Search

Uridine diphosphate N-acetylglucosamine (UDPAG) can be produced by chemical, enzymatic, chemoenzymatic, and fermentative methods. In this study, we used whole-cell catalysis method to produce UDPAG for the first time by Saccharomyces cerevisiae. In order to increase the ATP utilization efficiency and UDPAG conversion yield, the response surface methodology was applied to optimize the whole-cell catalytic conditions for UDPAG production. Firstly, effects of uridine 5′-monophosphate (5′-UMP), glucosamine, vitamin B1, glycerol, magnesium chloride, potassium chloride, temperature, sodium dihydrogen phosphate, sodium acetate, fructose, and pH on UDPAG production were evaluated by a fractional factorial design. Results showed that UDPAG production was mainly affected by sodium dihydrogen phosphate, temperature, and vitamin B1. Then, the concentrations of sodium dihydrogen phosphate and vitamin B1 and temperature were further investigated with a central composite design and response surface analysis. The cultivation conditions to obtain the optimal UDPAG production were determined: sodium dihydrogen phosphate, 31.2 g/L; temperature, 29°C, and vitamin B1, 0.026 g/L. This optimization strategy led to an enhancement of UDPAG production from 2.51 to 4.25 g/L, yield from 44.6% to 75.6% based on the initial 5′-UMP concentration, and ATP utilization efficiency from 7.43% to 12.6%. [ABSTRACT FROM AUTHOR]

The article informs considerable progress has been made in recent years to develop chemoenzymatic processes by the integration of environmentally friendly feature of biocatalysis with versatile reactivity of chemocatalysis. Mentions the current types of chemoenzymatic processes are relatively few and mostly rely on metal catalysts. Reports a previously unexplored cooperative chemoenzymatic system for the synthesis of N-heterocycles.

Attempts were made with success to produce uridine 5′-monophosphate (UMP) from orotic acid by a recombinant Saccharomyces cerevisiae strain pYX212-URA5/BJX12, using the whole cell biocatalytic process. URA5 and URA3 genes, encoding orotate phosphoribosytransferase (OPRTase) and orotidine monophosphate decarboxylase (ODCase), respectively were successfully overexpressed in the industrial yeast strain. As a result, S .cerevisae pYX212-URA5/BJX12 exhibited the highest biocatalytic ability, in contrast with the original industrial yeast strain and S. cerevisae pYX212/BJX12 that overexpressed ODCase only. It indicated that the first step of UMP production from orotic acid is a rate-limiting step. Effects of cultivation for the recombinant strain and biocatalytic reaction conditions on UMP production were also investigated. Cultivating the cells in malt extract medium for 36 h in the exponential phase of growth is in favor of converting orotic acid to UMP. To acquire a higher UMP yield, the conditions of the whole cell biocatalytic reaction were optimized and up to 3.8 g l UMP was produced in 24 h consequently. The yield was fivefold higher than the original UMP yield from the industrial yeast. In addition, the accumulation of 2.6 g l UDP (uridne 5′-diphosphate) in the process demonstrated the possibility for further genetic manipulation: deleting the UMPK (Uridylate Kinase, catalyzing UMP-UDP). [ABSTRACT FROM AUTHOR]

Ethyl ( S)-4-chloro-3-hydroxybutanoate ester (( S)-CHBE) is a precursor of enantiopure intermediates used for the production of chiral drugs, including the cholesterol-lowering 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitors (statins). The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate ester (COBE) to ( S)-CHBE by biocatalysis has several positive attributes, including low cost, mild reaction conditions, high yield, and a high level of enantioselectivity. During genome database mining of the yeast Pichia stipitis, our group found two novel carbonyl reductases (PsCRI and PsCRII) that have a promising future for the industrial production of ( S)-CHBE with >99% enantiomeric excess. This review covers the main process of biosynthesis of ( S)-CHBE: screening of microorganisms that catalyze the reduction of COBE to ( S)-CHBE (I); gene cloning, expression, and characterization of carbonyl reductases for the production of ( S)-CHBE in Escherichia coli (II); development of cofactor generation systems for regenerating cofactors (III); and biocatalysis of COBE to ( S)-CHBE by recombinant E. coli (IV). [ABSTRACT FROM AUTHOR]

Available molecular and genetic tools for the genetic manipulation of Arthrobacter species are limited until now. In gene engineering, a continuous set of promoters with various strengths are of importance for fine-tuning gene expression in metabolic optimization and control analysis. Here, for the first time, we constructed a promoter trap system using green fluorescence protein (GFP) as a reporter, for screening and characterizing functional Arthrobacter promoters. Twenty-three Arthrobacter transformants of various GFP fluorescence strengths were isolated and characterized through the analysis of DNA sequences. Among the 23 putative promoters, 2 were selected for deletion analysis of promoter elements. As a result, the deletion of the upstream of the putative promoter P8 and P13 caused a 43.8% decrease and a 29.1% increase in the fluorescence signals, respectively. Finally, we obtained the strongest promoter P13-3 which was 4.4 times more potent than the promoter of 6– hydroxyl–d–nicotine oxidase gene which was previously reported in Arthrobacter nicotinovorans, and the obtained promoter was used to improve the production of cyclic adenosine monophosphate in Arthrobacter sp. CGMCC 3584. The screening strategy together with obtained promoters in this study would contribute to the future engineering of Arthrobacter species. [ABSTRACT FROM AUTHOR]

Solvent-mediated transformation of disodium guanosine 5'-monophosphate (5'-GMPNa2) from amorphous to hydrated crystal phases was studied. The SMT process is dominated by the dissolution of the amorphous form and nucleation and growth of the crystal polymorph. The kinetic estimation based on population balance equation was carried out according to experimental data, including polymorphic fraction in the solid phase, solute concentration, and crystal size distribution. Furthermore, independent seeded batch experiments were carried out to estimate the kinetic parameters. The experimental data showed that the nucleation and growth of the crystal polymorph occurred shortly after the dissolution of the amorphous form. The estimated growth, nucleation, and dissolution rates indicated that the solution-mediated transformation was governed by the nucleation and growth of crystal polymorph. We believe that the results hold great importance toward understanding the transformation process of 5'-GMPNa2, which provides theoretical guidance for producing its specific forms, and possibly those for other pharmaceuticals and chemicals as well. [ABSTRACT FROM AUTHOR]

The solubility of biological chemicals in solvents provide important fundamental data and is generally considered as an essential factor in the design of crystallization processes. The equilibrium solubility data of inosine-5'-monophosphate disodium (5'-IMPNa2) in water, methanol, ethanol, acetone, as well as in the solvent mixtures (methanol + water, ethanol + water, acetone + water), were measured by an isothermal method at temperatures ranging from (293.15 to 313.15) K. The measured data in pure and mixed solvents were then modelled using the modified Apelblat equation, van't Hoff equation, λh equation, ideal model and the Wilson model. The modified Apelblat equation showed the best modelling results, and it was therefore used to predict the mixing Gibbs free energies, enthalpies, and entropies of 5'-IMPNa2in pure and binary solvents. The positive values of the calculated partial molar Gibbs free energies indicated the variations in the solubility trends of 5'-IMPNa2. Water and ethanol (in the binary mixture with water) were found to be the most effective solvent and anti-solvent, respectively. [ABSTRACT FROM AUTHOR]

With the aid of the highly active nitroxyl radical AZADO (2-azaadamantane N-oxyl), a simple method for the aerobic catalytic oxidation of alcohols is presented. The oxidations could typically proceed under practical ambient conditions (room temperature, air atmosphere, no moisture effect, metal-free, etc.) with a broad generality of the alcohol substrates, and especially for the oxidation of complex and highly functionalized alcohols. An ionic mechanism is proposed for the present system. [ABSTRACT FROM AUTHOR]

cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to ( S)-4-chloro-3-hydroxybutanoate ethyl ester [( S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/ n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles ( S)-CHBE formed per mole NAD+, was 13,980. [ABSTRACT FROM AUTHOR]

To develop an economical industrial medium, untreated cane molasses (UCM) was tested as a carbon source for fermentation culturing of Escherichia coli. To test the industrial application of this medium, we chose a strain co-expressing a carbonyl reductase (PsCR) and a glucose dehydrogenase (BmGDH). Although corn steep liquor (CSL) could be used as an inexpensive nitrogen source to replace peptone, yeast extract could not be replaced in E. coli media. In a volume of 40 ml per 1-l flask, a cell concentration of optical density (OD600) 15.1 and enzyme activities of 6.51 U/ml PsCR and 3.32 U/ml BmGDH were obtained in an optimized medium containing 25.66 g/l yeast extract, 3.88 g/l UCM, and 7.1% ( v/ v) CSL. When 3.88 g/l UCM was added to the medium at 6 h in a fed-batch process, the E. coli concentration increased to OD600 of 24, and expression of both PsCR and BmGDH were twofold higher than that of a batch process. Recombinant cells from batch or fed-batch cultures were assayed for recombinant enzyme activity by testing the reduction of ethyl 4-chloro-3-oxobutanoate to ethyl ( S)-4-chloro-3-hydroxybutanoate (CHBE). Compared to cells from batch cultures, fed-batch cultured cells showed higher recombinant enzyme expression, producing 560 mM CHBE in the organic phase with a molar yield of 92% and an optical purity of the ( S)-isomer of >99% enantiomeric excess. [ABSTRACT FROM AUTHOR]

A whole cell biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l−1 sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle. [ABSTRACT FROM AUTHOR]

A study was conducted to investigate the hydrolysis of cellulose-enriched corncob residue, a cellulosic waste from the xylo-oligosacchaddes industry, by two processes. The corncob residue was hydrolyzed by cellulases via direct hydrolysis and two-step hydrolysis. Cellulases were produced by Trichoderma reesei RutC-30 with kraft pulp as the substrate. When the cellulase dosage was above 8 FPU·g-1 of corncob residue and the corncob residue concentration was 3%, over 90% hydrolysis yield and 49.99% glucose yield were obtained at 48 h. To enhance the hydrolysis yield of corncob residue, a new process coupling enzymatic hydrolysis, separation, and acid treatment was investigated. The corncob residue was first hydrolyzed using cellulase for 24 h. Then the remaining solids of corncob residue was separated from the liquid containing soluble oligosaccharides, and allowed to subsequently hydrolyze, using the adsorbed enzyme for 24 h. Using this method, the total hydrolysis yield was up to 97.60%, which represents an increase by 7.5% in comparison to the direct 48 h enzymatic hydrolysis. When the hydrolysates of the two-step enzymatic process were subjected to the concentrated acid hydrolysis at 110 °C for 2 h, the glucose yield could be increased from 43% to 99%. [ABSTRACT FROM AUTHOR]