Effect of High Product Concentration in a Dual Fed-batch Asymmetric 3-oxo Ester Reduction by Baker's Yeast

Baker's-yeast-mediated asymmetric ethyl 3-oxobutanoate reduction using a fed-batch feeding strategy for both the 3-oxo ester and the electron donor, was explored as potential production system for enantiopure ethyl ( S )-3-hydroxybutanoate. The dual feed strategy was based on kinetic and stoichiometric data. One major aspect is the effect of high product concentrations on the progress of the reduction. According to initial rate experiments, product inhibition occurs at concentrations above 600 mM product causing a 10-fold decrease of the initial biomass-specific reduction rate. By using optimized feed rates and a biomass concentration of 43 g dw l &#109 1, a product concentration of 350 mM was reached within 80 h with a degree of conversion of 95%. The volumetric productivity was 0.58 g l &#109 1 h &#109 1, using 2.1 kg pressed yeast kg product &#109 1 and 0.52 kg glucose kg product &#109 1. During the fed-batch biotransformation the reduction rate continuously decreased and reduction ceased after 80 h, due to biocatalyst inactivation after prolonged use at increasing high product concentrations. The continuous decrease in reducing activity led to very high ethyl 3-oxobutanoate levels in the reactor resulting in an increase of the undesired specific ethyl ( R )-3-hydroxybutanoate production rate. Therefore, the enantiomeric excess of the product decreased, from initially 100 to ~75% at 80 h. It is concluded that the design of processes for efficient asymmetric bioreduction cannot solely be based on initial rate kinetics, but require detailed knowledge of the effects on activity and enantioselectivity upon long-term exposure to process conditions.     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

Microbial synthesis of ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate by asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate in an aqueous-organic solvent biphasic system

In this study, whole cells of Saccharomyces uvarum SW-58 were applied in an aqueous-organic solvent biphasic system for the asymmetric reduction of ethyl 4,4,4-trifluoroacetoacetate to ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate [(R)-2]. The results of reduction in different aqueous-organic solvent biphasic systems showed that dibutylphthalate provided the best compromise between the biocompatibility and the partition of substrate and product among the solvents tested. To optimize the reaction, several factors such as reaction pH, temperature, shaking speed, volume ratio of the aqueous phase to the organic phase and ratio of biomass/substrate were investigated. It was found that the change of these factors obviously influenced the conversion and initial reaction rate, and had a minor effect on the enatiomeric excess of the product. Under the optimal conditions, 85.0% of conversion and 85.2% of enatiomeric excess were achieved. The bioconversion in the biphasic system was more efficient compared with that in the monophasic aqueous system, and product concentration as high as 54.6 g/L was reached in the organic phase without addition of co-enzyme.     

Yeast Catalysed Reduction of β-Keto Esters (2): Optimisation of the Stereospecific Reduction by Zygosaccharomyces Rouxii

Zygosaccharomyces rouxii catalysed the reduction of ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) to the corresponding (S)-hydroxy ester (ethyl (S)-4-chloro-3-hydroxybutanoate) in high enantiomeric excess. The productivity of non-immobilised cells was compared to cells immobilised on a range of organic and inorganic supports. Cells immobilised in calcium alginate displayed a catalytic activity significantly higher than that of non-immobilised cells. A time dependent fall in the enantiomeric purity of the product was observed with the use of this matrix. This phenomenon was not seen in the reduction catalysed by non-immobilised cells.     

Microbial asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to optically active ethyl-4-chloro-3-hydroxybutanoate

Summary The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate through the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate with the NADPH-dependent aldehyde reductase ofSporobolomyces salmonicolor AKU 4429 is described. Under preparative scale reaction conditions with the acetone-fractionated aldehyde reductase, the amount of ethyl-4-chloro-3-hydroxybutanoate reached 33.1 mg/ml (85%ee; molar yield, 74.0%). Furthermore, conversion to ethyl (S)-4-chloro-3-hydroxybutanoate occurred on incubation with washed cells ofTrichosporon cutaneum AKU 4864 as the catalyst.     

Synthese von 3-Merkapto-3-methylpentan-5-lacton

An NADPH-dependent sorbose reductase from Candida albicans was identified to catalyze the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE). The activity of the recombinant enzyme toward COBE was 6.2 U/mg. The asymmetric reduction of COBE was performed with two coexisting recombinant Escherichia coli strains, in which the recombinant E. coli expressing glucose dehydrogenase was used as an NADPH regenerator. An optical purity of 99% (e.e.) and a maximum yield of 1240 mM (S)-4-chloro-3-hydroxybutanoate were obtained under an optimal biomass ratio of 1:2. A highest turnover number of 53,900 was achieved without adding extra NADP⁺/NADPH compared with those known COBE-catalytic systems.     

Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate by a Microbial Aldehyde Reductase in an Organic Solvent-Water Diphasic System

Enzyme-catalyzed asymmetric reduction of ethyl 4-chloro-3-oxobutanoate in an organic solvent-water diphasic system was studied. NADPH-dependent aldehyde reductase isolated from Sporobolomyces salmonicolor AKU4429 and glucose dehydrogenase were used as catalysts for reduction of ethyl 4-chloro-3-oxobutanoate and recycling of NADPH, respectively, in this system. In an aqueous system, the substrate was unstable. Inhibition of the reaction and inactivation of the enzymes by the substrate and the product were also observed. An n-butyl acetate-water diphasic system very efficiently overcame these limitations. In a 1,600-ml−1,600-ml scale diphasic reaction, ethyl (R)-4-chloro-3-hydroxybutanoate (0.80 mol; 86% enantiomeric excess) was produced from the corresponding oxoester in a molar yield of 95.4% with an NADPH turnover of 5,500 mol/mol.     

A novel reductase from Candida albicans for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP+/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.     

Yeast Catalysed Reduction of β-Keto Esters (1): Factors Affecting Whole-Cell Catalytic Activity and Stereoselectivity

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92–99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93–94% ee) compared with cells harvested in exponential phase (43–60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65–81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.     

Yeast Catalysed Reduction of β-Keto Esters (1): Factors Affecting Whole-Cell Catalytic Activity and Stereoselectivity

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92-99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93-94% ee) compared with cells harvested in exponential phase (43-60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65-81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.     

Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor

An NADH-dependent reductase (ScCR) from Streptomyces coelicolor was discovered by genome mining for carbonyl reductases. ScCR was overexpressed in Escherichia coli BL21, purified to homogeneity and its catalytic properties were studied. This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, α- and β-ketoesters, with high activity and excellent enantioselectivity (>99% ee) towards β-ketoesters. Among them, ethyl 4-chloro-3-oxobutanoate (COBE) was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene biphasic system. As much as 600 g/L (3.6 M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a co-substrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of >99% ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR for the industrial production of valuable chiral alcohols.     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

Asymmetric synthesis of (S)-ethyl-4-chloro-3-hydroxybutanoate using Candida parapsilosis ATCC 7330

Asymmetric reduction of ethyl-4-chloro-3-oxobutanoate to (S)-ethyl-4-chloro-3-hydroxybutanoate in aqueous medium by resting cells of Candida parapsilosis ATCC 7330 was optimized. The influence of culture parameters (inoculum size, inoculum age and biocatalyst harvest time) and reaction parameters (co-substrate, resting cell, pH and substrate concentrations) on the asymmetric reduction were studied. It was found that these parameters significantly influenced the rate of the asymmetric reduction. Under the optimum conditions, the final concentration of (S)-ethyl-4-chloro-3-hydroxybutanoate, enantiomeric excess and the isolated yield of (S)-ethyl-4-chloro-3-hydroxybutanoate were 1.38 M (230 g/l), >99 and 95%, respectively. The space time yield was 115 mmol/lh, which is significantly higher than other whole cell biocatalysts reported so far.     

Bioconversion of ethyl 4-chloro-3-oxobutanoate by permeabilized fresh brewer’s yeast cells in the presence of allyl bromide

Ethyl(R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) are obtained by cetyltrimetylammonium bromide (CTAB) permeabilized fresh brewer’s yeast whole cells bioconversion of ethyl 4-chloro-3-oxobutanoate (COBE ) in the presence of allyl bromide. The results showed that the activities of alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in CTAB permeabilized brewer’s yeast cells increased 525 and 7.9-fold, respectively, compared with that in the nonpermeabilized cells and had high enantioselectivity to convert COBE to (R)-CHBE. As one of co-substrates, glucose-6-phosphate was preprepared using glucose phosphorylation by hexokinase-catalyzed of CTAB permeabilized brewer’s yeast cells. In a two phase reaction system with n-butyl acetate as organic solvent and with 2-propanol and glucose-6-phosphate as co-substrates, the highest (R)-CHBE concentration of 447 mM was obtained with 110–130 g/l of the CTAB permeabilized cells at optimized pH, temperature, feeding rate and the shake speed of 125 r/min. The yield and enantiomeric excess (ee) of (R)-CHBE reached 99.5 and 99%, respectively, within 6 h.     

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.     

Hydrolytic activity in baker's yeast limits the yield of asymmetric 3-oxo ester reduction

Microbial reductions of ketones hold great potential for the production of enantiopure alcohols, as long as highly selective redox enzymes are not interfered with by competing activities. During reduction of ethyl 3-oxobutanoate by baker's yeast (Saccharomyces cerevisiae) to ethyl (S)-3-hydroxybutanoate, a high enantiomeric excess (> 99%) can be obtained. However, reported yields do not exceed 50-70%. In this article, three main causes are shown to be responsible for these low to moderate yields. These are evaporation of the substrate and product esters, absorption or adsorption of the two esters by the yeast cells and hydrolysis of the two esters by yeast enzymes. The hydrolysis products are further metabolized by the yeast. By reducing the evaporation and absorption losses, the reduction yield can easily be improved to about 85%. Improvement of the efficiency of the reduction and hence the reduction/hydrolysis ratio should lead to a further increase in yield.     

Effects of industrial storage on the bioreduction capacity of brewer’s yeast

The effects of industrial storage on the changes of the cell viability and the activities of intracellular alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) in brewer’s yeast, and the corresponding capacity for the bioconversion of ethyl-3-oxobutanoate (EOB) to ethyl (S)-3-hydroxybutanoate ((S)-EHB), were investigated. The viability of fresh brewer’s yeast cells stored in industrial circulating cooling water at 1–2°C showed 4 and 15% drop after the storage of 7 and 15 days, respectively, after which cells died rapidly. The pretreatment of the stored brewer’s yeast cells by washing and screening significantly enhanced cell viability during industrial storage. The intracellular levels of ADH and G6PDH after permeabilization of these stored cells with cetyltrimetylammonium bromide (CTAB) were much higher, which showed only slight decrease within 2 weeks during the industrial storage. When the stored cells after the permeabilization treatment was used as the biocatalyst at 90–120 g/L, EOB was converted almost completely into enantiopure (S)-EHB with an enantiomeric excess (ee) more than 99% and a yield of over 96%, by fed-batch bioconversion of 560 mM EOB within 6 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Novel Reductase from Candida albicans for the Production of Ethyl (S)-4-Chloro-3-hydroxybutanoate

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP⁺/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.     

High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium

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.     

Efficient bioreduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydrobutanoate by whole cells ofCandida magnoliae in water/n-butyl acetate two-phase system

The asymmetric biosynthesis of ethyl (S)-4-chloro-3-hydrobutanoate from ethyl 4-chloro-3-oxobutanoate was investigated by using whole cells ofCandida magnoliae JX120-3 without the addition of glucose dehydrogenase or NADP⁺/NADPH. In a one-phase system, the bioconversion yield was seriously affected on the addition of 12.1 g/L ethyl 4-chloro-3-oxobutanoate. In order to reduce this substrate inhibition, a water/n-butyl acetate two-phase system was developed, and the bioreduction conditions optimized with regard to the yield and product enantiometric excess value. The optimal conditions were as following: water ton-butyl acetate volume ratio of 1∶1, 4.0 g DCW/L active cells, 50 g/L glucose and 35°C. By adopting a dropwise substrate feeding strategy, high concentration of ethyl 4-chloro-3-oxobutanoate (60 g/L) could be asymmetrically reduced to ethyl (S)-4-chloro-3-hydrobutanoate with high yield (93.8%) and high enantiometric excess value (92.7%).