5-Hydroxymethylfurfural mediated developmental toxicity in Drosophila melanogaster.

5-hydroxymethylfurfural is a common byproduct in food. However, its effect on growth and development remains incompletely understood. This study investigated the developmental toxicity of 5-HMF to Drosophila larvae. The growth and development of Drosophila melanogaster fed with 5-50 mM 5-HMF was monitored, and its possible mechanism was explored. It was found that 5-HMF prolonged the developmental cycle of Drosophila melanogaster (25 mM and 50 mM). After 5-HMF intake, the level of reactive oxygen species in the third instar larvae increased by 1.23-1.40 fold, which increased the level of malondialdehyde and caused changes in antioxidant enzymes. Moreover, the nuclear factor erythroid-2 related factor 2 antioxidant signaling pathway and the expression of heat shock protein genes were affected. At the same time, 5-HMF disrupted the glucose and lipid metabolism in the third instar larvae, influencing the expression level of key genes in the insulin signal pathway. Furthermore, 5-HMF led to intestinal oxidative stress, and up-regulated the expression of the pro-apoptotic gene, consequently impacting intestinal health. In short, 5-HMF causes oxidative stress, disturbs glucose and lipid metabolism and induces intestinal damage, damaging related signaling pathways, and ultimately affecting the development of Drosophila melanogaster.

Nonlethal Furfural Exposure Causes Genomic Alterations and Adaptability Evolution in Saccharomyces cerevisiae.

Furfural is a major inhibitor found in lignocellulosic hydrolysate, a promising feedstock for the biofermentation industry. In this study, we aimed to investigate the potential impact of this furan-derived chemical on yeast genome integrity and phenotypic evolution by using genetic screening systems and high-throughput analyses. Our results showed that the rates of aneuploidy, chromosomal rearrangements (including large deletions and duplications), and loss of heterozygosity (LOH) increased by 50-fold, 23-fold, and 4-fold, respectively, when yeast cells were cultured in medium containing a nonlethal dose of furfural (0.6 g/L). We observed significantly different ratios of genetic events between untreated and furfural-exposed cells, indicating that furfural exposure induced a unique pattern of genomic instability. Furfural exposure also increased the proportion of CG-to-TA and CG-to-AT base substitutions among point mutations, which was correlated with DNA oxidative damage. Interestingly, although monosomy of chromosomes often results in the slower growth of yeast under spontaneous conditions, we found that monosomic chromosome IX contributed to the enhanced furfural tolerance. Additionally, terminal LOH events on the right arm of chromosome IV, which led to homozygosity of the SSD1 allele, were associated with furfural resistance. This study sheds light on the mechanisms underlying the influence of furfural on yeast genome integrity and adaptability evolution. IMPORTANCE Industrial microorganisms are often exposed to multiple environmental stressors and inhibitors during their application. This study demonstrates that nonlethal concentrations of furfural in the culture medium can significantly induce genome instability in the yeast Saccharomyces cerevisiae. Notably, furfural-exposed yeast cells displayed frequent chromosome aberrations, indicating the potent teratogenicity of this inhibitor. We identified specific genomic alterations, including monosomic chromosome IX and loss of heterozygosity of the right arm of chromosome IV, that confer furfural tolerance to a diploid S. cerevisiae strain. These findings enhance our understanding of how microorganisms evolve and adapt to stressful environments and offer insights for developing strategies to improve their performance in industrial applications.

Reasons for 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde resistance in Saccharomyces cerevisiae: current state of knowledge and perspectives for further improvements.

Common toxic compounds 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) are formed from dehydration of pentose and hexose, respectively, during decomposition of lignocellulosic biomass polymers. Furfural and HMF represent a major class of aldehyde toxic chemicals that inhibit microbial growth and interfere with subsequent fermentation for production of renewable fuels and chemicals. Understanding mechanisms of yeast tolerance aids development of tolerant strains as the most economic means to overcome the toxicity. This review updates current knowledge on yeast resistance to these toxic chemicals obtained from rapid advances in the past few years. Findings are largely exemplified by an adapted strain NRRL Y-50049 compared with its progenitor, the industrial yeast Saccharomyces cerevisiae type strain NRRL Y-12632. Newly characterized molecular phenotypes distinguished acquired resistant components of Y-50049 from innate stress response of its progenitor Y-12632. These findings also raised important questions on how to address more deeply ingrained changes in addition to local renovations for yeast adaptation. An early review on understandings of yeast tolerance to these inhibitory compounds is available and its contents omitted here to avoid redundancy. Controversial and confusing issues on identification of yeast resistance to furfural and HMF are further clarified aiming improved future research. Propositions and perspectives on research understanding molecular mechanisms of yeast resistance and future improvements are also presented. KEY POINTS: • Distinguished adapted resistance from innate stress response in yeast. • Defined pathway-based molecular phenotypes of yeast resistance. • Proposed genomic insight and perspectives on yeast resistance and adaptation.

Potential neurotoxicity of 5-hydroxymethylfurfural and its oligomers: widespread substances in carbohydrate-containing foods.

5-Hydroxymethylfurfural (5-HMF) is present in numerous carbohydrate-containing consumer products and is readily converted into two oligomers (II and III) by acid-catalyzed transformations. Previous studies have demonstrated various undesirable effects of 5-HMF at relatively high concentrations. In this study, we demonstrate that 5-HMF and its two oligomers exert neurotoxic effects in vivo and in vitro. All three substances blocked the proliferation of PC12 and HT22 cells at the S or G2-M phase in dose- and time-dependent manners. In addition, [Ca2+]i and reactive oxygen species levels were both significantly increased by treatment with these substances at 100 µM, individually, compared with the control group. Although no motor and cognitive deficits are observed, 5-HMF and III can induce anxiety- and depression-like behavior in adolescent mice at administered doses of 0.15 mg kg-1 and 1.5 mg kg-1in vivo, which are close to or less than the reported 24 h dietary intake of 5-HMF in humans. Together, our findings suggest the need for close monitoring of the content of these substances in food, as well as the need for studies on the effects of long-term exposure to them.

HMF causes anaphylactic symptoms by acting as a H1 receptor agonist.

5-Hydroxymethylfurfural (HMF) can readily form by acid-catalyzed transformations of various sugars such as fructose, sucrose and to a lesser degree glucose, and is known to widely exist in various sugar-containing consumer products. Thus the potential health effect of HMF has been a subject of intensive studies. There have been earlier reports of HMF's undesirable effects at or above high micromolar concentrations. In this study, HMF is found to stimulate the H1 receptor in vivo and in vitro. When assessed in cell culture and animal models, HMF was found to cause deformation of in cell culture studies of HUVECs at 50 µM, to increase the vascular permeability of paw skin at 1.0 mg/mL, and trigger symptoms of anaphylaxis in animal models at 32.5 µg/kg. At the molecular level, HMF was found to induce the release of NO and related cytokines, and trigger H1 receptor-mediated inflammatory responses. Mutation studies also suggest the binding sites for HMF on the H1 receptor. The findings described suggest the need for close monitoring of HMF contents in consumer products and their related side effects.

The Toxicological Aspects of the Heat-Borne Toxicant 5-Hydroxymethylfurfural in Animals: A Review.

The incidence of adverse reactions in food is very low, however, some food products contain toxins formed naturally due to their handling, processing and storage conditions. 5-(Hydroxymethyl)-2-furfural (HMF) can be formed by hydrogenation of sugar substances in some of manufactured foodstuffs and honey under elevated temperatures and reduced pH conditions following Maillard reactions. In previous studies, it was found that HMF was responsible for harmful (mutagenic, genotoxic, cytotoxic and enzyme inhibitory) effects on human health. HMF occurs in a wide variety of food products like dried fruit, juice, caramel products, coffee, bakery, malt and vinegar. The formation of HMF is not only an indicator of food storage conditions and quality, but HMF could also be used as an indicator of the potential occurrence of contamination during heat-processing of some food products such as coffee, milk, honey and processed fruits. This review focuses on HMF formation and summarizes the adverse effects of HMF on human health.

Formation and Identification of Two Hydroxmethylfurfural-Glycine Adducts and Their Cytotoxicity and Absorption in Caco-2 Cells.

Our previous research showed that thioacetal and Schiff base formed between 5-hydroxymethylfurfural (HMF) and cysteine or lysine considerably decreased the cytotoxicity of HMF. In this study, two adol condensation adducts, named 2ß-amino-3α-hydroxy-3-(5-(hydroxymethyl)furan-2-yl)propanoic acid (HGA) and 2α-amino-3ß-hydroxy-3-(5-(hydroxymethyl)furan-2-yl)propanoic acid (HGB), were prepared from the reaction products of glycine and HMF, and their cytotoxicities were investigated in Caco-2 cells. Compared with HMF, HGA and HGB displayed lower cytotoxicities against Caco-2 cells with IC50 values of 36.50 and 43.47 mM, respectively, versus 16.11 mM (HMF). In contrast to our findings in thioacetal and Schiff base products, HGA and HGB underwent a very high metabolism rate (99%) in Caco-2 cells. HGA and HGB may degrade to other products instead of HMF since no extracellular or intracellular HMF was detected.

Identification of a 5-Hydroxymethylfurfural-Lysine Schiff Base and Its Cytotoxicity in Three Cell Lines.

5-Hydroxymethylfurfural (HMF) can undergo the Maillard reaction with amino acids. However, the safety of the products remains unknown. In this study, a HMF-lysine Schiff base named (E)-N6-((5'-(hydroxymethyl)furan-2'-yl)methylene)lysine (HML) was identified and detected for the first time in baked foods. HML formation significantly decreased the cytotoxicity (IC50) of HMF against GES-1 cells (81.81 versus 5.02 mM and 73.76 versus 2.94 mM for HML versus HMF at 24 and 48 h, respectively), EA.hy926 cells (86.05 versus 4.85 mM and 77.22 versus 0.71 mM, respectively), and Caco-2 cells (155.77 versus 36.84 mM and 112.70 versus 18.51 mM, respectively). Exposure of Caco-2 cells to HMF at 10.0 mM triggered cell apoptosis of 14.02% (versus 8.54% in the control), whereas exposure to HML at 10-15 mM hardly increased cell apoptosis. Moreover, the absorption capacities of HMF and HML by Caco-2 cells were equivalent (p > 0.05) at 7.23-12.57% after incubation at 2 mM for 30-150 min.

Species-specific transcriptomic responses in Daphnia magna exposed to a bio-plastic production intermediate.

Hydroxymethylfurfural (HMF) is a plant-based chemical building block that could potentially substitute petroleum-based equivalents, yet ecotoxicological data of this compound is currently limited. In this study, the effects of HMF on the reproduction and survival of Daphnia magna were assessed through validated ecotoxicological tests. The mechanism of toxicity was determined by analysis of transcriptomic responses induced by exposure to different concentrations of HMF using RNA sequencing. HMF exerted toxicity to D. magna with an EC50 for effects on reproduction of 17.2 mg/l. HMF exposure affected molecular pathways including sugar and polysaccharide metabolism, lipid metabolism, general stress metabolism and red blood cell metabolism, although most molecular pathways affected by HMF exposure were dose specific. Hemoglobin genes, however, responded in a sensitive and dose-related manner. No induction of genes involved in the xenobiotic metabolism or oxidative stress metabolism pathway could be observed, which contrasted earlier observations on transcriptional responses of the terrestrial model Folsomia candida exposed to the same compound in a similar dose. We found 4189 orthologue genes between D. magna and F. candida, yet only twenty-one genes of those orthologues were co-regulated in both species. The contrasting transcriptional responses to the same compound exposed at a similar dose between D. magna and F. candida indicates limited overlap in stress responses among soil and aquatic invertebrates. The dose-related expression of hemoglobin provides further support for using hemoglobin expression as a biomarker for general stress responses in daphnids.

Quantitative assessment of DNA damage in the industrial ethanol production strain Saccharomyces cerevisiae PE-2.

Lignocellulosic hydrolysates remain one of the most abundantly used substrates for the sustainable production of second generation fuels and chemicals with Saccharomyces cerevisiae. Nevertheless, fermentation inhibitors such as acetic acid, furfural and hydroxymethylfurfural are formed during the process and can lead to slow or stuck fermentations and/or act as genotoxic agents leading to production strain genetic instability. We have developed a novel dominant deletion (DEL) cassette assay for quantification of DNA damage in both wild-type and industrial yeast strains. Using this assay, the ethanol production strain S. cerevisiae PE-2 was shown to be more resistant to hydrogen peroxide and furfural than the laboratory DEL strain RS112. Indeed, the PE-2 strain also showed a lower tendency for recombination, consistent with a more efficient DNA protection. The dominant DEL assay presented herein should prove to be a useful tool in the selection of robust yeast strains and process conditions for second generation feedstock fermentations.

Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance.

To fully exploit the microbial genome resources, a high-throughput experimental platform is needed to associate genes with phenotypes at the genome level. We present here a novel method that enables investigation of the cellular consequences of repressing individual transcripts based on the CRISPR interference (CRISPRi) pooled screening in bacteria. We identify rules for guide RNA library design to handle the unique structure of prokaryotic genomes by tiling screening and construct an E. coli genome-scale guide RNA library (~60,000 members) accordingly. We show that CRISPRi outperforms transposon sequencing, the benchmark method in the microbial functional genomics field, when similar library sizes are used or gene length is short. This tool is also effective for mapping phenotypes to non-coding RNAs (ncRNAs), as elucidated by a comprehensive tRNA-fitness map constructed here. Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks in a precise and high-throughput manner.

Furfural and 5-hydroxymethyl-furfural degradation using recombinant manganese peroxidase.

Biomass pretreatment-derived degradation compounds, such as furfural and 5-hydroxymethyl-furfural (HMF), inhibit the growth of fermentation microorganisms that utilize biomass to produce fuels and chemicals. Here we report that recombinant manganese peroxidase (rMnP) produced from the yeast Pichia pastoris can degrade furfural and HMF making them less toxic to microorganisms. Treatment with rMnP or manganese(III) acetate reduced furfural and HMF concentrations in a dose-dependent manner. Furfural disappearance was accompanied by malonate disappearance and accumulation of four distinct degradation products. Furfural was more readily degraded by rMnP and manganese(III) acetate than HMF. Growth assays using Saccharomyces cerevisiae indicated that rMnP treatment reduced the toxicity of furfural and HMF. This work provides an avenue to use rMnP to increase the growth of fermentation microorganisms that are inhibited by toxic compounds derived from pretreatment of biomass.

[Research progress of 5-hydroxymethylfurfural, a safety-related substance in traditional Chinese medicine injections].

Screening out the safety-related substances and establishing the corresponding standard has been a key research issue to improve the safety of traditional Chinese medicine injections(TCMIs). 5-HMF which widely exists in sugar-containing TCMIs has long been considered as an important safety-related substance. In this review, we summarizes the research progress on the toxicology of 5-HMF as well as the content and standards of 5-HMF in TCMIs.Therein, both literature summary and analysis results indicate that there are lack of toxicology researches of 5-HMF and its metabolites in TCMIs, although the potential toxicity of 5-HMF and its metabolites has been reported. Moreover, the content of 5-HMF largely varies from TCMIs to TCMIs, and even in the same TCMIs from different factories. To ensure the clinical efficacy of TCMIs, it urgent to carry out the study of the toxicology of 5-HMF in TCMIs comprehensively and systematically, so as to set up a relatively uniform standard as well as to develop process quality control method.

Formation of a Hydroxymethylfurfural-Cysteine Adduct and Its Absorption and Cytotoxicity in Caco-2 Cells.

Adducts of 5-hydroxymethylfurfural (HMF)-amino acids are formed during food processing and digestion; the elimination capacity of in vitro intestinal digests of biscuits, instant noodles, and potato crisps for HMF is 652, 727, and 540 µg/g, respectively. However, the safety of these adducts is unknown. In this study, an HMF-cysteine adduct named 1-dicysteinethioacetal-5-hydroxymehtylfurfural (DCH), which was found to be produced in the gastrointestinal tract after HMF intake, was prepared to test its effect toward Caco-2 cells. Compared with HMF, the adduct displayed lower cytotoxicity against Caco-2 cells with an IC50 value of 31.26 mM versus 14.95 mM (HMF). The DCH did not induce cell apoptosis, whereas HMF significantly increased the apoptosis rate after incubation at concentrations of 16, 32, and 48 mM for 72 h. DCH showed an absorption rate considerably lower than that of HMF by Caco-2 cells. Lower absorption of DCH may result in lower toxicity compared with HMF against Caco-2 cells. Intracellular transformation of DCH has been observed.

Biocatalytic Valorization of Furans: Opportunities for Inherently Unstable Substrates.

Biogenic furans (furfural and 5-hydroxymethylfurfural) are expected to become relevant building blocks based on their high degree of functionality and versatility. However, the inherent instability of furans poses considerable challenges for their synthetic modifications. Valorization routes of furans typically generate byproducts, impurities, wastes, and a cumbersome downstream processing, compromising their ecological footprint. Biocatalysis may become an alternative, given the high selectivity of enzymes, together with the mild reaction conditions applied. This Review critically discusses the options for enzymes in the upgrading of furans. Based on previous reports, a variety of biocatalytic transformations have been applied to furans, with successful cases both in aqueous and in water-free media. Options comprise the biodetoxification of toxic furans in hydrolysates, selective syntheses based on oxidation-reduction processes, solvent-free esterifications, or carboligations to afford C12 derivatives. Reported strategies show in general promising but still modest productivities (2-30 gproduct L-1 d-1 , depending on the example). There are opportunities with high potential and deserving of further development, scale-up, and technoeconomic assessment, to entirely validate them as realistic alternatives.

The yeast ADH7 promoter enables gene expression under pronounced translation repression caused by the combined stress of vanillin, furfural, and 5-hydroxymethylfurfural.

Lignocellulosic biomass conversion inhibitors such as vanillin, furfural, and 5-hydroxymethylfurfural (HMF) inhibit the growth of and fermentation by Saccharomyces cerevisiae. A high concentration of each fermentation inhibitor represses translation and increases non-translated mRNAs. We previously reported that the mRNAs of ADH7 and BDH2, which encode putative NADPH- and NADH-dependent alcohol dehydrogenases, respectively, were efficiently translated even with translation repression in response to severe vanillin stress. However, the combined effects of these fermentation inhibitors on the expression of ADH7 and BDH2 remain unclear. We herein demonstrated that exposure to a combined stress of vanillin, furfural, and HMF repressed translation. The protein synthesis of Adh7, but not Bdh2 was significantly induced under combined stress conditions, even though the mRNA levels of ADH7 and BDH2 were up-regulated. Additionally, adh7Δ cells were more sensitive to the combined stress than wild-type and bdh2Δ cells. These results suggest that induction of the ADH7 expression plays a role in the tolerance to the combined stress of vanillin, furfural, and HMF. Furthermore, we succeeded in improving yeast tolerance to the combined stress by controlling the expression of ALD6 with the ADH7 promoter. Our results demonstrate that the ADH7 promoter can overcome the pronounced translation repression caused by the combined stress of vanillin, furfural, and HMF, and also suggest a new gene engineering strategy to breed robust and optimized yeasts for bioethanol production from a lignocellulosic biomass.

Biocatalytic Reduction of HMF to 2,5-Bis(hydroxymethyl)furan by HMF-Tolerant Whole Cells.

Catalytic upgrading of 5-hydroxymethylfurfural (HMF), an important biobased platform chemical for high-value products, is currently of great interest. In this work, a new highly HMFtolerant yeast strain-Meyerozyma guilliermondii SC1103 was isolated, and biocatalytic reduction of HMF to 2,5-bis(hydroxymethyl)furan (BHMF) using its resting cells was reported. Cosubstrates exerted a significant effect on the catalytic activity and selectivity of microbial cells as well as their HMF-tolerant levels whereas the nitrogen source and mineral salts had no effects. In addition, M. guilliermondii SC1103 cells exhibited good catalytic performances within the range of pH 4.0-10.0. The yeast was highly tolerant to both HMF (up to 110 mm) and BHMF (up to 200 mm). In addition, 100 mm HMF could be selectively reduced to BHMF within 12 h by its resting cells in the presence of 100 mm glucose (as cosubstrate), with a yield of 86 % and selectivity of >99 %. The production of 191 mm of BHMF was realized within 24.5 h by using a fed-batch strategy, with a productivity of approximately 24 g L-1 per day. In addition, this new biocatalytic approach was applied for the reduction of furfural and 5-methylfurfural, affording the corresponding furfuryl alcohols with yields of 83 and 89 %, respectively.

Omics-based approaches reveal phospholipids remodeling of Rhizopus oryzae responding to furfural stress for fumaric acid-production from xylose.

In order to relieve the toxicity of furfural on Rhizopus oryzae fermentation, the molecular mechanism of R. oryzae responding to furfural stress for fumaric acid-production was investigated by omics-based approaches. In metabolomics analysis, 29 metabolites including amino acid, sugars, polyols and fatty acids showed significant changes for maintaining the basic cell metabolism at the cost of lowering fumaric acid production. To further uncover the survival mechanism, lipidomics was carried out, revealing that phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R. oryzae's adapting to furfural stress. Based on the above omics analysis, lecithin, inositol and soybean oil were exogenously supplemented separately with an optimized concentration in the presence of furfural, which increased fumaric acid titer from 5.78g/L to 10.03g/L, 10.05g/L and 12.13g/L (increased by 73.5%, 73.8% and 110%, respectively). These findings provide a methodological guidance for hemicellulose-fumaric acid development.

Determination of the main impurities formed after acid hydrolysis of soybean extracts and the in vitro mutagenicity and genotoxicity studies of 5-ethoxymethyl-2-furfural.

Soybean acid hydrolyzed extracts are raw-materials widely used for manufacturing of pharmaceuticals and cosmetics products due to their high content of isoflavone aglycones. In the present study, the main sugar degradation products 5-hydroxymethyl-2-furfural (HMF) and 5-ethoxymethyl-2-furfural (EMF) were quantitatively determined after acid hydrolysis of extracts from different soybean cultivars by a validated liquid chromatography method. The furanic compounds determined in samples cover the range of 0.16-0.21mg/mL and 0.22-0.33mg/mL for HMF and EMF, respectively. Complementarily, due to the scarce literature regarding the EMF toxicology, this study also assessed the EMF mutagenicity by the Salmonella/microsome test and genotoxicity by the comet assay. The results revealed that EMF did not show mutagenicity at the range of 50-5000µg/plate in S. typhimurium strains TA98, TA97a, TA100, TA102 and TA1535, but induced DNA damage in HepG2 cells at non-cytotoxic doses of 0.1-1.3mg/mL, mainly by oxidative stress mechanisms. Based on literature of HMF genotoxicity, and considering the EMF genotoxicity results herein shown, purification procedures to remove these impurities from extracts are recommended during healthcare products development to ensure the security of the products.

Precultivation of Bacillus coagulans DSM2314 in the presence of furfural decreases inhibitory effects of lignocellulosic by-products during L(+)-lactic acid fermentation.

By-products resulting from thermo-chemical pretreatment of lignocellulose can inhibit fermentation of lignocellulosic sugars to lactic acid. Furfural is such a by-product, which is formed during acid pretreatment of lignocellulose. pH-controlled fermentations with 1 L starting volume, containing YP medium and a mixture of lignocellulosic by-products, were inoculated with precultures of Bacillus coagulans DSM2314 to which 1 g/L furfural was added. The addition of furfural to precultures resulted in an increase in L(+)-lactic acid productivity by a factor 2 to 1.39 g/L/h, an increase in lactic acid production from 54 to 71 g and an increase in conversion yields of sugar to lactic acid from 68 to 88 % W/W in subsequent fermentations. The improved performance was not caused by furfural consumption or conversion, indicating that the cells acquired a higher tolerance towards this by-product. The improvement coincided with a significant elongation of B. coagulans cells. Via RNA-Seq analysis, an upregulation of pathways involved in the synthesis of cell wall components such as bacillosamine, peptidoglycan and spermidine was observed in elongated cells. Furthermore, the gene SigB and genes promoted by SigB, such as NhaX and YsnF, were upregulated in the presence of furfural. These genes are involved in stress responses in bacilli.