Construction of recombinant sake yeast containing a dominant FAS2 mutation without extraneous sequences by a two-step gene replacement protocol

A novel two-step gene replacement protocol was developed to construct a recombinant industrial yeast free of bacterial and drug-resistant marker sequences. A yeast strain exhibiting cerulenin resistance conferred by a dominant mutation of FAS2 was previously shown to produce high levels of a flavor component of Japanese sake. A N- and C-terminally truncated portion of the mutant FAS2 gene was subcloned to an integrating plasmid containing an aureobasidin A-resistant transformation marker and a galactose-inducible growth inhibitory sequence (GAL10p::GIN11). The plasmid was targeted into the chromosomal FAS2 locus of sake yeast Kyokai no. 7, resulting in a tandem repeat of inactive FAS2 sequences surrounding the integrated plasmid sequences. Cells containing the integrated plasmid were unable to grow on galactose medium due to the inhibitory effect of GAL10p::GIN11. This growth inhibition allowed efficient counter-selection for cells that had undergone homologous recombination between the FAS2 repeats by their growth on galactose medium. This recombination event resulted in loss of the integrated plasmid sequences and the resulting strains should contain a single copy of either wild-type or cerulenin-resistant FAS2. The selected cerulenin-resistant strains produced approximately 3.7-fold more ethyl caproate, a flavor component, than the Kyokai no. 7 strain. Southern blot and sequence analyses confirmed the presence of the FAS2 mutation and the absence of integrated plasmid sequences in the genome of the selected strain. This gene replacement method provides a straightforward approach for the construction of recombinant industrial yeasts free of undesirable DNA sequences.     

A high-efficiency method to replace essential genes with mutant alleles in yeast

Temperature-sensitive (TS), internally deleted and truncated alleles are important tools to facilitate the characterization of essential genes. We have developed a straightforward method to replace a wild-type gene with a mutant allele at the endogenous locus. This method is an efficient alternative to the two-step method for integration of alleles that are compromised in function or contain multiple mutations. A strain is constructed that has the essential gene of interest disrupted by a selectable marker. Strain viability is maintained by a plasmid carrying a copy of the essential wild-type gene and the ADE3 gene. The mutant allele is cloned into an integratable vector carrying a selectable/counter-selectable marker, such as URA3. The plasmid is linearized and transformed, directing integration to the 5' or 3' region flanking the essential open reading frame (ORF). Transformants that have integrated the mutant gene at the endogenous locus can lose the autonomous plasmid carrying the wild-type copy of the essential gene and the ADE3 gene. These transformants are identifiable as white sectoring colonies, display the mutant phenotype and may be characterized. An optional second selection step on 5-fluoroorotic acid (5-FOA) selects for popouts of the integrating vector sequences, leaves the mutant allele at the endogenous locus, and recycles selectable markers. We have used this method to integrate a TS allele of SPC110 that could not be integrated by standard methods.     

Random and targeted gene integrations through the control of non‐homologous end joining in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus DMKU3‐1042 is a thermotolerant yeast strain suitable for high‐temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 × 10⁶ transformants/µg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3‐1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non‐homologous end‐joining (NHEJ) pathway. In yeast lacking KU70, high‐frequency non‐homologous gene integration was abolished and the Kmku70 mutants showed 82–95% homologous gene targeting efficiencies using homologous sequences of 40–1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid‐free gene manipulations in K. marxianus. Copyright © 2009 John Wiley & Sons, Ltd.     

枯草芽孢杆菌基因的安全改良研究

为了构建能稳定表达外源基因且无抗性标记基因的枯草芽孢杆菌(Bacillus subtilis)工程菌。采用聚合酶链式反应(PCR)技术,分两段扩增定位于B.subtilis BJ3-2染色体上的谷氨酰胺酶基因(glsA)上下游基因片段作为同源臂。将同源臂及卡那霉素基因(Kan)克隆入温敏型载体pKSV7,构建了双交换载体pKUKD。并利用pKUKD质粒将kan基因定点整合入BJ3-2染色体上,通过Kan抗性筛选获得glsA敲除菌株BJ-kan。经检测BJ3-2菌株谷氨酰胺酶活力比重组菌株高3.2倍。结果显示:BJ3-2染色体上的基因可通过同源重组方式进行敲除与置换,为今后BJ3-2菌株以及野生型B.subtilis基因敲除与置换提供了更加便捷的方法。     

A positive selection for plasmid loss in Saccharomyces cerevisiae using galactose-inducible growth inhibitory sequences

Counter-selections for the loss of introduced plasmid sequences are useful for gene manipulations in yeast. We have used GAL10 promoter-mediated overexpression of GIN sequences, which inhibit the growth of cells, to develop a novel counter-selection system. Yeast cells carrying a GIN sequence grow normally on glucose medium but are unable to grow on galactose medium, whereas derivatives that have lost the GIN sequence are able to grow in the presence of galactose. We constructed autonomously replicating, integrating, and disruption plasmids carrying GIN sequences and tested their use to select for loss of the plasmid. The results showed that the GIN sequences provide a selection for efficient loss of plasmids or integrated constructs from yeast during growth on galactose medium, indicating that this system can be used for plasmid shuffling, gene replacements and marker gene recycling. This counter-selection system has wide application, because any Gal+ strain and a wide variety of marker genes can be used. In addition, counter-selection systems using growth-inhibitory sequences should be applicable to other yeasts and possibly to other organisms.     

ATP合成关键酶基因APT1在雅致放射毛霉中的克隆表达

为构建遗传稳定的ATP高产工程菌,利用PCR技术扩增酿酒酵母ATP合成关键酶基因APT1,并将其克隆至质粒pCB1004-Pgpd的相应位点,得到有强启动子Pgpd驱动的APT1基因超表达质粒pCB1004-Pgpd-APT1。在PEG-CaCl2介导下,超表达质粒转化雅致放射毛霉原生质体,获得ATP高产工程菌。其ATP产量及摩尔转化率比出发菌株提高44.04%。     

β_2肾上腺素受体基因在Sf9细胞中的表达及纯化

通过密码子优化、昆虫杆状病毒表达系统(Bac-to-Bac)构建、表达条件筛选及镍柱亲和层析,研究β_2肾上腺素受体(β_2 adrenergic receptor,β_2AR)基因(β_2AR)在昆虫细胞Sf9中的高效表达及纯化策略。方法:人工合成改造后的β_2AR基因,将其克隆至转移载体p Fast Bac1中,构建重组杆状病毒表达质粒p Fast Bac1-β_2AR’,转染昆虫细胞Sf9,优化表达条件,采用镍离子亲和层析法纯化重组蛋白并进行活性鉴定。结果:适宜的表达条件为感染细胞使用的感染复数5、感染后表达时间48 h,Western blot分析显示在47 k D左右处出现清晰的特异性条带,与预期结果一致。纯化的受体蛋白纯度大于90%,活性鉴定结果显示该受体蛋白可特异性吸附盐酸克伦特罗、沙丁胺醇及莱克多巴胺3种β激动剂的酶标记物,OD值分别为0.983、0.947和0.912。结论:本研究实现了β_2AR受体在Sf9细胞中的表达,且纯化后的受体蛋白保持了较好的β激动剂亲和活性,为利用β_2AR受体开展β激动剂多残留快速检测技术提供了依据。     

野油菜黄单胞菌产胶基因GumD克隆与重组质粒的构建

目的:建立野油菜黄单胞菌产胶基因GumD的克隆与重组质粒构建的方法。方法:以优化PCR实验条件扩增得到野油菜黄单胞菌产胶基因GumD片断,与抗药性质粒pMD18-T连接,获得重组质粒,并用CaCl2法转化JM109大肠杆菌细胞。结果:DNA测序结果表明,成功克隆产胶基因和构建重组质粒。结论:此方法可用于克隆野油菜黄单胞菌产胶基因和构建产胶基因重组质粒。     

Polyethyleneimine/chitosan hexamer-mediated gene transfection into intestinal epithelial cell cultured in serum-containing medium

In search of an efficient nonviral vector, polyethyleneimine (PEI)-based vectors were examined. In general, the transfection efficiency of nonviral vectors is suppressed by serum. Here we show that PEI based vectors, particularly, the chitosan hexamer-PEI vector, could perform efficient gene transfection into intestinal epithelial cells (IEC-6) in the presence of serum. The conjugation order of the two polymers with a plasmid (first, chitosan hexamer; second, PEI) was found to be an important factor in enhancing transfection efficiency.     

High fidelity segregation of a YEp vector in [cir0] strains of the yeast Saccharomyces cerevisiae

YEp vectors carrying the GPD promoter and PGK terminator for constitutive expression showed high partition efficiencies specifically in [cir(0)] strains, when DNA fragments were inserted into the cloning site. These plasmids appeared to be more stable than yeast centromeric plasmids, being lost in less than 10(-2) cells after each generation, and more than 90% of the cells carried the plasmids after 20 generations of growth in the absence of selective pressure. The REP3 region was essential together with the GPD promoter and PGK terminator for plasmid equipartitioning in [cir0] strains. The present results suggest that this host-vector system would be useful for astute observation of the phenotype caused by gene overexpression, and for heterogeneous protein production using natural medium, because of efficient partitioning of the plasmid without selective pressure.     

YHp as a highly stable,hyper-copy,hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir⁰ strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir⁰ ura3^- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.     

Effect of amplification of desensitized purF and prs on inosine accumulation in Escherichia coli

The effect of a phosphoribosylpyrophosphate (PRPP) synthetase gene (prs) that was desensitized to feedback inhibition by ADP on inosine accumulation was investigated using an inosine-producing mutant of Escherichia coli. At the same time, various types of plasmid having a PRPP amidotransferase gene (purF) that was desensitized to feedback inhibition by AMP and GMP were also investigated to improve inosine productivity using a compatible plasmid containing prs with a plasmid containing purF. The recombinant E. coli I-9 harboring a low-copy-number plasmid having the desensitized-purF (pMWKQ) accumulated 3.6 g/l inosine from 40 g/l glucose in a 2-d culture. Furthermore, desensitized-prs amplification, in addition to purF, resulted in the accumulation of 6.2 g/l inosine. Additionally, through these experiments, a spontaneous mutant with an enhanced inosine-producing ability compared with the parent strain I-9 was obtained. The spontaneous mutant I-9m harboring only pMWKQ and I-9m harboring both pMWKQ and pSTVDA (a plasmid having the desensitized-prs) accumulated 6.7 g/l and 7.5 g/l inosine, respectively, from 40 g/l glucose in a 3-d culture.     

Large-scale preparation of recombinant human calcitonin from a multimeric fusion protein produced in Escherichia coli

In order to develop a large-scale, high-yield production process for human calcitonin (hCT) in Escherichia coli, a stable expression plasmid was constructed and the expressed protein was modified for efficient cleavage by protease. Multiple copies of a synthetic gene encoding hCT-Leu-Arg, a substrate for C-terminal amidation by carboxypeptidase Y (CPY), were inserted into the stable expression plasmid. Using this plasmid, the expression of a multimeric fusion protein was induced by shifting the temperature from 34 to 38 degrees C. The multimeric fusion protein was accumulated as inclusion bodies. After the fermentation, the cells were harvested and disrupted by a homogenizer. The insoluble multimeric fusion protein was suspended in 6.6 M urea solution. At this time, however, the protein could not be solubilized and thus the efficiency of cleavage by protease was low. To solubilize the protein and protect Lys residues against digestion by trypsin, the protein was citraconilated with citraconic anhydride. Following S-sulfonation with Na2SO3-CuSO4, almost all the protein was solubilized. The solubilized, citraconilated, and S-sulfonated protein was digested with trypsin efficiently. By treatment with trypsin, the multimeric fusion protein was cleaved into monomers of citraconilated and S-sulfonated hCT-Leu-Arg (S-hCT-Leu-Arg). The subsequent decitraconilation was performed at low pH. S-hCT-Leu-Arg, isolated by preparative HPLC, was directly converted into S-hCT-HN2 by CPY without removal of the Arg residue. Finally, S-hCT-NH2 was desulfonated and converted into mature hCT. In this way, a large amount of recombinant mature hCT was obtained in a tank fermentation. To our knowledge, this is the first report of industrial-scale, human calcitonin production using a multimeric fusion protein expression system.     

Cloning of the bile salt hydrolase (bsh) gene from Enterococcus faecium FAIR-E 345 and chromosomal location of bsh genes in food enterococci

Enterococcus faecium strain FAIR-E 345 isolated from food was shown to possess bile salt hydrolase (Bsh) activity in a plate screening assay and by high-performance liquid chromatography analysis. The bsh gene was cloned and sequenced. DNA sequence analysis revealed that it encoded a protein of 324 amino acids, with pI 4.877. A bsh gene probe was prepared from the cloned bsh gene and was used for probing plasmid and total genomic DNA of Bsh-positive enterococci isolated from food to determine the genomic location of their bsh genes. This probe was able to detect the bsh gene among total genomic DNA preparations but not from plasmid preparations of 10 plasmid-bearing Enterococcus strains. However, the probe could detect the bsh gene from total genomic DNA preparations of 12 Enterococcus strains that did not contain detectable plasmid DNA. In no cases did the probe hybridize with plasmid DNA preparations, suggesting that the bsh gene among enterococci is probably generally chromosomally encoded. This presumptive chromosomal location of bsh genes among food enterococci suggests that transfer of this trait by conjugative plasmids is unlikely.     

乳酸乳球菌质粒pAG712的特性研究

乳酸乳球菌质粒pAG712 （15.5kb）赋予乳酸乳球菌细胞凝聚表型,它是以θ模式进行复制,复制蛋白与其它以θ模式进行复制的质粒的复制蛋白具有极高的同源性.复制区上的Bg/Ⅱ （4.2kb）片段决定质粒可以在乳酸乳球菌中进行复制.在本研究中,将抗生素抗性基因插入到质粒pAG712中,给予它选择性的标记,在研究中发现凝聚基因（aggregation gene）的存在导致了乳酸乳球菌质粒pAG712的分离不稳定性。     

Active lactonizing lipase (LipL) efficiently overproduced by Pseudomonas strains as heterologous expression hosts

Pseudomonas sp. strain 109 secretes lactonizing lipase (LipL), which catalyzes efficient intramolecular transesterification of omega-hydroxyfatty acid esters to form macrocyclic lactones. Because Escherichia coli was found to be unsuitable as an expression host due to the predominant formation of inactive LipL-inclusion bodies and a lack of proper secretion machinery which is also required for the formation of active LipL, Pseudomonas strains were surveyed as expression hosts. Pseudomonas sp. strain 109, an original LipL producer, showed a 7.1-fold higher level of active LipL when the lipL gene under the control of tac-lacUV5 tandem promoter was introduced together with a limL gene encoding a LipL-specific chaperon. Pseudomonas aeruginosa ADD 1976 containing a T7 RNA polymerase gene in the chromosome and plasmid-borne lipL-limL genes under the control of T7 promoter showed a 13-fold higher level of active LipL. Several combinations in the number of lipL and/or limL genes on the plasmid were investigated, and (lipL)3-limL was found to be most efficient, yielding a 67-fold greater production of active LipL than that obtained by the wild-type Pseudomonas sp. strain 109.