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Biopharmaceutical drugs have strong therapeutic effect in various diseases such as autoimmune diseases, rheumatism and cancer due to their target specific recognition and low side effects. In particular, the most representative antibodies among biopharmaceuticals are widely used for targeted drug delivery, diagnosis and treatment. Moreover, antibody-drug conjugate (ADC) have been developed by many researchers and biopharmaceutical companies. An antibody-drug conjugate, one of the new bio anticancer drugs is composed of an antibody, cytotoxic drug and chemical linker. Antibodies accurately recognize the target cancer, and drugs induce apoptosis or death of the cancer cells. Linkers stably connects the antibody and drug until ADC go to the target cancer cells. In order to develop an ADC, it is important to correctly configure each component. Recently, the 9th ADC was approved by food and drug administration (FDA) and a lot of development of new ADC is underway. The development of ADC is expected through the introduction of new antibodies including bi-specific and drugs with new anticancer mechanisms.

Mutant sugar transporter ScGAL2-N376F was overexpressed in Kluyveromyces marxianus for efficient utilization of xylose, which is one of the main components of cellulosic biomass. K. marxianus ScGal2_N376F, the ScGAL2-N376F-overexpressing strain, exhibited 47.04 g/l of xylose consumption and 26.55 g/l of xylitol production, as compared to the parental strain (24.68 g/l and 7.03 g/l, respectively) when xylose was used as the sole carbon source. When a mixture of glucose and xylose was used as the carbon source, xylose consumption and xylitol production rates were improved by 195% and 360%, respectively, by K. marxianus ScGal2_N376F. Moreover, the glucose consumption rate was improved by 27% as compared to that in the parental strain. Overexpression of both wild-type ScGAL2 and mutant ScGAL2-N376F showed 48% and 52% enhanced sugar consumption and ethanol production rates, respectively, when a mixture of glucose and galactose was used as the carbon source, which is the main component of marine biomass. As shown in this study, ScGAL2-N376F overexpression can be applied for the efficient production of biofuels or biochemicals from cellulosic or marine biomass.

The purpose of this study is to understand the effects of extracurricular program, and to draw implications for the subject curriculum environment through the case study by the biotechnology major among the interdisciplinary comparative course programs conducted by K University. The research data used for the study are student grade data gathered from three subjects in the biotechnology major from the second semester of 2018 and the first semester of 2019, and the survey results of participating students in the extracurricular program. As a result, professor’s intended instructional goal, learner’s extracurricular learning objectives, and core competencies were consistent, and participants’ academic achievement was higher than non-participants. In addition, it was shown that the experience of extracurricular program in connection with the major subject helped the participants to increase their interest in the major subjects and explore their career. Therefore, extracurricular program is composed of competency development education suitable for learner’s characteristics. However, it is necessary to study the effect of extracurricular program experiences related to major subjects on the learning continuity of participating students.

Background Simultaneous cofermentation of glucose and xylose mixtures would be a cost-effective solution for the conversion of cellulosic biomass to high-value products. However, most yeasts ferment glucose and xylose sequentially due to glucose catabolite repression. A well known thermotolerant yeast, Kluyveromyces marxianus, was selected for this work because it possesses cost-effective advantages over Saccharomyces cerevisiae for biofuel production from cellulosic biomass. Results In the present study, we employed a directed evolutionary approach using 2-deoxyglucose to develop a thermotolerant mutant capable of simultaneous cofermentation of glucose and xylose by alleviating catabolite repression. The selected mutant, K. marxianus SBK1, simultaneously cofermented 40 g/L glucose and 28 g/L xylose to produce 23.82 g/L ethanol at 40 °C. This outcome corresponded to a yield of 0.35 g/g and productivity of 0.33 g/L h, representing an 84% and 129% improvement, respectively, over the parental strain. Interestingly, following mutagenesis the overall transcriptome of the glycolysis pathway was highly downregulated in K. marxianus SBK1, except for glucokinase-1 (GLK1) which was 21-fold upregulated. Amino acid sequence of GLK1 from K. marxianus SBK1 revealed three amino acid mutations which led to more than 22-fold lower enzymatic activity compared to the parental strain. Conclusions We herein successfully demonstrated that the cofermentation of a sugar mixture is a promising strategy for the efficient utilization of cellulosic biomass by K. marxianus SBK1. Through introduction of additional biosynthetic pathways, K. marxianus SBK1 could become a chassis-type strain for the production of fuels and chemicals from cellulosic biomass. Electronic supplementary material The online version of this article (10.1186/s13068-019-1431-x) contains supplementary material, which is available to authorized users.

Multiple sclerosis is a chronic disorder commonly occurred in the central nervous system and is an autoimmune disease. Corticosteroids are used as a short-term treatment for multiple sclerosis, but they cause side effects. Therefore, interferon beta having antiviral and anti-inflammatory effects, is...

Antioxidant activities of blackberry juice and aronia juice were enhanced when fermentation was performed by acetic acid bacteria. Acetobacter pasteurianus exhibited 19.84% improvement of antioxidant activity (from 198.12 ± 2.03 to 237.42 ± 7.32 μmol TE/g) after 12 h fermentation of blackberry juice among four acetic acid bacteria. And A. pasteurianus sub sp. Pasteurianus exhibited 9.62% improvement of antioxidant activity (from 204.25 ± 3.98 to 223.89 ± 5.52 μmol TE/g) after 12 h fermentation of aronia juice. Metabolites of blackberry juice were analyzed to investigate the enhancement of antioxidant activity before and after fermentation. As results, Quercetin 7-(rhamnosylglucoside), nicotinic acid adenine dinucleotide, and quercetin 3-O-(6”-acetyl-glucoside) were significantly increased after fermentation by A. pasteurianus.

The objectives of this study were to analyze antioxidant activities and identify volatile compounds in mixed berry juice after fermentation by lactic acid bacteria (LAB). Antioxidant activity of the mixed berry juice increased significantly from 209.57±2.93 to 268.30±1.75 μmol TE/g after 24 h of fermentation. After LAB fermentation, 34 volatile compounds were identified. Among them, three compounds-benzoic acid, benzaldehyde, and vitispirane-showed significant changes in their concentrations. Peak areas of benzoic acid and benzaldehyde, which are known to possess antioxidant activities, increased by 64 and 188%, respectively, after fermentation. However, the peak area of vitispirane, which is the most abundant terpene compound in berry juices, decreased by 92% after fermentation.

The multiple sclerosis caused by multiple inflammatory disease or immune system disorder, is usually treated by interferon β through adjusting the abnormal immune reactions. For high production of human interferon β using recombinant E. coli, codon optimized and wild type genes were synthesized. When pET-15b or pET-21a vector was used as an expression vector with each gene, there was no target protein expression. When pQE30 vector was used as an expression vector, human interferon β was expressed by recombinant E. coli XL1-blue and E. coli JM109. Using the codon optimized gene, the expression of human interferon β was slightly increased as compared to that from wild type gene. However, most of expressed human interferon β was insoluble form.

Additional file 3: Fig. S3. Comparisons of xylose consumption rate and ethanol production rate of the selected 2-DG-resistant candidates from glucose and xylose mixture fermentation experiments at 96 h. Symbols: xylose consumption rate (green-filled square) and ethanol production rate (red-filled square).

A number of polysaccharides exhibit pharmacological activities. Polysaccharides derived from Inonotus obliquus (PLIO) appear to have various potential pharmacological properties, including anti‑tumor activity. However, the molecular mechanisms underlying these properties remain to be elucidated. The present study investigated the anti‑metastatic potential of PLIO and the underlying signaling pathways in B16‑F10 murine melanoma cells using the MTT colorimetric assay, in vitro migration and invasion assays, and flow cytometric and western blot analyses. PLIO inhibited the invasion of B16‑F10 cells and suppressed the expression of matrix metalloproteinases. PLIO treatment inhibited nuclear factor‑κB (NF‑κB) nuclear translocation in B16‑F10 cells. In addition, PLIO treatment inhibited the phosphorylation of c-Jun N‑terminal kinases and AKT. These results suggest that PLIO may suppress the invasion of highly metastatic melanoma cells via inhibition of the AKT/NF-κB signaling pathways.

The antibacterial activity from Tricholoma matsutake extracts were confirmed by minimum inhibitory concentration (MIC) test against various bacteria. Tricholoma matsutake extracts was applied for manufacturing low salted Jeot-gal with sodium DL-malate and sodium nitrite as food preservatives. Due to antibacterial activity from Tricholoma matsutake extracts, MIC of sodium DL-malate and sodium nitrite were significantly reduced to 0.025 g/L and 0.25 g/L, respectively. As a result of this study, a premium low salted Jeot-gal can be developed with low concentration of food preservatives by adding Tricholoma matsutake extracts.

Kluyveromyces marxianus is a well-known thermotolerant yeast. Although Saccharomyces cerevisiae is the most commonly used yeast species for ethanol production, the thermotolerant K. marxianus is more suitable for simultaneous saccharification and fermentation (SSF) processes. This is because enzymatic saccharification usually requires a higher temperature than that needed for the optimum growth of S. cerevisiae. In this study, we compared the fermentation patterns of S. cerevisiae and K. marxianus under various temperatures of fermentation. The results show that at a fermentation temperature of 45°C, K. marxianus exhibited more than two fold higher growth rate and ethanol production rate in comparison to S. cerevisiae. For SSF using starch or corn stover as the sole carbon source by K. marxianus, the high temperature (45°C) fermentations showed higher enzymatic activities and ethanol production compared to SSF at 30°C. These results demonstrate the potential of the thermotolerant yeast K. marxianus for SSF in the industrial production of biofuels.

Corn stover is one of the most promising lignocellulosic biomass that can be utilized for producing 1,3-propanediol and 2,3-butanediol. The pretreatment and enzymatic hydrolysis steps are essential for the bioconversion of lignocellulosic biomass to diols. For optimizing the pretreatment step, temperature, time, and NaOH concentration were evaluated based on total sugar recovery. Enzymatic hydrolysis for cellulose and hemicellulose were investigated at different solid-to-liquid ratios. The optimum conditions were found to be alkaline pretreatment with 0.25 mol dm−3 NaOH for 1 h at 60 °C followed by enzymatic hydrolysis at 50 °C for 48 h, with a solid slurry concentration of 100 g dm−3. Under these conditions, conversion rates of 92.55% and 78.82% were obtained from glucan and xylan, respectively. Diol production from fermentable sugars was 14.8 g dm−3, with a conversion yield and productivity of 0.46 g g−1, and 0.98 g dm−3 h−1, respectively. Our results are similar for diol production obtained using pure sugars under the same conditions. Therefore, mild alkaline pretreatment of corn stover facilitates delignification, significantly improving the rate of enzymatic saccharification and sugar recovery.

It is well known that Phellinus linteus, which produces hispidin and its derivatives, possesses antioxidant activities. In this study, we investigated whether hispidin has protective effects on palmitate-induced oxidative stress in C2C12 skeletal muscle cells. Our results showed that palmitate treatment in C2C12 myotubes increased ROS generation and cell death as compared with the control. However, pretreatment of hispidin for 8 h improved the survival of C2C12 myotubes against palmitate-induced oxidative stress via inhibition of intracellular ROS production. Hispidin also inhibited palmitate-induced apoptotic nuclear condensation in C2C12 myotubes. In addition, we found that hispidin can suppress cleavage of caspase-3, expression of Bax, and NF-κB translocation. Therefore, these results suggest that hispidin is capable of protecting C2C12 myotubes against palmitate-induced oxidative stress.

Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154 mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5–7 genes in K. marxianus.

Functional expression of a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa in Saccharomyces cerevisiae enables simultaneous co-fermentation of cellobiose and non-glucose sugars such as xylose. Here we investigate the functional role of aldose 1-epimerase (AEP) in engineered cellobiose utilization. One AEP (Gal10) and two putative AEPs (Yhr210c and Ynr071c sharing 50.6% and 51.0% amino acid identity with Gal10, respectively) were selected. Deletion of GAL10 led to complete loss of both AEP activity and cell growth on cellobiose, while GAL10 complementation restored the AEP activity and cell growth. In addition, deletion of YHR210C or YNR071C resulted in improved cellobiose utilization. These results suggest that the intracellular mutarotation of β-glucose to α-glucose might be a rate controlling step and Gal10 play a crucial role in cellobiose fermentation by engineered S. cerevisiae.

A gene (acas) designated as alpha-amylase was cloned from Arthrobacter chlorophenolicus A6. The multiple amino acid sequence analysis and functional expression of acas revealed that this gene really encoded an amylosucrase (ASase) instead of alpha-amylase. In fact, the recombinant enzyme exhibited typical ASase activity by showing both sucrose hydrolysis and glucosyltransferase activities. The purified enzyme has a molecular mass of 72 kDa and exhibits optimal hydrolysis activity at 45 degrees C and a pH of 8.0. The analysis of the oligomeric state of ACAS with gel permeation chromatography revealed that the ACAS existed as a monomer.

We demonstrate improved ethanol yield and productivity through cofermentation of cellobiose and galactose by an engineered Saccharomyces cerevisiae strain expressing genes coding for cellodextrin transporter ( cdt-1 ) and intracellular β-glucosidase ( gh1 - 1 ) from Neurospora crassa . Simultaneous fermentation of cellobiose and galactose can be applied to producing biofuels from hydrolysates of marine plant biomass.

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.

The presence of amylosucrase in 12 Alteromonas and Pseudoalteromonas strains was examined. Two Alteromonas species (Alteromonas addita KCTC 12195 and Alteromonas macleodii KCTC 2957) possessed genes that had high sequence homology to known amylosucrases. Genomic clones containing the ASase analogs were obtained from A. addita and A. macleodii, and the deduced amino acid sequences of the corresponding genes (aaas and amas, respectively) revealed that they were highly similar to the ASases of Neisseria polysaccharea, Deinococcus radiodurans, and Deinococcus geothermalis. Functional expression of amas in Escherichia coli was successful, and typical ASase activity was detected in purified recombinant AMAS, whereas the purified recombinant AAAS was nonfunctional. Although maximum total activity of AMAS was observed at 45 degrees C, the ratio of transglycosylation to total activity increased as the temperature decreased from 55 to 25 degrees C. These results imply that transglycosylation occurs preferentially at lower temperatures while hydrolysis is predominant at higher temperatures.

Aims: Isomaltulose (palatinose) is a slowly digestible sucrose isomer that can reduce both the glycemic and insulinemic response to foods. The aim of this study was to clone and express a sucrose isomerase (SIase) gene and characterize the protein that is responsible for the production of isomaltulose in the micro-organism Enterobacter sp. FMB-1. Methods and Results: A cosmid clone containing c. 6 kbp region encoding an SIase gene was identified. The 5969-bp chromosomal DNA fragment covering the SIase (esi) gene in Enterobacter sp. FMB-1 was sequenced. Although this DNA fragment contained several open reading frames other than esi, only the presence of esi was sufficient to produce isomaltulose in recombinant Escherichia coli. The esi gene was expressed in E. coli, leading to the characterization of its SIase activity. Conclusions: The Enterobacter sp. FMB-1 esi gene was successfully cloned and expressed in E. coli. This gene encoded a functional SIase that produced isomaltulose from sucrose. Significance and Impact of the Study: This is the first molecular analysis of an SIase gene in an Enterobacter strain. The functional expression of the Enterobacter sp. FMB-1 esi gene in E. coli offers an alternative choice for the industrial production of isomaltulose.

The purpose of the present study was to elucidate the cytoprotective effects of polysaccharides isolated from Inonotus obliquus. The polysaccharides were extracted from the fruiting body of I. obliquus (PFIO) and the liquid culture broth of I. obliquus (PLIO). The effects of PFIO and PLIO on hydrogen peroxide (H2O2)‑induced oxidative damage of RINm5F pancreatic β‑cells were comparatively investigated using an MTT assay, immunofluorescent staining, flow cytometry, and western blot analyses in vitro. The results of the present study demonstrated that treatment with PFIO and PLIO decreased DNA fragmentation and the rate of apoptosis. In addition, pretreatment of cells with PFIO and PLIO prior to H2O2 exposure resulted in increased insulin secretion and scavenging activity for intracellular reactive oxygen species, as compared with treatment with H2O2 alone. The results of the present study suggested that PFIO and PLIO may exert protective effects against H2O2‑induced oxidative stress via the regulation of mitogen‑activated protein kinases, nuclear factor‑κB and apoptotic proteins. Therefore, PFIO and PLIO may have potential merit as a medicinal food for the prevention of diabetes.

Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS.

The extract obtained from berries contains high amounts of anthocyanins, and this extract is used as a phytotherapeutic agent for different types of diseases. In this study, we examined the cytoprotective effects of cyanidin-3-glucoside (C3G) isolated from mulberry fruit against pancreatic β-cell apoptosis caused by hydrogen peroxide (H2O2)-induced oxidative stress. The MIN6 pancreatic β-cells were used to investigate the cytoprotective effects of C3G on the oxidative stress-induced apoptosis of cells. Cell viability was examined by MTT assay and lipid peroxidation was assayed by thiobarbituric acid (TBA) reaction. Immunofluorescence staining, flow cytometry and western blot analysis were also used to determine apoptosis and the expression of proteins associated with apoptosis. Our results revealed that H2O2 increased the rate of apoptosis by stimulating various pro-apoptotic processes, such as the generation of intracellular reactive oxygen species (ROS), lipid peroxidation, DNA fragmentation and caspase-3 activation. However, C3G reduced the H2O2-induced cell death in the MIN6N pancreatic β-cells. In addition, we confirmed that H2O2 activated mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 MAPK. C3G inhibited the phosphorylation of ERK and p38 without inducing the phosphorylation of JNK. Furthermore, C3G regulated the intrinsic apoptotic pathway-associated proteins, such as proteins belonging to the Bcl-2 family, cytochrome c and caspase-3. Taken together, our results suggest that C3G isolated from mulberry fruit has potential for use as a phytotherapeutic agent for the prevention of diabetes by preventing oxidative stress-induced β-cell apoptosis.

Polysaccharides isolated from the fruiting body of Inonotus obliquus (PFIO) are known to possess various pharmacological properties including antitumor activity. However, the anti-metastatic effect and its underlying mechanistic signaling pathway involved these polysaccharides in human non-small cell lung carcinoma remain unknown. The present study therefore aimed to determine the anti-metastatic potential and signaling pathways of PFIO in the highly metastatic A549 cells. We found that PFIO suppressed the migration and invasive ability of A549 cells while decreasing the expression levels and activity of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, PFIO decreased the phosphorylation levels of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) as well as the expression level of COX-2, and inhibited the nuclear translocation of nuclear factor κB (NF-κB) in A549 cells. These results suggested that PFIO could suppress the invasion and migration of human lung carcinoma by reducing the expression levels and activity of MMP-2 and MMP-9 via suppression of MAPKs, PI3K/AKT, and NF-κB signaling pathways.

Background Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown. This study investigated the effect of xylose on anaerobic CBP-mediated cellobiose fermentation by Saccharomyces cerevisiae. Results Yeast capable of fermenting cellobiose by the CBP pathway consumed cellobiose and produced ethanol at rates 61% and 42% slower, respectively, in the presence of xylose than in its absence. The system generated significant amounts of the byproduct 4-O-β-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose. In vitro competition assays identified xylose as a mixed-inhibitor for cellobiose phosphorylase activity. The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization. GX was also shown to be a substrate for cleavage by an intracellular β-glucosidase. Conclusions Xylose exerted negative impacts on CBP-mediated cellobiose fermentation by acting as a substrate for GX byproduct formation and a mixed-inhibitor for cellobiose phosphorylase activity. Future efforts will require efficient xylose utilization, GX cleavage by a β-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

Of the vitamins tested, inositol was the most effective for erythritol production. To increase erythritol production by Torula sp., inositol and a related compound, phytic acid (myoinositol hexaphosphate), were added to the culture media. Erythritol production in the presence of phytic acid was greater than that in the presence of inositol, due to the synergistic effects of phosphate and inositol. Supplementation with phosphate and inositol increased cell growth, erythritol production, and the activity of erythrose reductase in cells. Inositol was a more effective stimulator of cell growth and erythritol production than was phosphate.

The production of erythritol and the erythritol yield from glucose by Torula sp. were improved, in increasing order, by supplementing with 10 mg MnSO4 ⋅ 4H2O l−1, 2 mg CuSO4 ⋅ 5H2O l−1, and both 10 mg MnSO4 ⋅ 4H2O l−1 and 2 mg CuSO4 ⋅ 5H2O l−1. Mn2+ decreased the intracellular concentration of erythritol, whereas Cu2+ increased the activity of erythrose reductase in cells. These results suggest that Mn2+ altered the permeability of cells, whereas Cu2+ increased the activity of erythrose reductase in cells.

Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter ( cdt-1 ) and intracellular β-glucosidase ( gh1-1 ) genes from Neurospora crassa . Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae . However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1 . The rate of cellobiose fermentation by the HXT2.4 -expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher K m and 4-fold higher V max values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily glucose and xylose. However, strains of Saccharomyces cerevisiae presently used in bioethanol production ferment glucose but not xylose. Yeasts engineered to ferment xylose do so slowly, and cannot utilize xylose until glucose is completely consumed. To overcome these bottlenecks, we engineered yeasts to coferment mixtures of xylose and cellobiose. In these yeast strains, hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular β-glucosidase following import by a high-affinity cellodextrin transporter. Intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation allowing coconsumption of cellobiose and xylose. The resulting yeast strains, cofermented cellobiose and xylose simultaneously and exhibited improved ethanol yield when compared to fermentation with either cellobiose or xylose as sole carbon sources. We also observed improved yields and productivities from cofermentation experiments performed with simulated cellulosic hydrolyzates, suggesting this is a promising cofermentation strategy for cellulosic biofuel production. The successful integration of cellobiose and xylose fermentation pathways in yeast is a critical step towards enabling economic biofuel production.

A maltogenic amylase gene from Lactobacillus gasseri ATCC33323 (LGMA) was expressed in Lactococcus lactis MG1363 using the P170 expression system. The successful production of recombinant LGMA (rLGMA) was confirmed by the catalytic activity of the enzyme in liquid and solid media. The N-terminal amino acid sequencing analysis of the rLGMA showed that it was Met-Gln-Leu-Ala-Ala-Leu-, which was the same as that of genuine protein, meaning the signal peptide was efficiently cleaved during secretion to the extracellular milieu. The optimal reaction temperature and pH of rLGMA (55 degrees C and pH 5, respectively) and enzymatic hydrolysis patterns on various substrates (beta-cyclodextrin, starch, and pullulan) supported that rLGMA was not only efficiently secreted from the Lactococcus lactis MG1363 but was also functionally active. Finally, the branched maltooligosaccharides were effectively produced from liquefied corn starch, by using rLGMA secreted from Lactococcus lactis, with a yield of 53.1%.