Performance of a magnetically stabilized bed reactor with immobilized yeast cells

This paper is focused on the possibility to apply the magnetic stabilization technique in bioprocessing. The feasibility of a continuous ethanol fermentation process with immobilizedSaccharomyces cerevisiae cells in a magnetically stabilized bed (MSB) was demonstrated. The fermentation processes were carried out in an external magnetic field, transverse to the fluid flow. The flexibility to change the bed expansion owing to the independent change of the fluid flow and the field intensity (the “magnetization FIRST” mode) permitted the creation of fixed beds with different particle arrangements, which affected the bed porosity, the effective fluid-particle contact area, and the mass transfer processes on the particle-fluid interface. As a result, higher ethanol concentration, ethanol production, and glucose uptake rates than in conventional packed bed reactor were reached.     

活性酵母细胞不对称催化苯乙酮还原及树脂吸附对反应的促进作用

 以苯乙酮为模型底物,研究了水相体系中酵母细胞催化前手性芳香酮不对称还原生成相应手性醇的反应特性. 实验发现,酵母细胞催化苯乙酮不对称还原的产物以(S)-α-苯乙醇为主,反应的立体选择性很高,(S)-α-苯乙醇的对映体过量值可达99%左右. 在pH为7～8, 酵母细胞用量为0.2 g/ml的条件下能获得较高的产物收率(可达35%左右). 酵母细胞能选择性地氧化(S)-α-苯乙醇,而留下(R)-α-苯乙醇. 在反应体系中加入合适的吸附树脂,可以降低底物和产物对细胞的毒害作用,显著提高反应底物的初始浓度,从而提高产物收率.     

Ethanol production usingZymomonas mobilis in a cross-linked immobilized cell reactor

The bacteriumZymomonas mobilis may be utilized to produce ethanol from glucose in a cross-linked immobilized cell reactor. Reactor startup is much more rapid with cross-linkedZymomonas than with the yeastSaccharomyces cerevisiae. Volumetric ethanol productivities (based on liquid holdup) three times those obtained with cross-linked yeast, and comparable to those obtained withZymomonas immobilized by other methods, are possible.     

激光诱变选育面包酵母菌的研究

采用He-Ne激光对面包酵母菌进行诱变处理,筛选到3株低糖面团起发活力大幅度提高的菌株XD198、XD193和XD174,其低糖面团起发活力比出发菌株分别提高31.3%、32.7%和35.3%.海藻糖含量分别较出发菌株提高-1.37%、10.83%、5.10%.而且筛选到的高活力菌株遗传性能稳定.证明激光诱变效果明显,是进行面包酵母菌种选育的理想方法之一.     

Depletion of arginine in yeast cells decreases the resistance to hydrostatic pressure

High hydrostatic pressure (HP) inhibits growth and inactivates microorganisms by destabilizing non-covalent molecular interactions. Arginine contributes to stress resistance because it has a guanidine side chain, which assists in the refolding of aggregated proteins. We attempted to analyze the contribution of arginine to high HP stress using a pressure-sensitive mutant strain of Saccharomyces cerevisiae and a metabolomics approach. Our results showed that the content of 136 out of 250 detected metabolites differed in the mutant and parent strains. Decreased metabolites were involved in the tricarboxylic acid cycle and arginine biosynthesis. The expression of genes contributing to arginine biosynthesis was significantly lower in the mutant strain than in the parent strain. When arginine was supplemented to the medium, the mutant strain showed more tolerance to pressure. These results suggest that yeast cells survived due to the contribution of arginine to high pressure resistance. This indicates that depletion of arginine caused by decreased activity of the biosynthesis pathway confers sensitivity to HP.     

基于纤维小体展示技术的酿酒酵母纤维素乙醇研究进展

利用联合生物加工工艺生产第二代燃料乙醇(纤维素乙醇)是国内外的研究热点.前期的研究结果表明,酿酒酵母分泌或展示非复合型纤维素酶体系的应用效果并不理想,而复合型纤维素酶体系(即纤维小体)因对纤维素的降解能力比非复合型纤维素酶体系更强,所以其在酿酒酵母细胞表面的组装研究受到越来越多的关注.目前,单支架和双支架纤维小体在酵母细胞表面的完全自组装以及多细胞协同参与的非完全自组装均已实现.纤维小体展示型酿酒酵母已能直接利用结晶型纤维素发酵生产乙醇,但由于降解模块的结构缺陷,纤维素乙醇的产量仍然偏低.本文对纤维小体的酵母展示技术及其在纤维素乙醇发酵中的应用研究进行了论述,并对该领域的发展方向进行了展望.     

Metabolism of Deuterated erythro‐Dihydroxy Fatty Acids in Saccharomyces cerevisiae: Enantioselective Formation and Characterization of Hydroxylactones

Epoxides of fatty acids are hydrolyzed by epoxide hydrolases (EHs) into dihydroxy fatty acids which are of particular interest in the mammalian leukotriene pathway. In the present report, the analysis of the configuration of dihydroxy fatty acids via their respective hydroxylactones is described. In addition, the biotransformation of (±)‐erythro‐7,8‐ and ‐3,4‐dihydroxy fatty acids in the yeast Saccharomyces cerevisiae was characterized by GC/EI‐MS analysis. Biotransformation of chemically synthesized (±)‐erythro‐7,8‐dihydroxy(7,8‐²H₂)tetradecanoic acid ((±)‐erythro‐ 1 ) in the yeast S. cerevisiae resulted in the formation of 5,6‐dihydroxy(5,6‐²H₂)dodecanoic acid ( 6 ), which was lactonized into (5S,6R)‐6‐hydroxy(5,6‐²H₂)dodecano‐5‐lactone ((5S,6R)‐ 4 ) with 86% ee and into erythro‐5‐hydroxy(5,6‐²H₂)dodecano‐6‐lactone (erythro‐ 8 ). Additionally, the α‐ketols 7‐hydroxy‐8‐oxo(7‐²H₁)tetradecanoic acid ( 9a ) and 8‐hydroxy‐7‐oxo(8‐²H₁)tetradecanoic acid ( 9b ) were detected as intermediates. Further metabolism of 6 led to 3,4‐dihydroxy(3,4‐²H₂)decanoic acid ( 2 ) which was lactonized into 3‐hydroxy(3,4‐²H₂)decano‐4‐lactone ( 5 ) with (3R,4S)‐ 5 =88% ee. Chemical synthesis and incubation of (±)‐erythro‐3,4‐dihydroxy(3,4‐²H₂)decanoic acid ((±)‐erythro‐ 2 ) in yeast led to (3S,4R)‐ 5 with 10% ee. No decano‐4‐lactone was formed from the precursors 1 or 2 by yeast. The enantiomers (3S,4R)‐ and (3R,4S)‐3,4‐dihydroxy(3‐²H₁)nonanoic acid ((3S,4R)‐ and (3R,4S)‐ 3 ) were chemically synthesized and comparably degraded by yeast without formation of nonano‐4‐lactone. The major products of the transformation of (3S,4R)‐ and (3R,4S)‐ 3 were (3S,4R)‐ and (3R,4S)‐3‐hydroxy(3‐²H₁)nonano‐4‐lactones ((3S,4R)‐ and (3R,4S)‐ 7 ), respectively. The enantiomers of the hydroxylactones 4, 5 , and 7 were chemically synthesized and their GC‐elution sequence on Lipodex^® E chiral phase was determined.     

Effect of a nonmetabolizable analog of fructose-1,6-bisphosphate on glycolysis and ethanol production in strains ofSaccharomyces cerevisiae andEscherichia coli

Fructose-1,6-bisphosphate (F-1,6-P2) is an allosteric activator of two key enzymes of glycolysis: phosphofructokinase and pyruvate kinase. Regulation of glycolysis in a wild-typeSaccharomyces cerevisiae and a recombinantEscherichia coli by a dead-end structural analog of F-1,6-P2 was studied. 2,5-Anhydromannitol (2,5-AM), a structural analog of β-d-fructose, was used. On being taken up by the cells, 2,5-AM was converted into its monophosphate and diphosphate by the enzymes of the glycolytic pathway. The final product, 2,5-anhydromannitol-1,6-bisphosphate, could not be metabolized further and, therefore, accumulated inside the cells. Glucose and fructose were used as substrates. It was found that 2,5-AM at concentrations of 1 mM or less did not have any effect on either substrate consumption or ethanol production. At concentrations of 2,5-AM of 2.5 mM or greater, significant inhibition of both glucose and fructose was observed, with fructose inhibition much more severe. We discuss the possible mechanisms of glycolysis inhibition by 2,5-AM at high concentrations and the regulation of glycolysis by this compound.     

Design and 3D modeling investigation of a microfluidic electrode array for electrical impedance measurement of single yeast cells

High-resolution microscopic imaging may cause intensive image processing and potential impact of light irradiation on yeast replicative lifespan (RLS). Electrical impedance spectroscopy (EIS) could be alternatively used to perform high-throughput and label-free yeast RLS assays. Prior to fabricating EIS-integrated microfluidic devices for yeast RLS determination, systematic modeling and theoretical investigation are crucial for device design and optimization. Here, we report three-dimensional (3D) finite-element modeling and simulations of EIS measurement in a microfluidic single yeast in situ impedance array (SYIIA), which is designed by patterning an electrode matrix underneath a cell-trapping array. SYIIA was instantiated and modeled as a 5 × 5 sensing array comprising 25 units for cell immobilization, culturing, and time-lapse EIS recording. Simulations of yeast growing and budding in a sensing unit demonstrated that EIS signals enable the characterization of cell growth and daughter-cell dissections. In the 5 × 5 sensing array, simulation results indicated that when monitoring a target cell, daughter dissections in its surrounding traps may induce variations of the recorded EIS signals, which could cause mistakes in identifying target daughter-cell dissections. To eliminate the mis-identifications, electrode array pitch was optimized. Therefore, the results could conduct the design and optimization of microfluidic electrode-array-integrated devices for high-throughput and accurate yeast RLS assays.     

Tomography of functional organization in protein-protein interaction network

In recent years, after high throughput PPI data was available, studies have focused on unraveling how proteins organize their functionality from architecture of the PPI network. We examine the functional organization of a PPI network by dividing the network into layered structure around a protein according to shortest path length. We proposed an index, functional correlation, to assess the functional closeness of a specific protein with its l layer neighbors, i.e. proteins having l shortest path length from the center protein. Our results showed that functional correlation decays exponentially with the number of layers within a characteristic length l_c, and it becomes uncorrelated outside such a characteristic length. A simple model based on functional unit structure was proposed to explain this exponential decay of functional correlation.