BackgroundIsobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress.ResultsThe adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation.ConclusionsWe have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.

The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.

The molecular mechanism involved in tolerance and adaptation of ethanologenic Saccharomyces cerevisiae to inhibitors (such as furfural, acetic acid, and phenol) represented in lignocellulosic hydrolysate is still unclear. Here, 18 O-labeling-aided shotgun comparative proteome analysis was applied to study the global protein expression profiles of S. cerevisiae under conditions of treatment of furfural compared with furfural-free fermentation profiles. Proteins involved in glucose fermentation and/or the tricarboxylic acid cycle were upregulated in cells treated with furfural compared with the control cells, while proteins involved in glycerol biosynthesis were downregulated. Differential levels of expression of alcohol dehydrogenases were observed. On the other hand, the levels of NADH, NAD ؉ , and NADH/NAD ؉ were reduced whereas the levels of ATP and ADP were increased. These observations indicate that central carbon metabolism, levels of alcohol dehydrogenases, and the redox balance may be related to tolerance of ethanologenic yeast for and adaptation to furfural. Furthermore, proteins involved in stress response, including the unfolded protein response, oxidative stress, osmotic and salt stress, DNA damage and nutrient starvation, were differentially expressed, a finding that was validated by quantitative real-time reverse transcription-PCR to further confirm that the general stress responses are essential for cellular defense against furfural. These insights into the response of yeast to the presence of furfural will benefit the design and development of inhibitor-tolerant ethanologenic yeast by metabolic engineering or synthetic biology.

Continuing efforts have been made to explore novel exopolysaccharides (EPSs) for valuable applications. In this research, we report for the first time that a novel non-glucan EPS named EPS-605 can self-assemble to form spherical nanosize particles of ∼88 nm in diameter, expanding both the range of EPS type and structural type that EPSs self-assemble into. Characterization of EPS-605 shows that it is composed of mannose, glucose, and galactose with several modifications including acylation, phosphorylation, sulfation, and carboxylation, and a highly negative charge. EPS-605 showed a record biosorption capability for Pb, Cu, Cd, and methylene blue as compared to that of other reported EPSs, biosorbents, and nanosorbents. The adsorption ability of EPS-605 is affected by pH, temperature, the initial adsorbate concentration, the contact time, and the presence of background electrolytes. The mechanism of EPS-605 adsorbing heavy metals seems to be different to that for dyes. Moreover, EPS-605 can serve as the reductant in the synthesis of Au nanoparticles (AuNPs) and AgNPs enabling good monodispersity within the shortest time (of 30 min) compared to that from other EPSs and without any extra pretreatment. Our research advances the development of novel EPSs and provides a new, eco-friendly, and renewable platform for both the bioremediation and green synthesis of nanomaterials.

It is of great value to develop new antibacterial photodynamic therapy (PDT) strategies to improve antibacterial PDT efficacy of noncationic photosensitizers without introducing cytotoxicity, which is a great challenge for current leading efforts on antimicrobial PDT based on cell surface engineering. In this research, the hydrophobic and anionic photosensitizer rose bengal (RB) was chemically conjugated with bacterial exopolysaccharide (EPS) to generate an amphiphilic and negatively charged compound EPS-RB that could self-assemble into nanoparticles (NPs) in solution. These EPS-RB NPs possessed an increased singlet oxygen generation property in solution. As a result, EPS-RB exhibited improved photoinactivation for both Gram-negative and Gram-positive bacteria, leading to a record low RB working concentration, 8 μM or 500 nM for Escherichia coli or Staphylococcus aureus, respectively. Upon light irradiation, more EPS-RB bound to the cell surface and penetrated into bacteria than RB, with EPS-RB staying around the cell surface of the most irradiated E. coli while entering all irradiated S. aureus. Both scanning electron microscopy and fluorescence confocal imaging results show that the cell membrane of E. coli was damaged heavily but not S. aureus. All of these observations indicate that both the enhanced singlet oxygen production of EPS-RB NPs in solution and their consequently increased membrane binding and cellular penetration into the bacteria through the damaged cell membrane contribute to their significantly improved bacterial photoinactivation efficiency. In addition, EPS-RB has low cytotoxicity and negligible hemolytic activity, showing great biocompatibility. Therefore, the construction of EPS-RB provides a new strategy for the PDT effectiveness improvement of the separated cell/sensitizer systems and thus the design of next-generation antimicrobial agents.

Carbon dots (or carbon quantum dots) are small (less than 10 nm) and luminescent carbon nanoparticles with some form of surface passivation. As an emerging class of nanomaterials, carbon dots have found wide applications in medicine, bioimaging, sensing, electronic devices, and catalysis. In this review, we focus on the recent advancements of carbon dots for sensing and killing microorganisms, including bacteria, fungi, and viruses. Synthesis, functionalization, and a toxicity profile of these carbon dots are presented. We also discuss the underlying mechanisms of carbon dot-based sensing and killing of microorganisms.

Background and Aims The development and progression of hepatocellular carcinoma (HCC) is dependent on its local microenvironment. Tumor‐associated macrophages (TAMs) are deemed a key factor for the tumor microenvironment and attribute to contribute to tumor aggressiveness. However, the detailed mechanism underlying the pro‐metastatic effect of TAMs on HCC remains undefined. Approach and Results The present study proved that TAMs were enriched in HCC. TAMs were characterized by an M2‐polarized phenotype and accelerated the migratory potential of HCC cells in vitro and in vivo. Furthermore, we found that M2‐derived exosomes induced TAM‐mediated pro‐migratory activity. With the use of mass spectrometry, we identified that integrin, αMβ2 (CD11b/CD18), was notably specific and efficient in M2 macrophage–derived exosomes (M2 exos). Blocking either CD11b and/or CD18 elicited a significant decrease in M2 exos–mediated HCC cell metastasis. Mechanistically, M2 exos mediated an intercellular transfer of the CD11b/CD18, activating the matrix metalloproteinase‐9 signaling pathway in recipient HCC cells to support tumor migration. Conclusions Collectively, the exosome‐mediated transfer of functional CD11b/CD18 protein from TAMs to tumor cells may have the potency to boost the migratory potential of HCC cells, thus providing insights into the mechanism of tumor metastasis.

Because of the distinct surface structures of different cells (mammalian cells, fungi, and bacteria), surface labeling for these cells requires a variety of fluorescent dyes. Besides, fluorescent dyes (especially the commercial ones) for staining Gram-negative bacterial cell walls are still lacking. Herein, a conformation-adjustable glycol chitosan (GC) derivative (GC-PEG cholesterol-FITC) with “all-in-one” property was developed to realize universal imaging for plasma membranes of mammalian cells (via hydrophobic interaction) and cell walls of fungal and bacterial cells (via electrostatic interaction). By comparing the different staining behaviors of GC-PEG cholesterol-FITC and three other analogs (GC-PEG-FITC, GC-FITC, and cholesterol-PEG-FITC), we have elucidated the different roles the hydrophobic and electrostatic interactions play in the staining performance of these different cells. Such a simple, noncytotoxic, economic, and universal cell surface staining reagent will be very useful for investigating cell surface-related biological events and advancing cell surface engineering of various types of cells.