乳酸克鲁维酵母作为微生物细胞工厂的应用研究进展

乳酸克鲁维酵母（Kluyveromyces lactis）是一种典型的非常规酵母,在微生物基础研究和应用研究方面都有着非常重要的用途。该酵母具有食品安全级别高、蛋白分泌能力强、整合表达能力高效及大规模发酵能力优异等特点,因此,工业应用前景较为广阔。目前,乳酸克鲁维酵母作为蛋白表达系统已在食品和医药等行业中得到了广泛应用。近年来,国内外生物技术领域的科研人员以乳酸克鲁维酵母作为底盘细胞,利用合成生物学技术已经成功构建出了能够生产各种生化产品的微生物细胞工厂,该技术展示出了极大的发展潜力。本文主要对乳酸克鲁维酵母的菌种特点、合成生物学元件、遗传操作工具、基因编辑策略进行介绍,并综述乳酸克鲁维酵母作为细胞工厂的应用研究进展,可以为今后利用合成生物学方法在乳酸克鲁维酵母底盘中构建生产各种高附加值产品的高效微生物细胞工厂提供理论指导。     

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。     

解脂耶氏酵母产脂肪酶的研究进展

解脂耶氏酵母(Yarrowia lipolytica)是一种非常规酵母,能够利用多种底物分泌产生大量的脂肪酶,因此,该物种作为一种新型的工业用菌逐渐引起了学者的广泛关注。该文总结了解脂耶氏酵母产脂肪酶的特点、脂肪酶的编码基因、脂肪酶的活性及生产方法研究进展,并分别阐述了脂肪酶在环境治理、食品工业、生物柴油以及家禽养殖业四个方面的应用,目的在于阐明酵母生产脂肪酶的研究现状及应用前景,为探究脂肪酶新的生产方式及新用途提供依据。     

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。     

解脂耶氏酵母在食品工业中的应用

解脂耶氏酵母是一类非常规酵母,由于该酵母独特的理化性质、代谢特点被广泛应用于各个行业。因其属于广泛认为是安全的(generally regarded as safe,GRAS)微生物而被广泛应用于食品工业。文中主要对该酵母在食品工业中的应用进行总结,介绍其在生产奶类和肉制品中的应用,参与芳香族化合物生产,引起食品腐败,生产多元醇以及有机酸,生产表面活性剂和乳化剂,作为单细胞脂类及单细胞蛋白,食品垃圾的降解及转化。旨在阐明解脂耶氏酵母在食品工业中的重要性及广阔的应用前景。     

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=0~1.3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。     

重金属离子对耶氏解脂酵母油脂积累量及柠檬酸分泌量的影响

通过研究不同种重金属离子对生物量、细胞外柠檬酸含量、油脂含量及脂肪酸组成的影响,进一步探究不同浓度梯度下的金属离子对耶氏解脂酵母油脂积累量和柠檬酸分泌量的影响。结果表明,4种金属离子(Co2+、Cu2+、Mn2+、Ni2+)均能抑制耶氏解脂酵母细胞的生长,且随着浓度的增加抑制作用更加明显;其中Mn2+能够促进细胞柠檬酸分泌和油脂积累,油脂积累量和柠檬酸分泌量分别在1.0和2.0 mmol/L Mn2+时达到最大值,分别为17.6%(w/w)和6.9 g/L;其他3种金属离子则抑制柠檬酸分泌和油脂合成;Co2+、Ni2+、Cu2+影响脂肪酸组成,Co2+和Ni2+促进了饱和脂肪酸含量增加,单不饱和脂肪酸的含量急剧下降,而Cu2+则促进脂肪酸不饱和度的增加,亚油酸的含量从7%提高到40%以上。本研究首次探究了金属离子对耶氏解脂酵母微生物油脂积累和柠檬酸分泌的影响,为产油微生物油脂发酵生产提供有力的依据。     

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.     

泡椒凤爪中解脂耶氏酵母的分离鉴定及生长特性分析

本研究对涨袋泡椒凤爪产品中的主要腐败菌及其生长特性进行了分析。采用传统的分离培养方法,筛选出主要的污染菌群,通过美兰染色镜检对细胞形态进行检测,利用26S rDNA D1/D2区序列测定及系统发育分析对分离菌株进行鉴定,采取ERIC-RAPD技术对分离菌株的分子遗传多态性进行分析,最后对优势菌株的生长特性进行研究。结果表明:从涨袋产品中共分离出40株酵母菌,分别编号为PJ1~PJ40,镜检发现所有菌株的细胞形态相同,均为椭圆形。经分子鉴定发现所分离菌株都为解脂耶氏酵母,且为两个基因分型,说明解脂耶氏酵母是该产品中的一种优势腐败菌,且污染源不同。同时,研究发现解脂耶氏酵母PJ1的最适生长pH值为4.5、最适生长温度为25℃、并且NaCl浓度的升高对该菌的生长起到一定的抑制作用。     

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。     

Vectors for gene expression and amplification in the yeast Yarrowia lipolytica

New vector systems were developed for gene expression in Y. lipolytica. These plasmids contain: (a) as integration target sequences, either a rDNA region or the long terminal repeat zeta of the Y. lipolytica retrotransposon Ylt1; (b) the YlURA3 gene as selection marker for Y. lipolytica, either as the non-defective ura3d1 allele for single integration or the promotor truncated ura3d4 allele for multiple integration; (c) the inducible ICL1 or XPR2 promoters for gene expression; and (d) unique restriction sites for gene insertion. Multiple plasmid integration occurred as inserted tandem-repeats, which are present at 3-39 copies per cell. A correlation between gene copy number and the expressed enzyme activity was demonstrated with Escherichia coli lacZ as reporter gene under the control of the regulated ICL1 promoter. Increases in copy numbers from 5 to 13 for the lacZ expression cassettes resulted in an up to 10-11-fold linear increase of the beta-galactosidase activity in multicopy transformants during their growth on ethanol or glucose, compared with the low-copy replicative plasmid transformants (1.6 plasmid copies). These new tools will enhance the interest in Y. lipolytica as an alternative host for heterologous protein production.     

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.     

Cloning and characterization of the Yarrowia lipolytica squalene synthase (SQS1) gene and functional complementation of the Saccharomyces cerevisiae erg9 mutation

The squalene synthase (SQS) gene encodes a key regulatory enzyme, farnesyl-diphosphate farnesyltransferase (EC 2.5.1.21), in sterol biosynthesis. The SQS1 gene was isolated from a subgenomic library of the industrially important yeast Yarrowia lipolytica, using PCR-generated probes. Probes were based on conserved regions of homologues from different organisms. The complete nucleotide sequence of the coding region and the corresponding amino acid sequence were determined. The sequences showed extensive homologies with squalene synthase genes and enzymes from a number of other organisms and extreme amino acid conservation within the binding and catalytic domains. Direct cloning of a 4.3 kb genomic Y. lipolytica fragment, also comprising its own promoter and terminator sequences, into autonomously replicating plasmid YEp352 and subsequent transformation of a Saccharomyces cerevisiae mutant strain with relevant erg9: ura3-1 markers, resulted in functional complementation of these deficiencies, although Northern blot analyses did not reveal a unique full-length messenger. The availability of the Y. lipolytica SQS1 gene (GenBank Accession No. AF092497) offers prospects for metabolic engineering of the isoprenoid and sterol biosynthetic pathways.     

Gene editing and genetic engineering approaches for advanced probiotics: A review

The applications of probiotics are significant and thus resulted in need of genome analysis of probiotic strains. Various omics methods and systems biology approaches enables us to understand and optimize the metabolic processes. These techniques have increased the researcher's attention towards gut microbiome and provided a new source for the revelation of uncharacterized biosynthetic pathways which enables novel metabolic engineering approaches. In recent years, the broad and quantitative analysis of modified strains relies on systems biology tools such as in silico design which are commonly used methods for improving strain performance. The genetic manipulation of probiotic microorganisms is crucial for defining their role in intestinal microbiota and exploring their beneficial properties. This review describes an overview of gene editing and systems biology approaches, highlighting the advent of omics methods which allows the study of new routes for studying probiotic bacteria. We have also summarized gene editing tools like TALEN, ZFNs and CRISPR-Cas that edits or cleave the specific target DNA. Furthermore, in this review an overview of proposed design of advanced customized probiotic is also hypothesized to improvise the probiotics.     

微生物脂肪酶资源挖掘研究进展

脂肪酶广泛分布于动物、植物和微生物中,工业化脂肪酶主要来源于微生物。脂肪酶制剂的进一步工业化 应用受限于生产成本和脂肪酶的温度和pH值耐受性、活性、专一性和溶剂耐受性等酶学性质,虽然微生物脂肪酶 基因和蛋白相关资源信息已经非常丰富,但适合食品、药品和能源等工业应用的脂肪酶制剂品种依然较少,研究者 仍然在为新型和理想的脂肪酶制剂资源的挖掘而努力。本文重点综述了微生物脂肪酶资源挖掘的主要研究方法,主 要包括通过环境筛选和宏基因组筛选挖掘新的具有优良特性的脂肪酶;脂肪酶微生物生产菌株的改良、脂肪酶基因 在重组工程菌株中的重组表达和优化、蛋白质工程方法对脂肪酶蛋白进行改造、脂肪酶固定化和催化工艺的改良 等。     

代谢工程改造微生物合成单萜芳香产品的研究进展

单萜及其衍生物是重要的植物天然产物,且具有多种生物学功能。该类物质在多个领域中均表现出较高的开发利用价值,目前已被作为优质香精香料广泛应用于食品、饮料、化妆品和医药工业中,市场需求日益增长。从植物中提取这些单萜芳香产品存在着来源少、含量低和分离困难等缺点,很难满足市场需求。因此,开发生产单萜芳香产品可再生的微生物资源来补充甚至代替原有的植物资源就具有重要的理论意义和应用价值。近年来,研究人员利用代谢工程技术已经成功构建了合成单萜芳香产品的微生物细胞工厂,达到了利用微生物合成法生产该类工业产品的目的。本文主要从菌株改造、发酵优化及产物分离等角度总结了相关产物合成的代谢工程实例,并分析了目前利用代谢工程改造微生物合成单萜芳香产品所面临的瓶颈问题及其可能的解决方法,旨在为构建异源、廉价、高效生产单萜芳香产品的微生物细胞工厂并最终实现其绿色制造提供参考。     

Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster

The TRP1 gene encoding N-(5'-phosphoribosyl)-anthranilate isomerase was isolated from the yeast Yarrowia lipolytica, in which only a few genetic marker genes are available. The Y. lipolytica TRP1 gene (YlTRP1) cloned by complementation of Y. lipolytica trp1 mutation was found to be a functional homologue of Saccharomyces cerevisiae TRP1. Since YlTRP1 could be used for counterselection in medium containing 5-fluoroanthranilic acid (5-FAA), we constructed TRP blasters that contained YlTRP1 flanked by a direct repeat of a sequence and allowed the recycling of the YlTRP1 marker. Using the TRP blasters the sequential disruption of target genes could be carried out within the same strain of Y. lipolytica. The nucleotide sequence of the YlTRP1 gene has been deposited at GenBank under Accession No. AF420590.     

Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese

This work studied the qualitative and quantitative proteolytic and lipolytic activities of Yarrowia lipolytica strains isolated from two cheese types. Randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to compare the cheese strains of Y. lipolytica with strains isolated from other food products and with the type strain of the species in order to investigate the genetic diversity and occurrence of specific environmental groups. Diversity of proteolytic and especially lipolytic activity within Y. lipolytica strains isolated from dairy products was observed. In particular, the degree of specificity for saturated or unsaturated fatty acids as well as for even- or odd-numbered carbon free fatty acids (FFAs) varied among the strains. The RAPD-PCR profiles showed low genetic relatedness between many of the food isolates and the type strain of the species. Such genetic variability needs to be further evaluated. Most of the Y. lipolytica strains appeared to be specific to the particular environment from which they were isolated. However, phenotypic characteristics having technological importance in dairy products and, particularly, lipolytic activities did not correspond to the genetic differences observed by RAPD-PCR analysis.     

蛋氨酸特异性合成途径关键酶——高丝氨酸O-酰基转移酶的研究进展

在蛋氨酸生物合成过程中,蛋氨酸特异性合成反应的第一步是高丝氨酸酰基化,由高丝氨酸O-酰基转移酶催化,生成酰基高丝氨酸。高丝氨酸O-酰基转移酶受到末端产物蛋氨酸和S-腺苷蛋氨酸的反馈抑制和反馈阻遏,且高温下极易失活,严重影响碳流流向蛋氨酸,是蛋氨酸生物合成的关键酶,因此对高丝氨酸O-酰基转移酶的研究和改造具有重要意义。但目前关于高丝氨酸O-酰基转移酶的改造和研究较少,在微生物代谢途径中,碳流不能过多流入蛋氨酸合成途径,制约了微生物过量积累蛋氨酸,阻碍了发酵法生产蛋氨酸的工业进程。本文简述了高丝氨酸O-酰基转移酶在蛋氨酸生物合成途径中的作用,在介绍该酶的结构、催化机制和研究现状的基础上,提出该酶在反馈抑制和提高稳定性方面的分子改造策略。