Evaluation of the impact of lipid fouling during the chromatographic purification of virus‐like particles from Saccharomyces cerevisiae

BACKGROUND: Production of recombinant virus‐like particles (VLPs) in yeast expression systems for use as vaccines requires cell disruption and detergent‐mediated steps to liberate the product. Typically, these release high levels of cellular components such as lipids that foul chromatography columns. This study compares the impact of applying lipid‐rich and lipid‐depleted feedstocks to hydrophobic interaction chromatography columns to quantify the loss of performance caused by the presence of host lipids over a total of 40 operational cycles. RESULTS: VLP binding capacity in the lipid‐rich feed was significantly lower than for the lipid‐depleted feed, with greater than 24% of the lipids remaining in the column after each cycle. Triacylglycerol was found to be the major contaminant. The effectiveness of subsequent caustic clean‐in‐place was limited, resulting in column hydrophobicity increasing over repeated loading cycles. This improved the effective VLP binding capacity and affinity, but also made product elution more difficult, and recovery decreased by more than 70% over the 40 cycles. CONCLUSION: Host cell lipids cause major fouling problems during VLP purification. Instead of screening for better CIP conditions, priority should be given to identifying suitable upstream lipid removal strategies in order to maintain column performance and so yield more economically viable processes. Copyright © 2009 Society of Chemical Industry     

Bioconversion of corn stover hydrolysate to ethanol by a recombinant yeast strain

Three corn stover hydrolysates, enzymatic hydrolysates prepared from acid and alkaline pretreatments separately and hemicellulosic hydrolysate prepared from acid pretreatment, were evaluated in composition and fermentability. For enzymatic hydrolysate from alkaline pretreatment, ethanol yield on fermentable sugars and fermentation efficiency reached highest among the three hydrolysates; meanwhile, ethanol yield on dry corn stover reached 0.175 g/g, higher than the sum of those of two hydrolysates from acid pretreatment. Fermentation process of the enzymatic hydrolysate from alkaline pretreatment was further investigated using free and immobilized cells of recombinant Saccharomyces cerevisiae ZU-10. Concentrated hydrolysate containing 66.9 g/L glucose and 32.1 g/L xylose was utilized. In the fermentation with free cells, 41.2 g/L ethanol was obtained within 72 h with an ethanol yield on fermentable sugars of 0.416 g/g. Immobilized cells greatly enhanced the ethanol productivity, while the ethanol yield on fermentable sugars of 0.411 g/g could still be reached. Repeated batch fermentation with immobilized cells was further attempted up to six batches. The ethanol yield on fermentable sugars maintained above 0.403 g/g with all glucose and more than 92.83% xylose utilized in each batch. These results demonstrate the feasibility and efficiency of ethanol production from corn stover hydrolysates.     

Lipid Droplets and Their Autophagic Turnover via the Raft-Like Vacuolar Microdomains

Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs’ basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways.     

Cellular Models for Primary CoQ Deficiency Pathogenesis Study

Primary coenzyme Q₁₀ (CoQ) deficiency includes a heterogeneous group of mitochondrial diseases characterized by low mitochondrial levels of CoQ due to decreased endogenous biosynthesis rate. These diseases respond to CoQ treatment mainly at the early stages of the disease. The advances in the next generation sequencing (NGS) as whole-exome sequencing (WES) and whole-genome sequencing (WGS) have increased the discoveries of mutations in either gene already described to participate in CoQ biosynthesis or new genes also involved in this pathway. However, these technologies usually provide many mutations in genes whose pathogenic effect must be validated. To functionally validate the impact of gene variations in the disease’s onset and progression, different cell models are commonly used. We review here the use of yeast strains for functional complementation of human genes, dermal skin fibroblasts from patients as an excellent tool to demonstrate the biochemical and genetic mechanisms of these diseases and the development of human-induced pluripotent stem cells (hiPSCs) and iPSC-derived organoids for the study of the pathogenesis and treatment approaches.     

Detection of endogenous Snf1 and its activation state: application to Saccharomyces and Candida species

The stress-response Snf1 protein kinase of Saccharomyces cerevisiae serves as a powerful model for studies of the eukaryotic Snf1/AMP-activated protein kinase (AMPK) family. Central to studies of Snf1 are methods that determine its activation state under various physiological and genetic conditions. Here, we have developed a convenient and sensitive method for immunoblot analysis of endogenous yeast Snf1 and its activation-loop threonine (Thr210) phosphorylation. The method employs readily obtainable reagents and yields results that faithfully reflect the environmental and genetic conditions tested. Using our method, we have obtained evidence that Snf1 remains stress-regulated in reg1 Delta cells, revealing the existence of a Snf1 signalling mechanism(s) that is independent of Reg1-PP1 phosphatase. In addition to strains of common laboratory S. cerevisiae backgrounds, we have applied the method to two pathogenic Candida species, C. glabrata and C. albicans. We have detected proteins whose gel mobilities, immune properties and regulation patterns are consistent with those expected for the corresponding Snf1 homologues. Because Snf1 activation is a sensitive marker of several types of stress, including artifactual stresses associated with common cell harvesting and protein extraction procedures, the convenient and efficient protein extraction method described here should be advantageous for SDS-PAGE and immunoblot analyses of stress-regulated and other proteins from various yeast species.     

高生物量富硒酵母菌的选育

以酿酒酵母PT为出发菌株,采用梯度浓度筛选的方法对其进行驯化,得到1株生物量较高和对亚硒酸钠抗性较高的菌株GB-1。对其进行紫外诱变处理,当致死率达91.85%时,获得多株突变株。通过多次平板初筛和摇瓶复筛,得到1株高生物量富硒酵母UV-PT。采用响应面法对富硒酵母UV-PT发酵条件进行了优化。借助于SAS软件,首先利用Plackett-Burman试验设计筛选出影响富硒的3个主要因素,即转速、温度、初始pH值。在此基础上,再利用Box-Behnken试验设计及响应面分析法对发酵条件进行优化,确定最佳发酵条件:转速为203r/min,发酵温度为30.3℃,初始pH值为4.52。结果表明,优化后富硒酵母的生物量和含硒量分别为10.62g/L、1003.26μg/g,硒总含量为10654.62μg/L,为出发菌株的1.62倍。     

高产谷胱甘肽面包酵母菌种的筛选与发酵条件优化

通过摇瓶试验筛选出谷胱甘肽含量较高的面包酵母BY-14为试验菌株,对产谷胱甘肽的发酵条件进行了优化,其最适的培养条件为:转速160 r/min,发酵时间22 h,温度28℃,接种量10%,装液量50 mL.在优化的条件下,谷胱甘肽的含量达到16.04 mg/g,为初始条件下的1.48倍.     

啤酒废酵母酶法制备生物活性肽的工艺研究

以啤酒废酵母为原料,经菠萝蛋白酶酶解、分离、纯化、干燥等工序制得啤酒废酵母活性肽.试验通过单因素及响应面试验分析确定了菠萝蛋白酶对废酵母的最佳酶解条件:酶解温度40.61℃,pH6.58,酶用量2.62%(w/w).酶解后多肽得率可达到50.97%(w/w).     

浅谈啤酒废酵母的综合利用

啤酒废酵母富含蛋白质、核酸、维生素、矿物质等多种营养成分,搞好综合利用,具有重要的意义。主要论述了啤酒废酵母综合利用现状及前景。