771 in Z. mobilis significantly increased the conversion rate to both furfural and HMF and resulted in an accelerated cell growth and improved ethanol productivity in corn stover hydrolysate. Further, the ethanol fermentation performance was enhanced again by co-expression of the transhydrogenase gene udhA with ZMO1771 by elevating the NADPH availability. CONCLUSIONS: A genetically modified Z. mobilis by co-expressing alcohol dehydrogenase gene ZMO1771 with transhydrogenase gene udhA showed enhanced conversion rate of furfural and HMF and accelerated ethanol fermentability from lignocellulosic hydrolysate. The results presented in this study provide an important method on constructing robust strains for efficient ethanol fermentation from lignocellulose feedstock.

Inhibitor degradation and lipid accumulation potentials of oleaginous yeast Trichosporon cutaneum using lignocellulose feedstock.

Oleaginous yeast Trichosporon cutaneum is robust to high levels of lignocellulose derived inhibitor compounds with considerable lipid accumulation capacity. The potential of lipid accumulation of T. cutaneum ACCC 20271 was investigated using corn stover hydrolysates with varying sugar and inhibitor concentrations. Biodiesel was synthesized using the extracted lipid and the product satisfied the ASTM standards. Among the typical inhibitors, T. cutaneum ACCC 20271 is relatively sensitive to furfural and 4-hydroxybenzaldehyde, but strongly tolerant to high titers of formic acid, acetic acid, levulinic acid, HMF, vanillin, and syringaldehyde. It is capable of complete degradation of formic acid, acetic acid, vanillin and 4-hydroxybenzaldehyde. Finally, the inhibitor degradation pathways of T. cutaneum ACCC 20271 were constructed based on the newly sequenced whole genome information and the experimental results. The study provided the first insight to the inhibitor degradation of T. cutaneum and demonstrated the potentials of lipid production from lignocellulose.

Genome sequence of Trichosporon cutaneum ACCC 20271: An oleaginous yeast with excellent lignocellulose derived inhibitor tolerance.

Oleaginous yeast Trichosporon cutaneum demonstrated excellent lipid accumulation performance and inhibitor tolerance derived from lignocellulose pretreatment. Here we firstly report a 30.45Mb assembly genome of T. cutaneum ACCC 20271 for understanding the microbial lipid biosynthesis and mechanism of inhibitor tolerance and degradation.

Engineering wild-type robust Pediococcus acidilactici strain for high titer L- and D-lactic acid production from corn stover feedstock.

Pediococcus acidilactici TY112 producing L-lactic acid and P. acidilactici ZP26 producing D-lactic acid, were engineered from the wild-type P. acidilactici DQ2 by ldhD or ldh gene disruption, and the robustness of the wild-type strain to the inhibitors derived from lignocellulose pretreatment was maintained well. In simultaneous saccharification and fermentation (SSF), 77.66 g L(-1) of L-lactic acid and 76.76 g L(-1) of D-lactic acid were obtained at 25% (w/w) solids content of dry dilute acid pretreated and biodetoxified corn stover feedstock. L- and D-Lactic acid yield and productivity were highly dependent on the inhibitor removal extent due to the significant down-regulation on the expressions of ldh and ldhD encoding lactate dehydrogenase by inhibitor, especially syringaldehyde and vanillin at the low concentrations. This study provided a prototype of industrial process for high titer L- and D-lactic acid production from lignocellulose feedstock.

Transcriptional analysis of Amorphotheca resinae ZN1 on biological degradation of furfural and 5-hydroxymethylfurfural derived from lignocellulose pretreatment.

BACKGROUND: Furfural and 5-hydroxymethylfurfural (HMF) are the two major inhibitor compounds generated from lignocellulose pretreatment, especially for dilute acid, steam explosion, neutral hot water pretreatment methods. The two inhibitors severely inhibit the cell growth and metabolism of fermenting strains in the consequent bioconversion step. The biodetoxification strain Amorphotheca resinae ZN1 has demonstrated its extraordinary capacity of fast and complete degradation of furfural and HMF into corresponding alcohol and acid forms. The elucidation of degradation metabolism of A. resinae ZN1 at molecular level will facilitate the detoxification of the pretreated lignocellulose biomass and provide the metabolic pathway information for more powerful biodetoxification strain development. RESULTS: Amorphotheca resinae ZN1 was able to use furfural or HMF as the sole carbon source for cell growth. During the detoxification process, A. resinae ZN1 firstly reduced furfural or HMF into furfuryl alcohol or HMF alcohol, and then oxidized into furoic acid or HMF acid through furan aldehyde as the intermediate at low concentration level. The cell mass measurement suggested that furfural was more toxic to A. resinae ZN1 than HMF. In order to identify the degradation mechanism of A. resinae ZN1, transcription levels of 137 putative genes involved in the degradation of furfural and HMF in A. resinae ZN1 were investigated using the real-time quantitative PCR (qRT-PCR) method under the stress of furfural and HMF, as well as the stress of their secondary metabolites, furfuryl alcohol and HMF alcohol. Two Zn-dependent alcohol dehydrogenase genes and five AKR/ARI genes were found to be responsible for the furfural and HMF conversion to their corresponding alcohols. For the conversion of the two furan alcohols to the corresponding acids, three propanol-preferring alcohol dehydrogenase genes, one NAD(P)(+)-depending aldehyde dehydrogenase gene, or two oxidase genes with free oxygen as the substrate were identified under aerobic condition. CONCLUSIONS: The genes responsible for the furfural and HMF degradation to the corresponding alcohols and acids in A. resinae ZN1 were identified based on the analysis of the genome annotation, the gene transcription data and the inhibitor conversion results. These genetic resources provided the important information for understanding the mechanism of furfural and HMF degradation and modification of high tolerant strains used for biorefinery processing.

Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment.

BACKGROUND: Phenolic aldehydes generated from lignocellulose pretreatment exhibited severe toxic inhibitions on microbial growth and fermentation. Numerous tolerance studies against furfural, 5-hydroxymethyl-2-furaldehyde (HMF), acetate, and ethanol were reported, but studies on inhibition of phenolic aldehyde inhibitors are rare. For ethanologenic strains, Zymomonas mobilis ZM4 is high in ethanol productivity and genetic manipulation feasibility, but sensitive to phenolic aldehyde inhibitors. Molecular mechanisms of tolerance for Z. mobilis toward phenolic aldehydes are not known. RESULTS: We took the first insight into genomic response of Z. mobilis ZM4 to the phenolic aldehyde inhibitors derived from lignocellulose pretreatment. The results suggest that the toxicity to cells is caused by the functional group of phenolic aldehyde, similar to furfural and HMF, rather than aromatic groups or phenolic hydroxyl groups. Transcriptome response against 4-hydroxybenzaldehyde, syringaldehyde, and vanillin, representing phenolic groups H, S, and G, respectively, was investigated. The atlas of the important genes responsible for significantly enhanced and repressed genes at the genomic level was illustrated. 272 genes with twofold greater expressions than non-treated controls and 36 gene clusters in response to challenges of these phenolic aldehydes were identified. Several reductases encoded by ZMO1116, ZMO1696, and ZMO1885 were found to play the key roles in reducing phenolic aldehydes into the corresponding phenolic alcohols. Reduction of phenolic aldehydes by overexpression of ZMO1116, ZMO1696, and ZMO1885 in Z. mobilis ZM4 resulted in the increased inhibitor conversion and ethanol productivity, especially for 4-hydroxybenzaldehyde and vanillin. Several transporter genes such as ZMO0282, ZMO0283, ZMO0798, ZMO0799, and ZMO0800 was also displayed significantly increased expressions against the phenolic aldehydes. CONCLUSIONS: The genes encoding reductases are with potentials on phenolic aldehydes-tolerant genes contributing to the reduction of phenolic aldehydes into the corresponding phenolic alcohols forms for Z. mobilis ZM4. Overexpression of the key genes improved the conversion ratio and ethanol productivity of 4-hydroxybenzaldehyde and vanillin with high toxicity. New knowledge obtained from this research aids understanding the mechanisms of bacterial tolerance and the development of the next-generation biocatalysts for advanced biofuels production.