工业微生物解脂耶氏酵母及其应用研究*

解脂耶氏酵母是一种可利用多种底物发酵生产多种产品的非常规酵母,环境适应性强、易培养、安全性高。因此,该物种作为一种新型的生物工程菌株引起了科学界的广泛关注。近年来,工业生物技术因绿色、循环、低碳等优势成为新兴工业技术,在国内外得到了快速发展。介绍了解脂耶氏酵母的特征及其代谢生产各类化合物的方法,并通过对工业生物技术与传统化学化工技术的比较分析,阐述了工业生物技术的特点、研究现状及应用前景。     

工业微生物解脂耶氏酵母及其应用研究*

解脂耶氏酵母是一种可利用多种底物发酵生产多种产品的非常规酵母,环境适应性强、易培养、安全性高。因此,该物种作为一种新型的生物工程菌株引起了科学界的广泛关注。近年来,工业生物技术因绿色、循环、低碳等优势成为新兴工业技术,在国内外得到了快速发展。介绍了解脂耶氏酵母的特征及其代谢生产各类化合物的方法,并通过对工业生物技术与传统化学化工技术的比较分析,阐述了工业生物技术的特点、研究现状及应用前景。     

解脂耶氏酵母tsr1突变体的构建*

将解脂耶氏酵母与蛋白质分泌有关的ＴＳＲ１基因编码区部分缺失的ＤＮＡ片段转化一株解脂耶氏酵母。通过体内同源重组,部分缺失的外源ｔｓｒ１片段取代了酵母染色体上的正常的ＴＳＲ１基因,从而获得ｔｓｒ１的转化子。Ｓｏｕｔｈｅｒｎ杂交结果表明,用该法成功地构建了ｔｓｒ１突变体,这为进一步研究解脂耶氏酵母ＴＳＲ１基因的功能奠定了基础。     

解脂耶氏酵母TSR1基因的定点诱变*

对解脂耶氏酵母与蛋白质分泌有关的TSR1基因进行寡核苷酸介导的定点诱变 ,限制性内切酶切割和拼接 ,得到了该基因的一系列缺失突变体。这为进一步研究TSR1基因不同结构域的功能奠定了基础。     

代谢工程改造解脂耶氏酵母合成羧酸的研究进展

解脂耶氏酵母是一种具有独特生理代谢特征的非常规酵母.它具有可以利用多种廉价碳源、低pH值耐受性好、分泌能力强等优点,因此非常适合用于各种工业产品的微生物发酵.目前,解脂耶氏酵母已被证实具有高效生产多种(同源或异源)有机羧酸的能力.本文对近年来利用代谢工程及合成生物学技术改造解脂耶氏酵母生产羧酸的实例进行了总结,并重点介...     

代谢工程改造解脂耶氏酵母合成植物萜类化合物的研究进展

萜类化合物是一类广泛存在于植物中的天然产物,其在食品、药品和化工等多个领域中均有广泛的用途,市场潜力巨大.因此,开发生产萜类化合物等植物天然产物可再生的微生物资源来补充甚至代替原有稀少和珍贵的植物资源,具有重要的理论意义和潜在的应用价值.解脂耶氏酵母是目前使用最广泛的非常规酵母底盘细胞之一.近年来,利用代谢工程及合成生...     

解脂耶氏酵母表达系统研究进展

解脂耶氏酵母（Yarrowia lipolytica）是非常规酵母中具代表性的一种,它底物广泛,尤其能利用有机酸（柠檬酸、异柠檬酸）,蛋白类（蛋白酶、脂肪酸、酯酶、磷酸酶、α-甘露糖苷酶、RNase）。烷烃类廉价物质作为底物分泌大量的代谢产物,自上世纪40年代被发现以来,越来越受到研究者的重视,并于上世纪90年代被开发成为一种新的酵母表达系统,用于42种异源蛋白的高效表达。综述了解脂耶氏酵母表达系统及其特点,有利于研究者从转录和翻译的水平研究异源蛋白在此菌中的表达分泌路径以及寻找到调控型启动子。     

代谢工程构建重组耶氏解脂酵母生产中长链聚羟基脂肪酸酯

中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。     

解脂耶氏酵母合成萜烯的进展:见微知著的改造策略

萜烯是一类基于五碳单元类异戊二烯的天然化合物,种类繁多且具有多种生物活性,被广泛应用于食品、医药和化工领域.传统萜类物质生产依赖于化学合成或植物组织提取,存在产率低、资源浪费的缺点.近年来,代谢工程和合成生物学的发展促进了微生物细胞工厂的高效构建,为化学品的微生物合成提供了新的选择.解脂耶氏酵母因前体甲羟戊酸途径的内源...     

解脂耶氏酵母表达调控工具的开发及天然产物合成的研究进展

解脂耶氏酵母是一种重要的产油酵母,由于其能利用多种疏水性底物,具有良好的耐酸、耐盐等胁迫耐受性,具有高通量的三羧酸循环,可提供充足的乙酰辅酶A前体等特点,被认为是生产萜类、聚酮类和黄酮类等天然产物的理想宿主,在代谢工程领域有着广泛的应用。近年来,越来越多的基因编辑、表达和调控工具被逐渐开发,这促进了解脂耶氏酵母合成各种天然产物的研究。文中综述了近年来解脂耶氏酵母中基因表达和天然产物合成方面的研究进展,并探讨了在该酵母中异源合成天然产物所面临的挑战和可能的解决方案。     

Cloning of the gene encoding the catalytic subunit of casein kinase II from the yeast Yarrowia lipolytica

Casein kinase II from the yeast Yarrowia lipolytica is a heterotetramer of the form αα′β₂. We report on the cloning and sequencing of a partial cDNA and of the complete genomic DNA coding for the catalytic α subunit of the casein kinase II from this yeast species. The sequence of the gene coding for this enzyme has been analyzed. No intron was found in the gene, which is present in a single copy. The deduced amino acid sequence of the gene shows high similarity with those of α subunit described in other species, although, uniquely, Y. lipolytica CKIIα lacks cysteines. We find that the α subunit sequence of Y. lipolytica CKII is shown greater homology with the corresponding protein from S. pombe than with that from S. cerevisiae. We have analyzed CKIIα expression and CKIIα activity. We show that expression of this enzyme is regulated. The catalytic subunit is translated from a single mRNA, and the enzyme is present at a very low level in Y. lipolytica, as in other yeasts. Received: 20 December1997 / Accepted: 19 June 1997     

解脂耶氏酵母中囊泡蛋白YlSec15的鉴定及功能研究

解脂耶氏酵母(Yarrowia lipolytica)进行出芽繁殖时,决定未来分裂平面的出芽位点不是随机选取的,而是选择在前一次细胞分裂位置的对侧出芽,即进行双极出芽。目前对解脂耶氏酵母双极出芽的分子调控机制并不清楚。通过观察蛋白定位及过量表达的方法研究了解脂耶氏酵母中囊泡蛋白YlSec15的功能。结果表明:YlSec15在细胞中有明显的极性定位,在细胞的小芽内以及大中芽的芽颈处富集,过量表达YlSec15抑制了菌丝的形成并使得部分细胞的出芽位点选择方式由双极出芽转变为随机出芽,而引起这一变化的原因可能是由于过量的YlSec15在细胞中不能进行正常的极性定位。此外,YlSec15可能是通过YlRas2介导的信号通路参与调控细胞的菌丝形成及双极出芽。这一发现丰富了解脂耶氏酵母中双极出芽的分子调控机制,也证明了极性生长与囊泡运输之间是相互影响的。     

Effect of redox potential on the growth of Yarrowia lipolytica and the biosynthesis and activity of heterologous hydroperoxide lyase

The aim of this study was to investigate the effect of redox potential (Eh) on the growth of the yeast Yarrowia lipolytica in both oxidizing (Eh = +350 mV) and reducing (Eh = −150 mV) media and its effect on the expression and activity of hydroperoxide lyase (HPL). HPL activity was assayed in media with Eh values ranging from −250 to +720 mV. In order to change the Eh value of the media, reducing agents including dithiotreitol (1 g/L) and hydrogen (4%) as well as oxidants such as potassium ferricyanide (1 g/L) and oxygen (100%), were used. The experimental findings showed that oxidizing conditions, with Eh of +350 mV, were favorable for the growth of the yeast, whereas reducing conditions, with Eh values of −150 mV, resulted in a higher expression of HPL. In addition, the results showed that the enzymatic activity of the purified HPL was enhanced in the presence of 0.5 mM dithiotreitol but decreased with 1 mM potassium ferricyanide and bubbling O₂. However, HPL activity increased 1.5 times in the presence of 4% hydrogen with an Eh value of −170 mV.     

解脂耶氏酵母细胞表面展示乳糖水解酶高效水解乳糖

乳糖是婴幼儿获取能量的重要碳源之一,但乳糖需要在乳糖酶的作用下水解成半乳糖与葡萄糖后才能被吸收。缺少乳糖分解酶的婴幼儿在摄入含乳糖的食品后,未被消化的乳糖会直接进入大肠,刺激大肠蠕动加快,造成一系列不适应症状即乳糖不耐症,我国属于乳糖不耐症高发国家。因此,解决乳糖的体外水解问题对减轻该症状有重要的意义。研究通过将β-半乳糖苷酶(也称为乳糖水解酶)表面展示在食品安全微生物解脂耶氏酵母(Yarrowia lipolytica)细胞表面,通过培养获得该酵母,然后直接利用酵母细胞来水解乳糖生成半乳糖与葡萄糖。采用该工程酵母细胞(HCY10),能在24小时内完全水解50g/L的乳糖,生成半乳糖与葡萄糖。该方法具有高效、简便的优点,能为乳糖的高效水解提供一条新的途径。     

Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica

Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Combined effects of 13-hydroperoxides and dithiotreitol were optimised by a central composite design and a model was proposed. Finally, 6 mM (600 mg/L) of hexanal was obtained when 119 mM of 13-hydroperoxides (37 g/L) and 50 mM of dithiotreitol were introduced directly in the biocatalytic medium of the yeast Y. lipolytica.     

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica

Summary The mating type gene MA TA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.     

Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.