Yeon-Woo Ryu

Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae

Abstract. 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.

Biotechnological production and applications of coenzyme Q10

Coenzyme Q10 is widely used as an essential component of ATP generation in the oxidative phosphorylation process and as an antioxidant preventing lipid peroxidation and scavenging superoxide. It is also recommended as a supplement to 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Research efforts on the production of coenzyme Q10 by microorganisms focus on the development of potent strains by conventional mutagenesis and metabolic engineering, analysis and modification of the key metabolic pathways and optimization of fermentation strategies. Especially, random mutants with drugs resistance show a high coenzyme Q10 concentration. Metabolic engineering techniques have been applied to improve coenzyme Q10 production. The key enzymes involved in the coenzyme Q10 biosynthesis pathway have been cloned and expressed in Escherichia coli. The rational design of metabolic pathways in combination with engineering optimization of fermentation processes could facilitate the development of viable bioconversion processes.

Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene

Abstract Fermentation characteristics of recombinant Saccharomyces cerevisiae containing a xylose reductase gene from Pichia stipitis were investigated in an attempt to convert xylose to xylitol, a natural five-carbon sugar alcohol used as a sweetener. Xylitol was produced with a maximum yield of 0.95 g g −1 xylitol xylose consumed in the presence of glucose used as a co-substrate for co-factor regeneration. Addition of glucose caused inhibition of xylose transport and accumulation of ethanol. Such problems were solved by adopting glucose-limited fed-batch fermentations where a high ratio of xylose to glucose was maintained during the bioconversion phase. The optimized two-substrate fed-batch fermentation carried out with S. cerevisiae EH13.15:pY2XR at 30°C resulted in 105.2 g l −1 xylitol concentration with 1.69 g l −1 h −1 productivity.

Optimization of erythritol production by Candida magnoliae in fed-batch culture

A two-stage fed-batch process was designed to enhance erythritol productivity by the mutant strain of Candida magnoliae. The first stage (or growth stage) was performed in the fed-batch mode where the growth medium was fed when the pH of the culture broth dropped below 4.5. The second stage (or production stage) was started with addition of glucose powder into the culture broth when the cell mass reached about 75 g dry cell weight l−1. When the initial glucose concentration was adjusted to 400 g l−1 in the production stage, 2.8 g l−1 h−1 of overall erythritol productivity and 41% of erythritol conversion yield were achieved, which represented a fivefold increase in erythritol productivity compared with the simple batch fermentation process. A high glucose concentration in the production phase resulted in formation of organic acids including citrate and butyrate. An increase in dissolved oxygen level caused formation of gluconic acid instead of citric acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 100–103.

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

ABSTRACT Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (Km = 7.9 mM; kcat/Km = 0.73 mM−1 s−1). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (kcat/Km = 450 mM−1 s−1) than with NADPH (kcat/Km = 5.5 mM−1 s−1), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu2+ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l−1) and less than 200 g l−1 total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l−1 xylitol concentration, 0.75 g xylitol g xylose−1 xylitol yield and 3.9 g xylitol l−1 h−1 volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257

Stable expression of xylose reductase gene enhances xylitol production in recombinant Saccharomyces cerevisiae

Abstract Effects of the expression mode of the xylose reductase gene ( XYL1 ) on xylitol production in recombinant Saccharomyces cerevisiae strains were investigated in batch and fed-batch cultures. The gene coding for xylose reductase (XR) was introduced into S. cerevisiae in two different ways: by using a δ-integration vector for chromosome-integration and a YRp-based episomal plasmid vector. The two expression systems showed the different pattern of xylitol production in a glucose-limited fed-batch culture as opposed to the similar profile in a batch culture. The recombinant S. cerevisiae strain harboring the XR gene in the chromosome yielded a 1.70-fold enhancement in xylitol productivity in the fed-batch culture compared with the YRp-based xylose reductase expression strain. Such an improvement for the integrated recombinant strain was supported by the fact that the mitotic stability of the XR gene along with its high expression level worked in a cooperative manner.

Analysis and optimization of a two-substrate fermentation for xylitol production using Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose using Candida tropicalisATCC 13803. A two-substrate fermentation was designed in order to increase xylitol yield and volumetric productivity. Glucose was used initially for cell growth followed by conversion of xylose to xylitol without cell growth and by-product formation after complete depletion of glucose. High glucose concentrations increased volumetric productivity by reducing conversion time due to high cell mass, but also led to production of ethanol, which, in turn, inhibited cell growth and xylitol production. Computer simulation was undertaken to optimize an initial glucose concentration using kinetic equations describing rates of cell growth and xylose bioconversion as a function of ethanol concentration. Kinetic constants involved in the equations were estimated from the experimental results. Glucose at 32 g L−1 was estimated to be an optimum initial glucose concentration with a final xylose concentration of 86 g L−1 and a volumetric productivity of 5.15 g-xylitol L−1 h−1. The two-substrate fermentation was performed under optimum conditions to verify the computer simulation results. The experimental results were in good agreement with the predicted values of simulation with a xylitol yield of 0.81 g-xylitol g-xylose−1 and a volumetric productivity of 5.06 g-xylitol L−1 h−1.

Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester

The ester-hydrolyzing enzyme families, including lipase and esterase, mediated a broad range of reactions and, thus, were able to act on a variety of ester compounds that are found naturally or exploited industrially. With the increasing demand for pharmacological use, attempts to produce an enantiomer (S)-ketoprofen from the corresponding ethyl ester have recently been proliferating, but information about the structure and function of related enzymes has not been reported to date in detail. Here, we reported the construction, expression, and one-step purification of a potential esterase in Escherichia coli with a hexahistidine tag at its N-terminus. The expression level of the enzyme was more than 20% of the total protein in E. coli, resulting in approximately 1.2mg of the purified proteins by an affinity resin, Ni-NTA, from a 0.2L of bacterial culture in a single step. As typical properties, its innate traits that revealed favorable reactions at alkaline pH and high activity to the triglycerides composed of short chain fatty acids (<C(6)) supported the enzyme to be an esterase. The enzyme was determined to be a monomer with a calculated molecular mass of 42 kDa and showed quite a high activity to rac-ketoprofen ethyl ester (27,000 U), with strict selectivity to (S)-enantiomer (>99% ee(p)). The small-scale conversion using the recombinant enzyme strongly suggested the enzyme to be useful for enzyme-mediated chiral resolution of (S)-ketoprofen.

Cloning and Characterization of Thermostable Esterase from Archaeoglobus fulgidus

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86∼90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Aspl98 and His226. The esterase activity test with various acyl chain length of ρ-nitrophenol resulted that Est-AF showed highest specific activity with ρ-nitrophenylbutyrate (PNPC4) and rapidly decrease with ρ-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with ρ-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with ρ-nitrophenylbutyrate (pNPC4) was 80°C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70°C, and 10% of activity still remains after 3 h of incubation at 90°C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 µmole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.