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Although engineered Saccharomyces cerevisiae fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with S. cerevisiae expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered S. cerevisiae fermenting cellobiose. From the series of cellobiose fermentation using S. cerevisiae expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1. As GH1-1 does not have a secretion signal peptide, non-conventional protein secretion might facilitate the secretion of GH1-1. In cellobiose fermentations with S. cerevisiae expressing only GH1-1, knockout of TLG2 gene involved in non-conventional protein secretion pathway significantly delayed cellodextrin formation by reducing the secretion of GH1-1 by more than 50%. However, in cellobiose fermentations with S. cerevisiae expressing both GH1-1 and CDT-1, TLG2 knockout did not show a significant effect on cellodextrin formation, although secretion of GH1-1 was reduced by more than 40%. These results suggest that the development of new intracellular β-glucosidase, not influenced by non-conventional protein secretion, is required for better cellobiose fermentation performances of engineered S. cerevisiae fermenting cellobiose.

This study is aimed to explore the impact of fermentation temperature on laver kombucha by profiling the accumulation and degradation of metabolites and elucidating their related pathways of metabolism. Laver kombucha was produced through ultrasound-assisted extraction and fermentation using a biofilm called SCOBY at 25 and 30 °C (hereafter named K-25 and K-30, respectively) for 14 days. Overall, organic acids, soluble sugars, amino acids, and phenolic compounds were found to participate in the biosynthesis pathway. The level of amino acids showed a decreasing trend, except taurine in the K-30. At day 14, phenolic compounds (pyrogallol, ρ-hydroxybenzoic acid, ρ-coumaric acid, salicylic acid, rutin, and naringin) were accumulated in both samples. Although it showed a similar trend, K-25 exhibited a higher metabolite accumulation tendency than K-30. This comprehensive characterization of the dynamic changes of metabolites and pathway prediction can pinpoint the influence of the fermentation conditions on the biosynthesis of secondary metabolites.

Until recently, four types of cellobiose-fermenting

We investigated the feasibility of producing bioethanol from mixed softwood pretreated with the ionic liquid 1-butyl-3-methylimidazolium acetate ([Bmim]Ac). The optimal pretreatment conditions were determined by response surface methodology to be 100°C for 15 h, and the fermentable sugar yield was estimated to be 92.5%. Efficient pretreatment of softwood was maintained even after reutilizing [Bmim]Ac up to four times. Through the enzymatic saccharification and subsequent fermentation, bioethanol was produced with 0.42 g/g of yield and 0.24 g/L/h of productivity, which clearly suggests that efficient and economical bioethanol production can be achieved by optimizing pretreatment processes and reutilizing ionic liquid.

In a previously engineered Saccharomyces cerevisiae recombinant, the cellobiose fermentation rate was significantly lower than the glucose fermentation rate. Thus, we implemented a genome-wide perturbation library to find gene targets for improving the cellobiose fermentation capability of the yeast strain. Unexpectedly, we discovered a transformant that contained an additional β-glucosidase gene (gh1-1), possibly through homologous recombination between the plasmids. The additional β-glucosidase led to the fastest cellobiose fermentation activity among all the transformants evaluated, and the strain demonstrated significantly higher β-glucosidase activity than the control strain, especially during the initial exponential growth phase. The present work revealed the benefit of the extra gh1-1 copy for efficient cellobiose fermentation in the engineered S. cerevisiae strain.

2'-Fucosyllactose (2-FL) is one of most abundant functional oligosaccharides in human milk, which is involved in many biological functions for human health. To date, most microbial systems for 2-FL production have been limited to use Escherichia coli JM strains since they cannot metabolize lactose. In this study, E. coli BL21star(DE3) was engineered through deletion of the whole endogenous lactose operon and introduction of the modified lactose operon containing lacZ△M15 from E. coli K-12. Expression of genes for guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic enzymes and heterologous α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori allowed the engineered E. coli BL21star(DE3) to produce 2-FL with 3-times enhanced yield than the non-engineered E. coli BL21star(DE3). In addition, the titer and yield of 2-FL were further improved by adding the three aspartate molecules at the N-terminal of FucT2. Overall, 6.4 g/L 2-FL with the yield of 0.225 g 2-FL/g lactose was obtained in fed-batch fermentation of the engineered E. coli BL21star(DE3) expressing GDP-l-fucose biosynthetic enzymes and three aspartate tagged FucT2.

In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular β-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular β-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution. Nine rounds of a serial subculture of S. cerevisiae expressing CDT-2 and cellobiose phosphorylase on cellobiose led to the isolation of an evolved strain capable of fermenting cellobiose to ethanol 10-fold faster than the original strain. After sequence analysis of the isolated CDT-2, a single point mutation on CDT-2 (N306I) was revealed to be responsible for enhanced cellobiose fermentation. Also, the engineered strain expressing the mutant CDT-2 with cellobiose phosphorylase showed a higher ethanol yield than the engineered strain expressing CDT-1 and intracellular β-glucosidase under anaerobic conditions, suggesting that CDT-2 coupled with cellobiose phosphorylase may be better choices for efficient production of cellulosic ethanol with the engineered yeast.

Hwa Young Choi, Ling Li, Seung Kee Cho, Won-Heong Lee, Jin-Ho Seo, and Nam Soo Han* Department of Food Science and Technology, BK21 Education and Research Center for Advanced Bio-Agriculture Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea Department of Bioenergy Science and Technology, Chonman National University, Gwangju 500-757, Republic of Korea Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Republic of Korea

Saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate cellodextrin utilization by S. cerevisiae, a fungal cellodextrin-utilizing pathway from Neurospora crassa consisting of a cellodextrin transporter and a cellodextrin hydrolase has been introduced into S. cerevisiae. Two cellodextrin transporters (CDT-1 and CDT-2) were previously identified in N. crassa, but their kinetic properties and efficiency for cellobiose fermentation have not been studied in detail. In this study, CDT-1 and CDT-2, which are hypothesized to transport cellodextrin with distinct mechanisms, were introduced into S. cerevisiae along with an intracellular β-glucosidase (GH1-1). Cellobiose transport assays with the resulting strains indicated that CDT-1 is a proton symporter while CDT-2 is a simple facilitator. A strain expressing CDT-1 and GH1-1 (DCDT-1G) showed faster cellobiose fermentation than the strain expressing CDT-2 and GH1-1 (DCDT-2G) under various culture conditions with different medium compositions and aeration levels. While CDT-2 is expected to have energetic benefits, the expression levels and kinetic properties of CDT-1 in S. cerevisiae appears to be optimum for cellobiose fermentation. These results suggest CDT-1 is a more effective cellobiose transporter than CDT-2 for engineering S. cerevisiae to ferment cellobiose.

Simultaneous saccharification and fermentation (SSF) has been considered a promising and economical process for cellulosic ethanol production. Further cost savings could be gained by reducing enzyme loading and engineering host strain for ethanol production. In this study, we demonstrate efficient ethanol production by SSF without supplementation of β-glucosidase using an engineered Saccharomyces cerevisiae strain expressing a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa. Ethanol production profiles by the engineered yeast without supplementation of β-glucosidase and by a parental strain with supplementation of β-glucosidase were examined under various fermentation conditions. When initial cell mass concentrations were low, the traditional SSF with supplementation of β-glucosidase showed better ethanol production than SSF with the engineered strain without supplementing β-glucosidase. However, the engineered strain without supplementation of β-glucosidase showed almost the same or even better ethanol productivity than the parental strain with supplementation of β-glucosidase when initial cell mass concentrations were elevated. Our results suggest that efficient ethanol production by SSF could be achieved by engineered yeast capable of fermenting cellobiose without addition of extracellular β-glucosidase, leading to economic production of cellulosic ethanol.

The noise map can be applied to predict the effect of noise and establish the noise reduction measure. But the predicted value in the noise map can vary depending on the input variables. Thus, we surveyed the several prediction models and analyzed the changes corresponding to the variables for obtaining the coherency and accuracy of prediction results. As a result, we know that the Schall03 and CRN model can be applied to predict the railway noise in Korea and the correction value, such as bridges correction, multiple reflection correction, curve correction must be used for reflecting the condition of the prediction site. Also, we know that the prediction guideline is an essential prerequisite in order to obtain the unified and accurate predicted value for railway noise.

Efficient regeneration of NADPH is one of the limiting factors that constrain the productivity of biotransformation processes. In order to increase the availability of NADPH for enhanced biotransformation by engineered Escherichia coli, modulation of the pentose phosphate pathway and amplification of the transhydrogenases system have been conventionally attempted as primary solutions. Recently, other approaches for stimulating NADPH regeneration during glycolysis, such as replacement of native glyceradehdye-3-phosphate dehydrogenase (GAPDH) with NADP-dependent GAPDH from Clostridium acetobutylicum and introduction of NADH kinase catalyzing direct phosphorylation of NADH to NADPH from Saccharomyces cerevisiae, were attempted and resulted in remarkable impacts on NADPH-dependent bioprocesses. This review summarizes several metabolic engineering approaches used for improving the NADPH regenerating capacity in engineered E. coli for whole-cell-based bioprocesses and discusses the key features and progress of those attempts.

Recently, the impact on hearing induced by using of portable audio equipment have been actively studied. In general, Because they turn the volume up with loud background noise, they may expose to louder noise. In this study, we investigated the maximum noise level of 20 the domestic potable audio equipment and estimated the impact of the hearing induced by portable audio equipment in according to exposure time. As a result, the use of portable audio equipment is assumed to be more three hours when the level of more than 50 % of volume is most likely to affect the hearing.

The focus of this work was to develop a combined acid and alkaline hydrothermal pretreatment of lignocellulose that ensures high recovery of both hexose and pentose. Dilute sulfuric acid and lime pretreatments were employed sequentially. Process performance was optimized in terms of catalyst concentration, retention time, and temperature using response surface methodology. Medium operational conditions in the acid stage and harsh conditions in the alkaline stage were desirable with optimal performance at 0.73 wt% H(2)SO(4), 150 °C, 6.1 min in the first stage, and 0.024 g lime/g biomass, 202 °C, 30 min in the second stage. In comparison to single-stage pretreatments with high recovery of either glucose or xylose, two-stage process showed great promises with80 % glucose and70 % xylose recovery. In addition, the method greatly improved ethanol fermentation with yields up to 0.145 g/g Miscanthus, due to significantly reduced formation of inhibitory by-products such as weak acids, furans, and phenols. Supplementing biomimetic acids would further increase glucose yield by up to 15 % and xylose yield by 25 %.

As Saccharomyces cerevisiae cannot utilize xylose as a carbon source, expression of XYL1 coding for xylose reductase (XR) from Scheffersomyces (Pichia) stipitis enabled production of xylitol from xylose with a high yield. However, insufficient supply of NAD(P)H for XR and inhibition of xylose uptake by glucose are identified as major constraints for achieving high xylitol productivity. To overcome these problems, we engineered S. cerevisiae capable of converting xylose into xylitol through simultaneous utilization of xylose and cellobiose. An engineered S. cerevisiae (D-10-BT) expressing XR, cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) produced xylitol via simultaneous utilization of cellobiose and xylose. The D-10-BT strain exhibited 40% higher volumetric xylitol productivity with co-consumption of cellobiose and xylose compared to sequential utilization of glucose and xylose. Furthermore, the overexpression of S. cerevisiae ALD6, IDP2, or S. stipitis ZWF1 coding for cytosolic NADP(+)-dependent dehydrogenases increased the intracellular NADPH availability of the D-10-BT strain, which resulted in a 37-63% improvement in xylitol productivity when cellobiose and xylose were co-consumed. These results suggest that co-utilization of cellobiose and xylose can lead to improved xylitol production through enhanced xylose uptake and efficient cofactor regeneration.

Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant cellobiose transporter [CDT-1 (F213L) exhibiting higher VMAX than CDT-1] and GH1-1 in this study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases mixture in SSF could lead the further engineered strain to produce ethanol considerably better than the parental strain with β-glucosidase. Probably, better production of ethanol by the further engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to not only 2-times lower Monod constant (KS) for cellobiose than KS of the parental strain for glucose but also 5-times lower KS than Michaelis-Menten constant (KM) of the extracellular β-glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the engineered yeast to transport lower level of cellobiose enables a more efficient SSF for producing ethanol from cellulose.

Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-L-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E. coli producing GDP-L-fucose resulted in a maximum GDP-L-fucose concentration of 291.5 mg/l, which corresponded to a 51 % enhancement compared with the control strain. In a fed-batch Baeyer-Villiger (BV) oxidation of cyclohexanone using recombinant E. coli expressing Pos5p, a maximum ε-caprolactone concentration of 21.6 g/l was obtained, which corresponded to a 96 % enhancement compared with the control strain. Such an increase might be due to the enhanced availability of NADPH in recombinant E. coli expressing Pos5p. These results suggested that efficient regeneration of NADPH was possible by functional expression of Pos5p in recombinant E. coli, which can be applied to other NADPH-dependent biotransformation processes in E. coli.

Guanosine 5'-triphosphate (GTP) is the key substrate for biosynthesis of guanosine 5'-diphosphate (GDP)-L-fucose. In this study, improvement of GDP-L-fucose production was attempted by manipulating the biosynthetic pathway for guanosine nucleotides in recombinant Escherichia coli-producing GDP-L-fucose. The effects of overexpression of inosine 5'-monophosphate (IMP) dehydrogenase, guanosine 5'-monophosphate (GMP) synthetase (GuaB and GuaA), GMP reductase (GuaC) and guanosine-inosine kinase (Gsk) on GDP-L-fucose production were investigated in a series of fed-batch fermentations. Among the enzymes tested, overexpression of Gsk led to a significant improvement of GDP-L-fucose production. Maximum GDP-L-fucose concentration of 305.5 ± 5.3 mg l(-1) was obtained in the pH-stat fed-batch fermentation of recombinant E. coli-overexpressing Gsk, which corresponds to a 58% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes. Such an enhancement of GDP-L-fucose production could be due to the increase in the intracellular level of GMP.

Genes encoding GDP-mannose 4, 6-dehydratase (GMD) and GDP-l-fucose synthetase (GFS) were cloned from Bacteroides thetaiotaomicron and overexpressed in recombinant Escherichia coli by constructing isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression vectors. GMD and GFS genes from B. thetaiotaomicron were 60 and 45%, respectively, identical to those from E. coli K12 over their entire lengths. An optimum expression condition of 30oC and 0.1 mM IPTG was chosen for maximum soluble expression of B. thetaiotaomicron GMD and GFS in recombinant E. coli BL21(DE3). Functional expression of B. thetaiotaomicron GMD and GFS in recombinant E. coli strains was confirmed by measuring intracellular GDP-l-fucose content.

The biocatalytic efficiency of recombinant Corynebacterium glutamicum expressing the chnB gene encoding cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871 was investigated. Optimization of an expression system and induction conditions enabled the recombinant biocatalyst to produce CHMO to a specific activity of ca. 0.5 U mg −1 protein. Tight control of feeding of an energy source (i.e., glucose) and dissolved oxygen tension during fed-batch culture-based biotransformation allowed the cells to produce ɛ-caprolactone to a concentration of 16.0 g l −1 . The specific and volumetric productivity for cyclohexanone oxidation were 0.12 g g dry cells −1 h −1 (17.5 U g −1 of dry cells) and 2.3 g l −1 h −1 (330 U l −1 ), respectively. These values correspond to over 5.4- and 2.7-fold of recombinant Escherichia coli expressing the same gene under similar reaction conditions. It could be concluded that the recombinant C. glutamicum is a promising biocatalyst for Baeyer–Villiger oxidations.

Sepiapterin is a precursor for the synthesis of tetrahydrobiopterin (BH4), which is a wellknown cofactor for aromatic amino acid hydroxylation and nitric oxide synthesis in higher mammals. In this study, a recombinant Escherichia coli BL21(DE3) strain harboring cyanobacterial guanosine 5'-triphosphate cyclohydrolase 1 (GCH1) and human 6- pyruvoyltetrahydropterin synthase (PTPS) genes was constructed to produce sepiapterin. The optimum conditions for T7 promoter-driven expression of GCH1 and PTPS were 30°C and 0.1 mM isopropyl-β-D-thioglucopyranoside (IPTG). The maximum sepiapterin concentration of 88.1 ± 2.4 mg/l was obtained in a batch cultivation of the recombinant E. coli, corresponding to an 18-fold increase in sepiapterin production compared with the control condition (37°C and 1 mM IPTG).

Zero trans-influx assays of glucose and xylose were performed using Saccharomyces cerevisiae to investigate transport characteristics under high and low glucose conditions. Under high glucose conditions, most glucose was transported by the low-affinity transporter. The high-affinity transporter was expressed under low glucose conditions, transporting over 50% glucose. Inhibition kinetics revealed that xylose was transported by both high- and low-affinity glucose transporters. Affinities of both glucose transporters for xylose were very low under high glucose condition but increased to a similar level to glucose under low glucose condition. The maximum rate of xylose transport increased by 85%, while an overall maximum glucose transport rate decreased by 42% under low glucose condition, indicating the presence of other transport system for sugars except for glucose. It was suggested that expression of the high-affinity transporter and increased affinity of glucose transporters for xylose under low glucose condition would provide a fermentation strategy for enhancing the productivity of xylitol by recombinant S. cerevisiae harboring the xylose reductase gene.

Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

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 (0.25 g xylose h−1 g cells−1) as fast as YSX3 and accumulated a small amount of xylitol (0.03 g g xylose−1) as low as SX3, resulting in an improved ethanol yield (0.27 g g xylose−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.

Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

Guanosine 5’-diphosphate (GDP)- l -fucose, an activated form of a nucleotide sugar, plays an important role in a wide range of biological functions. In this study, the enhancement of GDP- l -fucose production was attempted by supplementation of mannose, which is a potentially better carbon source to be converted into GDP- l -fucose than glucose, and combinatorial overexpression of the genes involved in the biosynthesis of GDP- d -mannose, a precursor of GDP- l -fucose. Supply of a mannose and glucose led to a 1.3-fold-increase in GDP- l -fucose concentration (52.5 ± 0.8 mg l −1 ) in a fed-batch fermentation of recombinant E. coli BL21star(DE3) overexpressing the gmd and wcaG genes, compared with the case using glucose as a sole carbon source. A maximum GDP- l -fucose concentration of 170.3 ± 2.3 mg l −1 , corresponding to a 4.4-fold enhancement compared with the control strain overexpressing gmd and wcaG genes only, was achieved in a glucose-limited fed-batch fermentation of a recombinant E. coli BL21star(DE3) strain overexpressing manB , manC , gmd and wcaG genes. Further improvement of GDP- l -fucose production was not obtained by additional overexpression of the manA gene.

Whole-cell conversion of cyclohexanone to epsilon-caprolactone was attempted by recombinant Escherichia coli BL21(DE3) expressing cyclohexanone monooxygenase (CHMO) of Acinetobacter calcoaceticus NCIMB 9871. High concentrations of cyclohexanone and epsilon-caprolactone reduced CHMO-mediated bioconversion of cyclohexanone to epsilon-caprolactone in the resting recombinant E. coli cells. Metabolically active cells were employed by adopting a fed-batch culture to improve the production of epsilon-caprolactone from cyclohexanone. A glucose-limited fed-batch Baeyer-Villiger oxidation where a cyclohexanone level was maintained less than 6 g/l resulted in a maximum epsilon-caprolactone concentration of 11.0 g/l. The maximum epsilon-caprolactone concentration was improved further to 15.3 g/l by coexpression of glucose-6-phosphate dehydrogenase, an NADPH-generating enzyme encoded by the zwf gene which corresponded to a 39% enhancement in epsilon-caprolactone concentration compared with the control experiment performed under the same conditions.

A recombinant Escherichia coli strain was developed to produce guanosine 5'-diphosphate (GDP)-L-fucose, donor of L-fucose, which is an essential substrate for the synthesis of fucosyloligosaccharides. GDP-D: -mannose-4, 6-dehydratase (GMD) and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase (WcaG), the two crucial enzymes for the de novo GDP-L-fucose biosynthesis, were overexpressed in recombinant E. coli by constructing inducible overexpression vectors. Optimum expression conditions for GMD and WcaG in recombinant E. coli BL21(DE3) were 25 degrees C and 0.1 mM isopropyl-beta-D-thioglucopyranoside. Maximum GDP-L-fucose concentration of 38.9 +/- 0.6 mg l(-1) was obtained in a glucose-limited fed-batch cultivation, and it was enhanced further by co-expression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 +/- 0.5 mg l(-1) GDP-L-fucose under the same cultivation condition.

Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce epsilon-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. A fed-batch process was designed to obtain high cell density for improving production of epsilon-caprolactone. The fed-batch SPO process gave the best results, 10.2 g/L of epsilon-caprolactone and 0.34 g/(L.h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.

Background 2'-Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5′-diphosphate (GDP)-l-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP- l-fucose biosynthetic enzymes and heterologous fucosyltransferase. Results The gene for FucT2 from Helicobacter pylori was introduced to the GDP- l-fucose producing recombinant E. coli BL21 star(DE3) strain. However, only small amount of 2-FL was produced in a batch fermentation because the E. coli BL21star(DE3) strain assimilated lactose instead of converting to 2-FL. As an alternative host, the E. coli JM109(DE3) strain which is incapable of assimilating lactose was chosen as a 2-FL producer. Whole cell biosynthesis of 2-FL from lactose was investigated in a series of batch fermentations using various concentrations of lactose. The results of batch fermentations showed that lactose was slowly assimilated by the engineered E. coli JM109(DE3) strain and 2-FL was synthesized without supplementation of another auxiliary sugar for cell growth. A maximum 2-FL concentration of 1.23 g/l was obtained from a batch fermentation with 14.5 g/l lactose. The experimentally obtained yield (g 2-FL/g lactose) corresponded to 20% of the theoretical maximum yield estimated by the elementary flux mode (EFM) analysis. Conclusions The experimental 2-FL yield in this study corresponded to about 20% of the theoretical maximum yield, which suggests further modifications via metabolic engineering of a host strain or optimization of fermentation processes might be carried out for improving 2-FL yield. Improvement of microbial production of 2-FL from lactose by engineered E. coli would increase the feasibility of utilizing 2-FL as a prebiotic in various foods.