葡萄糖氧化酶基因在解脂耶氏酵母中的表达及发酵优化

葡萄糖氧化酶(β-D-glucose:oxygen 1-oxidoreductase;EC 1.1.3.4,简称GOD)是生物领域中至关重要的工具酶之一,被广泛应用于食品工业、饲料工业、纺织、医药等行业中。作者构建了一株重组解脂耶氏酵母Yarrowia lipolytica 1-28,分泌GOD,并进一步运用单因素实验与正交实验在摇瓶水平对其发酵培养基进行了优化。实验结果显示,最佳发酵培养基配方为:甘油20 g/L,酵母膏2.64 g/L,氯化铵2.64 g/L,无水硫酸镁0.13 g/L,磷酸二氢钾0.32 g/L,维生素B13.34×10-4g/L,初始pH值6.0。优化后GOD发酵产量达到11.0 U/mL,较初始发酵培养基GOD产量提高了72%。在此基础上进行3 L罐发酵,重组菌在甘油补料30 g/L,pH为5.0时,28℃发酵190h,发酵液酶活达到81.6 U/mL。     

常压室温等离子体诱变选育高产脂肪酶解脂耶氏酵母

采用常压室温等离子体(Atmospheric room temperature plasma,ARTP)对产脂肪酶的解脂耶氏酵母菌株YL1进行诱变;通过三丁酸甘油酯平板法、p-NPP法以及酸碱滴定法等筛选得到高产脂肪酶的目标菌株C4,并研究其遗传稳定性。结果表明,解脂耶氏酵母菌株YL1的最佳诱变时间为60s,菌株致死率达97.45%;突变株C4的脂肪酶酶活为13.4 U/mL,较出发菌株提高了82.6%,多代培养后遗传稳定;与出发菌株相比,突变株脂肪酶可使维生素A棕榈酸酯的合成转化率提高36.9%。     

解脂耶氏酵母lip2脂肪酶水解谷维素产生阿魏酸的研究

对解脂耶氏酵母(Yarrowia lipolytica)脂肪酶水解谷维素产生阿魏酸的酶反应体系进行了研究。实验发现解脂耶氏酵母全脂肪酶粉(105U/mg)在50mmol/L p H7.0 Tris-HCl(含7.5mmol/L黄胆酸钠),100mmol/L p H6.0磷酸钠缓冲液(含1000U脂肪酶)的体系中,水解产生阿魏酸的得率为2.94%。为了进一步提高脂肪酶水解效率,对解脂耶氏酵母脂肪酶中主要组分lip2脂肪酶基因进行了克隆,整合至毕赤酵母GS115基因组后发酵制取lip2脂肪酶粉(70.1U/mg),于上述酶解体系中进行水解谷维素实验。实验结果表明阿魏酸产率为2.87%。获得的lip2脂肪酶催化效率略低于全脂肪酶粉催化效率,但是获得了单一的脂肪酶基因,为进一步采取分子进化技术提高其催化能力奠定了基础。     

解脂耶氏酵母URA3基因的敲除

构建了营养缺陷型解脂耶氏酵母菌株,使之用于遗传标记和高产香味物质γ-癸内酯.作者利用基因同源重组的方法敲除掉尿嘧啶合成酶关键基因URA3基因,用尿嘧啶营养缺陷型培养基(SD-URA)添加一定浓度的5-氟乳清酸(5-FOA)和尿嘧啶筛选获得转化子.实验表明:尿嘧啶营养缺陷型菌株在含有5-FOA和尿嘧啶的培养基上生长而野生型菌株不生长,从而建立了一种快速获得营养缺陷型解脂耶氏菌株的方法.     

不同碳源培养下解脂耶氏酵母的转录组差异分析

以葡萄糖为碳源生长的解脂耶氏酵母（Yarrowia lipolytica）为对照组,以油酸为碳源生长的Y. lipolytica为实验组,利用Illumina高通量测序平台对两者进行转录组测序,通过多种生物信息学方法对数据进行分析处理。对照组的测序结果共得到17 923 921 clean reads;实验组的测序结果共得到22 656 852 clean reads。两个样品差异表达基因的分析显示共有536 个显著性差异表达基因,376 个基因表达上调,160 个基因表达下调。通过GO和KEGG富集分析可以了解这些差异基因参与的生物学调控。本研究为解脂酵母分子生物学的进一步研究提供了参考。     

尼罗红荧光光谱法快速测定解脂耶氏酵母油脂含量的研究

为了建立快速简便检测解脂耶氏酵母油脂含量的方法,探讨了尼罗红-光谱法测定解脂耶氏酵母油脂含量的检测条件。通过研究解脂耶氏酵母最佳发射光波长、细胞密度、尼罗红染液用量、染色时间、不同助溶剂效能及最佳体积分数对荧光强度的影响,确定了最佳检测条件,得到细胞油脂含量与荧光强度的线性关系。解脂耶氏酵母在激发光560 nm、发射光650 nm处有最高荧光值,每毫升菌液加入质量分数为0.1 mg/m L的尼罗红染液20μL,加入体积分数为15%的异丙醇,黑暗染色5 min,在细胞OD600=0¹.3的范围内,菌液的油脂含量（X）与荧光值（Y）呈较好的线性关系,线性关系式为Y=6.3651X+10.097,R2=0.9902,灵敏度达0.0001 g。该方法能够准确地反映出解脂耶氏酵母胞内油脂含量。尼罗红-荧光法可成为一种快速检测解脂耶氏酵母胞内油脂含量的新方法。      

代谢工程改造解脂耶氏酵母生产油脂研究进展北大核心CSCD

微生物油脂是可再生能源发展的重要方向,近年来通过合成生物学方法和代谢工程技术改造,解脂耶氏酵母的产油水平提高迅速,展现了良好的应用发展前景。从代谢途径关键酶调控、负反馈调节解除、代谢途径关键酶异源表达、乙酰辅酶A和NADPH替代途径构建、强化氧化应激保护、促进脂肪酸分泌、适应性进化和计算机辅助模拟8个方面,梳理总结了代谢工程改造解脂耶氏酵母生产油脂的最新研究进展。通过对现有研究分析发现,廉价底物中的毒性成分影响细胞生长和油脂合成,以及油脂调控网络机制的不完全明晰,是限制解脂耶氏酵母油脂产量提升的主要障碍。为此,可通过设计引入解毒途径,添加解毒剂,或筛选毒性化合物耐受菌,以及利用多组学技术和计算机辅助优化进一步解析代谢调控机制解决此问题。此外,在“双碳”背景下,可在解脂耶氏酵母中引入高效的人造光合作用或碳固定途径,利用二氧化碳生产油脂。     

欧盟就解脂耶氏酵母富硒生物质作为新型食品安全性发布意见

2020年1月31日,欧洲食品安全局就解脂耶氏酵母富硒生物质(selenium-enriched biomass of Yarrowia lipolytica)作为新型食品的安全性发布意见。应欧盟委员会的要求,欧盟膳食、营养和过敏症科学小组(NDA)根据法规(EU)2015/2283就解脂耶氏酵母生物质作为新型食品提供科学意见。     

共轭亚油酸合成相关基因在耶氏解脂酵母中异源表达及活性研究

为了探究植物乳杆菌(Lactobacillus plantarum)中与共轭亚油酸(CLA)生物合成相关的3个基因:(亚)油酸水合酶基因(mcra)、短链脱氢酶/氧化还原酶基因(dh)、乙酰乙酸脱羧酶基因(dc)在耶氏解脂酵母(Yarrowia lipolytica,Y.lipolytica)中异源表达后能否具有活性,利用两个耶氏解脂酵母整合表达质粒(p INA 1269和p INA 1312),将3个基因分别导入耶氏解脂酵母营养缺陷型宿主菌Polf(Ura~-,Leu~-)中,构建了重组菌株。在不同重组菌中添加相应的底物:亚油酸(LA)和10-羟基-顺12-十八碳烯酸(10-HOE),然后对反应体系进行脂肪酸检测,得到基因对应的不同产物:10-HOE和10-氧代-反11-十八碳烯酸(10-oxo-trans 11-octadecenoic acid),证明mcra、dh、dc在耶氏解脂酵母中进行了异源表达并且具有活性。     

溶氧对耶氏解脂酵母油脂积累和柠檬酸分泌的影响

产油耶氏解脂酵母能将培养基中过量的碳源转化为油脂储存于细胞内并分泌大量的柠檬酸。在耶氏解脂酵母发酵过程中通过关联搅拌转速和通气量调控培养基中的溶氧含量处于5%、10%、20%、30%和不控制5种水平,来研究溶氧对其油脂积累和柠檬酸分泌的影响。结果表明,随着发酵培养基中溶氧的增加,耶氏解脂酵母细胞内油脂含量和柠檬酸分泌量均有所增加,且不控制培养基中溶氧时,细胞内油脂含量和柠檬酸产量均最高,油脂含量达到细胞干重的11.62%(w/w),柠檬酸产量达到21.0 g/L。培养基中不同的溶氧含量还会影响油脂的脂肪酸组成,高溶氧能够促进油酸含量的增加,溶氧不控制时油酸的含量最高,达到48.62%。本研究为耶氏解脂酵母产脂发酵培养过程中溶氧的控制提供了重要的实验依据。     

重组解脂亚罗酵母合成菜油甾醇

菜油甾醇是一种具有重要生理活性的植物甾醇。为构建合成菜油甾醇的工程菌株,通过敲除解脂亚罗酵母polf菌株中麦角固醇合成基因ERG5,促进麦角-5,7,24-三烯醇的体内积累,进而表达经密码子优化的非洲爪蟾DHCR7基因,使麦角-5,7,24-三烯醇在DHCR7和polf胞内ERG4的共同催化作用下,转化成菜油甾醇,最后利用GC-MS对重组菌株的发酵产物进行检测分析。结果表明,重组菌株polf-ERG5--DHCR7+(PED)合成菜油甾醇的产量达0.485 mg/g(以细胞干重计)。重组解脂亚罗酵母合成菜油甾醇菌株的成功构建,为生物合成法合成植物甾醇提供了新思路。     

Yarrowia lipolytica发酵生产γ-癸内酯工艺的初步研究

目的:研究生物转化方法生产天然γ-癸内酯。方法:在单因素实验的基础上,对Yarrowia lipolytica AS2.1405菌株以蓖麻油为底物发酵生产γ-癸内酯的培养基配方进行优化。结果:在较为适宜的培养基中培养48h后,γ-癸内酯发酵产量达到0.6g/L。结论:实验结果可为γ-癸内酯发酵工艺改进提供依据。     

Cell surface characterization of Yarrowia lipolytica IMUFRJ 50682

In the present work, the surface characteristics of a wild-type strain of Yarrowia lipolytica (IMUFRJ50682) were investigated. Six different methods to characterize cell surfaces--adhesion to polystyrene; hydrophobic interaction chromatography (HIC); microbial adhesion to solvents (MATS) test; zeta potential; microbial adhesion to hydrocarbons (MATH) test; and contact angle measurement (CAM)--were employed to explain the cell surface behaviour of Y. lipolytica (IMUFRJ50682). This Y. lipolytica strain presents significant differences at the cell surface compared with another Y. lipolytica strain (W29) previously reported in the literature. The main difference is related to the higher cell adhesion to non-polar solvents. The proteins present on the cell wall of Y. lipolytica IMUFRJ50682 seem to play an important role in these particular surface characteristics because of the consistent reduction of this yeast hydrophobic character after the action of pronase on its cell wall.     

Yarrowia lipolytica

The yeast Yarrowia lipolytica presents specific physiological, metabolic and genomic characteristics, which differentiate it from the model yeast Saccharomyces cerevisiae. These properties have led several research groups to use this yeast as a model for basic knowledge. Thanks to the development of advanced genetic tools and ‐omic approaches, significant progress has been achieved in the understanding of specific biological processes. This review, after a short presentation of this model yeast, will briefly highlight the different use of Y. lipolytica for basic knowledge and the advantages gained by exploiting this non‐conventional yeast. Future perspectives in employing this yeast for basic knowledge in the field of RNA splicing and genome evolution, and for the study of lipid metabolism, are also discussed. Copyright © 2012 John Wiley & Sons, Ltd.     

Yarrowia lipolytica SRP54 Homolog and Translocation of Kar2p

To investigate the role of Srp54p in protein translocation, the Yarrowia lipolytica SRP54 homolog was cloned. Sequencing revealed an open reading frame of 536 amino acids coding for a 57·2 kilodalton polypeptide with 55 to 57% sequence identity to Srp54ps of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mouse. Like these Srp54ps, Y. lipolytica Srp54p has an N-terminal domain with a highly conserved GTP-binding site and a methionine-rich C-terminal domain. Differing results regarding the essentiality of SRP subunits were obtained. SRP54 is important but not essential for growth, but it was reconfirmed that at least one SRP RNA gene is essential. Cells with SRP54 deleted grow about six times more slowly than wild type; faster-growing colonies, still growing much slower than wild type, appeared quite frequently. In srp54Δ cells, no untranslocated alkaline extracellular protease precursor was detected. Therefore, to develop another reporter molecule the Y. lipolytica KAR2 homolog was cloned and Kar2p antibodies were produced. For Kar2p an untranslocated precursor was detected in srp54Δ but not in wild-type cells, suggesting that its translocation was defective in the srp54Δ cells. These results confirm an in vivo role for SRP in protein translocation in Y. lipolytica, suggest that SRP RNA or an SRP core-particle has functions not shared by Srp54p, and show that, as in S. cerevisiae and Sz. pombe, reporter molecules differ in their dependency on SRP for translocation. The SRP54 and KAR2 sequences have been deposited in GenBank under Accession Numbers U42418 and U63136.     

A single FKS homologue in Yarrowia lipolytica is essential for viability

The synthesis of beta-1,3-glucan, the structural component of the yeast cell wall which gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of beta-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role. FKS1 has been characterized in Saccharomyces cerevisiae, where its function is at least partially redundant with that of FKS2/GSC2. FKS homologues have also been identified in several other fungal species, including Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Cryptococcus neoformans and Paracoccidiodes brasiliensis. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Fks1ps to isolate the possible FKS homologue genes of the strictly aerobic non-conventional yeast Yarrowia lipolytica. Using this approach, we have isolated a single FKS homologue which we have named YlFKS1; this codes a 1961 amino acid protein that shows a high degree of homology with other Fksps. Expression analysis of YlFKS1 under different conditions affecting the cell wall did not reveal significant differences. Finally, attempts to obtain a Y. lipolytica strain containing a disrupted YlFKS1 allele failed, despite having used two different techniques. Taken together, these results suggest that, unlike S. cerevisiae, YlFKS1 is the only FKS1 homologue in Y. lipolytica and is essential for growth.     

解脂耶罗威亚酵母筛选方法的改良及紫外诱变选育

解脂耶罗威亚酵母(Yarrowia lipolytica)对油脂类化合物、甘油酯有很强的代谢能力,也是工业上较为广泛应用的菌种之一.通过对油脂降解菌的筛选方法进行了改良研究,结果表明,初筛培养基pH值为6.0,维多利亚蓝B溶液加入量为1％,并且将培养基中油脂用组织捣碎机搅拌2min～3min,使培养基呈均匀的白色乳浊液,灭菌后倒出的平板对油脂降解圈的表现效果最好.为进一步提高保藏菌种的油脂降解能力,采用紫外诱变的方法对该菌种进行改良,确定最佳诱变时间为50s,经诱变筛选后将菌株的油脂降解能力从63％提高为79％,该突变菌株在对数期表现出较强的油脂降解能力,并具有良好的遗传稳定性,为生物法处理餐饮废水提供了有用的微生物资源.     

Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin. © 1998 John Wiley & Sons, Ltd.     

An efficient method for production of kynurenic acid by Yarrowia lipolytica

Kynurenic acid (KYNA) is a compound derived from the tryptophan catabolic pathway. Antioxidant and neuroprotective properties have been confirmed for KYNA, which makes it an interesting and important metabolite of biomedical significance. In the present study, the yeast Yarrowia lipolytica was tested for KYNA biosynthesis. The results showed that Y. lipolytica strain S12 is able to produce KYNA in high concentrations (up to 21.38 μg/ml in culture broth and 494.16 μg/g cell dry weight in biomass) in optimized conditions in a medium supplemented with tryptophan. Different conditions of culture growth, including the source of carbon, its concentration and pH value of the medium, as well as the influence of an inhibitor or precursor of KYNA synthesis, were analysed. The obtained data confirmed the presence of KYNA metabolic pathway in the investigated yeast. To our best knowledge, this is the first study that reports KYNA production in the yeast Y. lipolytica in submerged fermentation.