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。     

解脂酵母中POX3基因的敲除研究

对耶氏酵母（Yarrowia lipolytica）中编码与γ-癸内酯合成相关的乙酰辅酶A氧化酶基因（POX1-POX5）中的POX3基因用Cre/loxP同源重组系统进行敲除。设计含有60个核苷酸与POX3基因的ORF两侧序列同源的长引物,以pUG6为模板构建带有loxP-KanMX-loxP系统的敲除组件,之后转化耶氏酵母,通过PCR确定阳性克隆子。将质粒pSH65转入阳性克隆子,半乳糖诱导表达Cre酶以切除KanMX基因。最后在YPD培养基中传代丢失pSH65质粒,获得POX3缺陷型菌株。由于在解脂酵母中表达生产γ-癸内酯直接牵扯到若干个需要抑制的基因,单独缺失其中的一个基因理论上可能实现不了产量的极大值。本研究为进一步构建组合敲除准备了条件。      

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。     

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。     

解脂耶氏酵母β氧化基因敲除菌的构建及挥发性脂肪酸的利用

为了抑制解脂耶氏酵母长链脂肪酸的β氧化,使其稳定地积累,通过基因调控途径构建了4株脂肪酸β氧化基因敲除菌株,并研究了重组菌株利用挥发性脂肪酸作为碳源合成长链脂肪酸的能力。结果表明:分别敲除pox2、pox3和pex10的3株单基因敲除菌株及同时敲除pox2和pox3的组合基因敲除菌株构建成功,4株重组菌株均可以利用挥发性脂肪酸进行生长。与原始菌株相比,polf-Δpox2Δpox3和polf-Δpex10总脂产量(长链脂肪酸总和)在发酵后期并未因脂肪酸的β氧化而出现明显下降,是可以利用挥发性脂肪酸且可实现脂质积累的重组菌株。     

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。     

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。     

耶氏酵母菌POX5基因的敲除

对于耶氏酵母(Yarrowia lipolytica)中编码与γ–癸内酯合成相关的乙酰辅酶A氧化酶基因(POX1-POX5)中的POX5基因用Cre/loxP同源重组系统进行敲除。设计含有60个核苷酸与POX5基因的ORF两侧序列同源的长引物,以pUG6为模板构建带有Cre/loxP系统的敲除组件,之后转化耶氏酵母YL(Yarrowia lipolytica),通过PCR确定阳性克隆子。将质粒pSH65转入阳性克隆子,半乳糖诱导表达Cre酶以切除KanMX基因并经过PCR确认。最后在YPD培养基中连续传代培养丢失pSH65质粒,成功获得POX5缺陷型菌株。     

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。      

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.     

Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556

The URA3 gene, coding for orotidine-5′-phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29·3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identifies of 81% and 88%, respectively. Using contour-clamped homogeneous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.     

Cloning and chromosomal mapping of URA3 genes of Pichia farinosa and P. sorbitophila encoding orotidine-5'-phosphate decarboxylase

The PfURA3 gene, which encodes orotidine-5'-phosphate decarboxylase, of osmotolerant yeast Pichia farinosa NFRI 3,621, was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of the PfURA3 gene and its deduced amino acid sequence indicated that the gene encodes a protein (PfUra3p) of 267 amino acids. Pulsed-field gel electrophoresis and subsequent Southern blot analysis showed that the genome of P. farinosa NFRI 3621 consisted of seven chromosomes, each approximately 1.1-2.2 Mb in size (11.8 Mb in total) and that PfURA3 was located on chromosome V. Pichia sorbitophila is considered as a synonym of P. farinosa. The genome of P. sorbitophila IFO10021 may consist of 12 chromosomes, each approximately 1.2-2.2 Mb in size. P. sorbitophila has two copies of URA3 genes, termed PsURA3 and PsURA30, which were located on chromosome VIII and III, respectively. The difference between PfURA3 and PsURA3 was only two amino acid substitutions, whereas that between PsURA3 and PsURA30 was six amino acid substitutions and the deletion of the C-terminal amino acid by a stop codon insertion. The sequences of PfURA3, PsURA3 and PsURA30 have been deposited in the DDBJ data library under Accession Nos AB071417, AB109042 and AB109043, respectively.     

Isolation and characterization of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Torulaspora delbrueckii

A DNA fragment containing the URA3 gene from Torulaspora delbrueckii was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an ORF of 795 bp, encoding a 264 amino acid protein, which shares a high similarity to the Saccharomycetaceae Ura3 proteins. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the TdUra3 protein. The GeneBank Accession No. for TdURA3 is AF518402.     

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.     

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。     

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。