Weissella koreensis and Pediococcus pentosaceus bacterial ghosts induce inflammatory responses as immunostimulants.

In this study, bacterial ghosts (BGs) were generated from Weissella koreensis LKS42 (WKorGs) and Pediococcus pentosacues KA94 (PPGs) by chemically inducing lysis using substances such as hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), acetic acid (CH3COOH), sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium carbonate (Na2CO3), n-butanol, and C6H8O7. HCl-induced WKorGs and PPGs exhibited complete removal of DNA and displayed transverse membrane dissolution tunnel structures under scanning electron microscopy (SEM). Cell viability assays showed high viability of RAW 264.7 cells exposed to HCl-induced WKorGs and PPGs. Additionally, treatment with HCl-induced WKorGs and PPGs elevated mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, iNOS) and the anti-inflammatory cytokine IL-10 in RAW 264.7 cells. These findings suggest that HCl-induced WKorGs and PPGs have the potential to be used as inactivated bacterial immunostimulants, highlighting their promising applications in immunization and immunotherapy.

A bacteria-based carbon sequestration and waste recycling system.

Achieving carbon neutrality requires a variety of technological approaches. In the present study, we confirmed the applicability of a carbon cycle system in several industrial fields using sulphur-oxidising bacteria. This system produces a nitrogen fertiliser, which decreases carbon emissions by recycling H2S and NH3 pollutants discharged into the atmosphere or wastewater. It should be considered in industrial fields as a carbon reduction strategy.

The Quinazoline Otaplimastat (SP-8203) Reduces the Hemorrhagic Transformation and Mortality Aggravated after Delayed rtPA-Induced Thrombolysis in Cerebral Ischemia.

Acute ischemic stroke is the leading cause of morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only agent clinically approved by FDA for patients with acute ischemic stroke. However, delayed treatment of rtPA (e.g., more than 3 h after stroke onset) exacerbates ischemic brain damage by causing intracerebral hemorrhage and increasing neurotoxicity. In the present study, we investigated whether the neuroprotant otaplimastat reduced delayed rtPA treatment-evoked neurotoxicity in male Sprague Dawley rats subjected to embolic middle cerebral artery occlusion (eMCAO). Otaplimastat reduced cerebral infarct size and edema and improved neurobehavioral deficits. In particular, otaplimastat markedly reduced intracerebral hemorrhagic transformation and mortality triggered by delayed rtPA treatment, consequently extending the therapeutic time window of rtPA. We further found that ischemia-evoked extracellular matrix metalloproteases (MMPs) expression was closely correlated with cerebral hemorrhagic transformation and brain damage. In ischemic conditions, delayed rtPA treatment further increased brain injury via synergistic expression of MMPs in vascular endothelial cells. In oxygen-glucose-deprived endothelial cells, otaplimastat suppressed the activity rather than protein expression of MMPs by restoring the level of tissue inhibitor of metalloproteinase (TIMP) suppressed in ischemia, and consequently reduced vascular permeation. This paper shows that otaplimastat under clinical trials is a new drug which can inhibit stroke on its own and extend the therapeutic time window of rtPA, especially when administered in combination with rtPA.

Hydrochloric acid-treated Bacillus subtilis ghosts induce IL-1 beta, IL-6, and TNF-alpha in murine macrophage.

Background: Bacterial ghosts (BGs) are empty cell envelopes commonly generated using Gram-negative bacteria; they represent a potential platform for efficient adjuvant and vaccine delivery systems. However, the efficient production of BGs from bacteria in a short period of time is challenging. Objective: The purpose of this study was to investigate the possibility of producing BGs in the Gram-positive Bacillus subtilis using various chemicals, and the potential application of BGs as a novel immunomodulatory agent. Results: In this study, Bacillus subtilis ghosts (BSGs) were generated, for the first time to the best of our knowledge, using the minimum inhibitory concentration (MIC) of hydrochloric acid (HCl; 6.25 mg/mL), sulfuric acid (H2SO4; 3.125 mg/mL), and nitric acid (HNO3; 6.25 mg/mL). Among the BSGs generated using these chemicals, HCl-induced BSGs were completely DNA-free as confirmed by real-time polymerase chain reaction. Scanning electron microscopy showed the formation of transmembrane lysis tunnel structures in HCl-induced BSGs. Murine macrophages exposed to the HCl-induced BSGs at a concentration of 1 × 105 CFU/mL showed a cell viability of 97.8%. Additionally, HCl-induced BSGs upregulated the expression of pro-inflammatory cytokines including interleukin (IL)-1ß, tumor necrosis factor alpha, and IL-6. Furthermore, we found differences in the protein expression profiles between intact live bacteria and BSGs using two-dimensional electrophoresis coupled with peptide mass fingerprinting/matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Conclusion: These data suggest that the HCl-induced BSGs may be potentially safe and effective candidates for inactivated bacterial vaccines and/or immunostimulants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13273-022-00221-5.

A Salmonella typhimurium ghost vaccine induces cytokine expression in vitro and immune responses in vivo and protects rats against homologous and heterologous challenges.

Salmonella enteritidis and Salmonella typhimurium are important food-borne bacterial pathogens, which are responsible for diarrhea and gastroenteritis in humans and animals. In this study, S. typhimurium bacterial ghost (STG) was generated based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH). Experimental studies performed using in vitro and in vivo experimental model systems to characterize effects of STG as a vaccine candidate. When compared with murine macrophages (RAW 264.7) exposed to PBS buffer (98.1%), the macrophages exposed to formalin-killed inactivated cells (FKC), live wild-type bacterial cells and NaOH-induced STG at 1 × 108 CFU/mL showed 85.6%, 66.5% and 84.6% cell viability, respectively. It suggests that STG significantly reduces the cytotoxic effect of wild-type bacterial cells. Furthermore, STG is an excellent inducer for mRNAs of pro-inflammatory cytokine (TNF-α, IL-1ß) and factor (iNOS), anti-inflammatory cytokine (IL-10) and dual activities (IL-6) in the stimulated macrophage cells. In vivo, STG vaccine induced humoral and cellular immune responses and protection against homologous and heterologous challenges in rats. Furthermore, the immunogenicity and protective efficacy of STG vaccine were compared with those of FKC and non-vaccinated PBS control groups. The vaccinated rats from STG group exhibited higher levels of serum IgG antibody responses, serum bactericidal antibodies, and CD4+ and CD8+ T-cell populations than those of the FKC and PBS control groups. Most importantly, after challenge with homologous and heterologous strains, the bacterial loads in the STG group were markedly lower than the FKC and PBS control groups. In conclusion, these findings suggest that the STG vaccine induces protective immunity against homologous and heterologous challenges.

A study on detection of glucose concentration using changes in color coordinates.

Glucose concentration is closely related to the metabolic activity of cells and it is the most important substance as the energy source of a living body which plays an important role in the human body. This paper proposes an optical method that can measure the concentration of glucose. The change in glucose concentration was observed by using CIE diagram, and wavelength and purity values were detected. Also, even small changes in glucose concentration can be evaluated through mathematical modeling. This system is simple, economical, and capable of quantifying optical signals with numerical values for glucose sensing. This method can be applicable to the clinical field that examines diabetes mellitus or metabolic syndrome.

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs).

Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 106 CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1ß and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

A new optical technique to monitor joint motion using position sensitive detector.

BACKGROUND: Evaluation of joint movements is essential to choose an appropriate rehabilitation protocol for a patient. OBJECTIVE: The aim of this study is to suggest an alternative optical technique for measurement of joint angle which is convenient, inexpensive, and can be operated in real time. METHODS: According to the principle of the triangulation method, position sensitive detector (PSD) converts reflected light signals into distance-related voltages. Various parameters were investigated to increase detection range and resolution of joint angle measurements. RESULTS: The accuracy of the suggested optical sensor was verified by comparing with a commercial goniometer and 3D motion capture system. CONCLUSIONS: This method can be applied to monitor recovery progress for the patient in rehabilitation and sport science.

Construction of an efficient xylose-fermenting diploid Saccharomyces cerevisiae strain through mating of two engineered haploid strains capable of xylose assimilation.

Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain. Specifically, we obtained two engineered haploid strains (YSX3 and SX3). The YSX3 strain consumed xylose rapidly and produced a lot of xylitol. On the contrary, the SX3 strain consumed xylose slowly with little xylitol production. After converting the mating type of SX3 from alpha to a, the resulting strain (SX3-2) was mated with YSX3 to construct a heterozygous diploid strain (KSM). The KSM strain assimilated xylose (0.25gxyloseh(-1)gcells(-1)) as fast as YSX3 and accumulated a small amount of xylitol (0.03ggxylose(-1)) as low as SX3, resulting in an improved ethanol yield (0.27ggxylose(-1)). We found that the improvement in xylose fermentation by the KSM strain was not because of heterozygosity or genome duplication but because of the complementation of the two xylose-metabolic pathways. This result suggested that mating of suboptimal haploid strains is a promising strategy to develop engineered yeast strains with improved xylose fermenting capability.

Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus.

Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) >> P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the cassettes were integrated into the chromosome, the stochastic behavior was markedly reduced. These combined results imply that the gene expression stochasticity should be overcome in order to use this strain for delicate metabolic engineering, which would require the co-expression of several genes.

Olfactomedin 4 suppresses tumor growth and metastasis of mouse melanoma cells through downregulation of integrin and MMP genes.

Olfactomedin 4 (OLFM4) is highly expressed in gastrointestinal cancers and has an anti-apoptotic function. The roles of OLFM4 in tumor growth and metastasis and how it functions in these processes remain elusive. We investigated the function of OLFM4 in tumor growth and metastasis using B16F10 mouse melanoma cells as an experimental system. Our results showed that OLFM4 had no positive effect on cell viability or cell cycle progression in B16F10 cells. However, it significantly suppressed the tumorigenicity of B16F10 cells, i.e., intradermal primary tumor growth and lung metastasis. OLFM4 also suppressed the migration and invasion of B16F10 cells in vitro. For further insight into the mechanisms underlying OLFM4-mediated suppression of tumor progression, we examined the effect of OLFM4 on the expression of integrin and matrix metalloproteinase (MMP), both of which are involved in tumor progression. Overexpression of OLFM4 clearly reduced the expression levels of integrin α1, integrin α4, integrin α5, integrin α6, and MMP9. Moreover, forced expression of MMP9 attenuated the inhibitory activity of OLFM4 on migration and invasiveness. Our findings provide the experimental evidence that OLFM4 may function as a tumor suppressor and an anti-metastatic gene during tumor progression.

SP-8203 shows neuroprotective effects and improves cognitive impairment in ischemic brain injury through NMDA receptor.

The extracts of earth worms, Eisenia andrei, have been used as a therapeutic agent for stroke in the traditional medicine. It is also reported that the protease fraction separated from the extracts has strong anti-thrombotic activity. Besides anti-thrombotic actions, we found that SP-8203, N-[3-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)propyl]-N-{4-[3-(2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)propylamino]butyl}acetamide, derived from the extracts of earth worms blocked N-methyl-(D)-aspartate (NMDA) receptor-mediated excitotoxicity in a competitive manner. The neuroprotective effects of SP-8203 were attributable to prevention of Ca(2+) influx through NMDA receptors. The systemic administration of SP-8203 markedly reduced neuronal death following middle cerebral artery occlusion in rats. SP-8203 significantly improved spatial learning and memory in the water maze test. These results provided strong pharmacological basis for its potential therapeutic roles in cerebral ischemia.

Bacterial persisters tolerate antibiotics by not producing hydroxyl radicals.

In a phenomenon called persistence, small numbers of bacterial cells survive even after exposure to antibiotics. Recently, bactericidal antibiotics have been demonstrated to kill bacteria by increasing the levels of hydroxyl radicals inside cells. In the present study, we report a direct correlation between intracellular hydroxyl radical formation and bacterial persistence. By conducting flow cytometric analysis in a three-dimensional space, we resolved distinct bacterial populations in terms of intracellular hydroxyl radical levels, morphology and viability. We determined that, upon antibiotic treatment, a small sub-population of Escherichia coli survivors do not overproduce hydroxyl radicals and maintain normal morphology, whereas most bacterial cells were killed by accumulating hydroxyl radicals and displayed filamentous morphology. Our results suggest that bacterial persisters can be formed once they have transient defects in mediating reactions involved in the hydroxyl radical formation pathway. Thus, it is highly probable that persisters do not share a common mechanism but each persister cell respond to antibiotics in different ways, while they all commonly show lowered hydroxyl radical formation and enhanced tolerance to antibiotics.

The TSP motif in AP180 inhibits phospholipase D1 activity resulting in increased efficacy of anticancer drug via its direct binding to carboxyl terminal of phospholipase D1.

Phospholipase D (PLD) has two isoforms, PLD1 and PLD2. Both isoforms are possible candidates for the development of anticancer drugs, since PLDs in several cancer cells act as survival factors. The aim of this study was to elucidate the inhibitory mechanism of PLD1 by AP180 in human cancer cells. Transfection of the human AP180 (hAP180) gene markedly inhibited phobol-12-myristate 13-acetate-induced PLD activity resulting in exacerbation of anticancer drug-induced cell death. Experiments using deletion mutants of hAP180 showed that three amino acids (Thr312-Pro314) are critical for inhibition of PLD1 activity by binding directly to PLD1, and, of these, Ser313 was the most important residue for both binding to and inhibiting PLD1. However, this inhibitory relationship did not exist between hAP180 and PLD2. In addition, the C-terminal region of PLD1 is important for the interaction with hAP180. These results indicated that Thr312-Pro314 (especially Ser313 as a phosphorylation residue) of hAP180 can regulate hPLD1 activity through binding with the C-terminal region of PLD1.

The progesterone receptor as a transcription factor regulates phospholipase D1 expression through independent activation of protein kinase A and Ras during 8-Br-cAMP-induced decidualization in human endometrial stromal cells.

Decidualization is a biological and morphological process occurring in hES (human endometrial stromal) cells. Previously, we reported that PLD1 (phospholipase D1) plays an important role in cAMP-induced decidualization of hES cells. In the present study, we focused on how PLD1 expression is up-regulated during decidualization. Treatment with PKA (protein kinase A) inhibitors (Rp-cAMP or H89) or a Ras inhibitor (manumycin) partially inhibited PLD1 expression and decidua formation in response to cAMP treatment. Interestingly, dual inhibition of PKA and Ras completely inhibited PLD1 expression and cAMP-induced decidualization. These results suggest that PLD1 expression during decidualization is controlled additively by PKA and Ras. The use of inhibitors showed that extracellular-signal-regulated kinase, a downstream effector of Ras, was required for PLD activation and the morphological changes during decidualization, but not for the increase in PLD1 protein. Next, to investigate the regulator of the PLD1 gene at the transcriptional level, a promoter assay using deletion mutants of the PLD1 promoter was performed; the result indicated that PR (progesterone receptor) was a possible regulator of the PLD1 gene. In addition, chromatin immunoprecipitation assays on the PLD1 promoter identified PR as a transcription factor for PLD1 expression during 8-Br-cAMP-induced decidualization. Taken together, our findings demonstrate that PKA and Ras are novel regulators of PLD1 expression and also identify PR as a transcription factor for PLD1 expression during the decidualization of hES cells.

Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering.

Although Saccharomyces cerevisiae is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, and then fast galactose-fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of tTUP1 significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C-terminal repression domain might bring in earlier and higher expression of GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses.

[Spontaneous dissolution of isolated superior mesenteric vein thrombosis in acute pancreatitis].

Acute pancreatitis can result in many vascular complications in both artery and vein. Venous complication usually occurs as a form of splenic or portal vein thrombosis, and also can simultaneously occur in superior mesenteric vein as well. Rarely, isolated superior mesenteric vein thrombosis occurs as a venous complication. Although it is uncommon, mesenteric vein thrombosis is an important clinical entity because of the possibility of mesenteric ischemia and infarction of small bowel. The treatments of mesenteric venous thrombosis include anticoagulation therapy, transcatheter therapy and surgical intervention. We report a case of 45-year- old man who had acute pancreatitis with isolated superior mesenteric vein thrombosis, which was spontaneously dissolved with the resolution of underlying inflammation without anticoagulation or surgical intervention.

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism.

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.

Restoration of growth phenotypes of Escherichia coli DH5alpha in minimal media through reversal of a point mutation in purB.

A point mutation (E115K) resulting in slower growth of Escherichia coli DH5alpha and XL1-Blue in minimal media was identified in the purB gene, coding for adenylosuccinate lyase (ASL), through complementation with an E. coli K-12 genomic library and serial subcultures. Chromosomal modification reversing the mutation to the wild type restored growth phenotypes in minimal media.

Identification of gene targets eliciting improved alcohol tolerance in Saccharomyces cerevisiae through inverse metabolic engineering.

The economic production of biofuels from renewable biomass using Saccharomyces cerevisiae requires tolerance to high concentrations of sugar and alcohol. Here we applied an inverse metabolic engineering approach to identify endogenous gene targets conferring improved alcohol tolerance in S. cerevisiae. After transformation with a S. cerevisiae genomic library, enrichment of the transformants exhibiting improved tolerance was performed by serial subculture in the presence of iso-butanol (1%). Through sequence analysis of the isolated plasmids from the selected transformants, four endogenous S. cerevisiae genes were identified as overexpression targets eliciting improved tolerance to both iso-butanol and ethanol. Overexpression of INO1, DOG1, HAL1 or a truncated form of MSN2 resulted in remarkably increased tolerance to high concentrations of iso-butanol and ethanol. Overexpression of INO1 elicited the highest ethanol tolerance, resulting in higher titers and volumetric productivities in the fermentation experiments performed with high glucose concentrations. In addition, the INO1-overexpressing strain showed a threefold increase in the specific growth rate as compared to that of the control strain under conditions of high levels of glucose (10%) and ethanol (5%). Although alcohol tolerance in yeast is a complex trait affected by simultaneous interactions of many genes, our results using a genomic library reveal potential target genes for better understanding and possible engineering of metabolic pathways underlying alcohol tolerance phenotypes.