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.     

The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme-deficient cells

ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol Δ⁵ desaturase that introduces the C5=6 unsaturation in ergosterol biosynthesis. The ERG3 gene has been mapped on chromosome XII, 13·7 centimorgans from GAL2 toward SPT8. The essentially of the gene is dependent on the conditions used for the cultivation of the mutants. Insertionally inactivated mutants of ERG3 fail to grow without ‘sparking’ levels of Δ⁵ sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol.     

Fatty acid synthase inhibitors separated from oiltea camellia by high-speed counter-current chromatography

Abstract: The inhibitory effects of oiltea camellia extracts (OCEs) and its active components on fatty acid synthase (FAS) were investigated. OCE potently inhibited the activity of FAS with an half‐maximal inhibitory concentration (IC₅₀) value of 2.30 μg/mL. The inhibition kinetic results showed that OCE and acetyl‐CoA competitively inhibited FAS but these compounds exhibited mixed inhibition against malonyl‐CoA and NADPH. Further study uncovered that the active components of OCE, ellagic acid (EA) and 3‐O‐methylellagic acid 4′‐O‐β‐D‐glucopyranoside (MEAG), which were isolated and purified by high‐speed counter‐current chromatography (HSCCC) using a 2‐phase solvent system of chloroform‐ethanol‐water‐acetic acid (4:3:2:0.01, v/v/v/v), inhibited FAS with IC₅₀ of 2.50 and 37.73 μg/mL, respectively. Their inhibition kinetics were different from that of OCE. Both of them exhibited uncompetitive inhibition for nicotinamide adenine dinucleotide phosphate (NADPH) and decreased the FAS activity through inactivation of acetyl/malonyl transferase on FAS. These results suggest that OCE could be a valuable resource for bioactive substances. Practical Application: With the gradual increase in tea oil production, it was in urgent need of dealing with Camellia fruit hull, which was always discarded because of low economic benefits. Camellia fruit hull has been shown to have significant antioxidant effects including 1, 1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging ability and ferric‐reducing antioxidant power. This study found that the ethanolic extract of Camellia fruit hull at low concentration efficiently inhibited FAS activity, which is a potential therapy target of both obesity and cancer. These results suggest that OCE could be a valuable resource for bioactive substances.     

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.     

高效表达木糖醇脱氢酶基因酿酒酵母的构建及木酮糖发酵的初步研究

通过RT-PCR方法克隆得到Candida tropicalis木糖醇脱氢酶基因xyl2,将该基因连入酵母表达载体pYES2的诱导型启动子GAL1下,构建表达质粒pYES2-xyl2;同时用从Pichia pastoris中克隆获取的甘油醛磷酸脱氢酶基因GAP换下GAL1基因,构建含组成型启动子GAP基因的表达质粒pYES2-GAP-xyl2;通过电转化法将其依次转入酿酒酵母S.cerevisiae INVSc1,山梨醇培养基上筛选的转化子经木糖醇梯度驯化培养,筛选出1株耐木糖醇浓度为20%的酿酒酵母重组菌株ZCX4和1株在半乳糖诱导下耐木糖醇浓度为15%的重组菌株YDX2。酶活测定表明,重组菌株ZCX4比酶活0.621 U/mg(蛋白),是YDX2比酶活的2.29倍。摇瓶发酵结果显示,重组菌株ZCX4木糖醇消耗76.46 g/L,木糖醇消耗率为76.46%,是重组菌株YDX2木糖醇消耗率的1.63倍,说明木糖醇脱氢酶实现了高效表达。     

巴斯德毕赤酵母PEP4基因的剔除及对基因工程菌外源蛋白质表达的影响

目的构建蛋白酶缺失菌株,提高外源蛋白质表达的分泌率与产量。方法构建质粒pRS306/PEP4 L&R,利用基因重组原理剔除巴斯德毕赤酵母中的PEP4基因。比较基因剔除前后外源蛋白质表达量的差异。结果剔除毕赤酵母中的PEP4基因后外源蛋白质表达量平均提高9．5%。结论剔除毕赤酵母中的PEP4基因可有效地控制外源蛋白质MIP表达时的降解。     

Over-expression of GSH1 gene and disruption of PEP4 gene in self-cloning industrial brewer's yeast

Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.     

废纸脱墨浆的甲脒亚磺酸漂白

介绍了用甲脒亚磺酸（ＦＡＳ）漂白彩色混合废纸脱墨浆和高白度办公废脱墨浆的实验情况,探讨了ＦＡＳ用量,ＮａＯＨ和ＦＡＳ用量比,浆浓,漂白温度和时间对漂后纸浆白度的影响,并进行了Ｈ２Ｏ２－ＦＡＳ两段漂白,比较了ＦＡＳ、Ｈ２Ｏ２单段漂白和Ｈ２Ｏ２－ＦＡＳ、ＦＡＳ－Ｈ２Ｏ２两段漂白的效果。实验结果表明,ＦＡＳ是一种有效的废纸浆漂白剂和脱色剂。     

Extraction,isolation, characterisation,antioxidant and anti-fatigue activities of Pleurotus eryngii polysaccharides

Pleurotus eryngii polysaccharide (PEP) was obtained using hot water extraction and ethanol precipitation. Three purified polysaccharide fractions (namely PEP1-A, PEP2-A and PEP3-A) were obtained from PEP using DEAE-cellulose-52 chromatography and a gel permeation Sephadex G-100 column. Firstly, this paper examined the characterisation of PEP1-A, PEP2-A and PEP3-A. The corresponding molecular weights were 5.378 × 10⁵, 9.506 × 10⁶ and 4.975 × 10⁵ Da, respectively. PEP1-A, PEP2-A and PEP3-A had similar monosaccharide compositions. PEP1-A was β-configuration, and PEP2-A and PEP3-A were α-configuration. PEP1-A, PEP2-A and PEP3-A had pyran-type rings, (1 → 3) glucose and (1 → 6) galactose linkages. Secondly, PEP, PEP1-A, PEP2-A and PEP3-A possessed antioxidant activities, and PEP was best. Therefore, only PEP was used to study its anti-fatigue activity in vivo. The result proved that PEP had anti-fatigue activity. PEP could be used as a valuable natural food supplement for preventing anti-fatigue or functional food.     

填饲对鹅肥肝脂肪酸合成酶基因mRNA表达的影响

以朗德鹅为对象,采用实时荧光定量PCR法研究不同填饲周龄脂肪酸合成酶基因(FAS基因)表达的规律性。结果显示,FAS基因相对表达量(FAS与β-actin 基因表达量的比值)在填饲前、填饲1周、填饲2周、填饲3周和填饲4周相对表达量逐渐增高,分别为0.035、0.128、0.253、0.876、1.009,说明肥肝组织中FAS基因表达丰度与填饲期呈显著正相关,FAS基因在鹅肥肝形成过程及体脂沉积过程中具有一定的调控作用,为促进鹅肝内脂肪沉积的主要调控基因。     

Detection of a point mutation in FAS2 gene of sake yeast strains by allele-specific PCR amplification

To identify yeast mutants with a point mutation, detection of the specific mutant alleles is necessary. For this purpose, we applied allele-specific polymerase chain reaction (PCR) to detect the FAS2-1250S dominant mutant allele that encodes an altered fatty acid synthase in Japanese brewer's yeast strains. These strains are known to produce a higher amount of ethyl caproate in Japanese sake. The mutant strains were supposed to be diploid and to contain heterozygous alleles, including wild-type FAS2 and a dominant FAS2-1250S. A set of oligonucleotide primers was designed to contain different nucleotides at their 3' termini: one type was identical to the wild type and the other to the mutant FAS2. Another set of primers was designed to have an additional mismatch at the second nucleotide from their 3' termini. By testing with control strains, we established PCR conditions for specific amplification. Using these conditions and a simple template preparation procedure with SDS, the presence of the allele was detected in commercially used sake yeast strains. The method presented here will be useful for the identification of specific yeast strains.     

重组鲍鱼肌肉脯氨酰内肽酶的性质研究

为研究皱纹盘鲍（Haliotis discus hannai）中脯氨酰内肽酶（Prolyl endopeptidase,Hdh-PEP)的酶学特性与结构特性,利用基因工程技术重组并在大肠杆菌中高效表达了皱纹盘鲍PEP。原核表达的Hdh-PEP分子量为85 kDa,在pH2～6、温度20～60 ℃条件下,Hdh-PEP的表面疏水性明显升高。氨基酸序列同源性分析结果表明,Hdh-PEP催化结构域中有三个高度保守的氨基酸序列:Seq 1:K-D-G-T-K/R-I-P、Seq 2:Y-G-Y-G-G-F和Seq 3:I-R-G-G-E-Y/F。酶动力学研究表明,Hdh-PEP的米氏常数K_m为5.32 μmol/L,催化常数k_cat值为15.7 s?1。PEP的特异性抑制剂SUAM-14746和ZPP对Hdh-PEP酶活力具有强抑制作用,丝氨酸蛋白酶抑制（PMSF）对Hdh-PEP酶活力也有较大程度的抑制作用。本实验制备了高特异性抗Hdh-PEP多克隆抗体,可检测鲍鱼肌肉中天然PEP的存在情况。Hdh-PEP的体外高效表达和特异性多克隆抗体制备为后续深入研究Hdh-PEP的性质提供了重要参考。     

The shock of vacuolar PrA on glycolytic flux,oxidative phosphorylation,and cell morphology by industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> WZ65

Proteinase A (PrA) is one of the most significant vacuolar proteinase in S. cerevisiae, and it plays an important role in S. cerevisiae physiology and metabolism, especially under unfavorable environment. In this study, the differences in pyruvate kinase (PYK) level under fructose-1,6-diphosphate (FDP) induction and ATP synthesis block among SC1 (the wild-type yeast that was industrial Saccharomyces cerevisiae WZ65), SC2 (PEP4 partial deletion) and SC3 (PEP4 complete deletion) were examined. Results showed that the induction caused by FDP clearly increased PYK expression no matter for which strain, but the increasing effect is more significant for SC2 (P < 0.05). The comparative results of intracellular ATP accumulation showed that the induction by FDP may be affected at the presence of PrA. The block experiment of ATP synthesis showed that PYK activities in PEP4-modified strains are lower than that of the wild type, but the intracellular ATP levels in the wild-type one are generally higher than the PEP4-modified strains after rotenone treatment (P < 0.01). This implies that the effect of PrA deficiency on intracellular ATP accumulation was much more pronounced than the effect of rotenone on oxidative phosphorylation. The cell morphology of three strains was comparatively examined by means of transmission electron microscopy (TEM). The PEP4-modified strains possessed more vacuoles, and cell structure were more integrated than the wild-type strain. Current data preliminarily indicated that the deletion of PEP4 gene in industrial S. cerevisiae WZ65 may not only affected PYK expression but also modulated the oxidative phosphorylation flux.     

油茶籽提取物对脂肪酸合酶的抑制作用

脂肪酸合酶(FAS)被认为是治疗肥胖症的潜在靶点.研究表明:油荼壳提取物对FAS具有很强的快结合可逆抑制作用.萃取的最佳溶剂为40%乙醇水溶液,此提取物的半抑制质量浓度为2.02μg/mL.而油茶饼70%乙醇提取物则具有很强的慢结合不可逆抑制作用,在0.31 mg/mL下2 min内FAS失活超过85%.结果表明:油荼壳提取物与油荼饼提取物分别在快结合抑制与慢结合抑制上显示出很强的抑制能力,预计为新的抑制剂,对研究FAS的作用机理与防治肥胖症的应用上可能具有重要的价值.     

Short communication: Principal components and factor analytic models for test-day milk yield in Brazilian Holstein cattle

A total of 46,089 individual monthly test-day (TD) milk yields (10 test-days), from 7,331 complete first lactations of Holstein cattle were analyzed. A standard multivariate analysis (MV), reduced rank analyses fitting the first 2, 3, and 4 genetic principal components (PC2, PC3, PC4), and analyses that fitted a factor analytic structure considering 2, 3, and 4 factors (FAS2, FAS3, FAS4), were carried out. The models included the random animal genetic effect and fixed effects of the contemporary groups (herd-year-month of test-day), age of cow (linear and quadratic effects), and days in milk (linear effect). The residual covariance matrix was assumed to have full rank. Moreover, 2 random regression models were applied. Variance components were estimated by restricted maximum likelihood method. The heritability estimates ranged from 0.11 to 0.24. The genetic correlation estimates between TD obtained with the PC2 model were higher than those obtained with the MV model, especially on adjacent test-days at the end of lactation close to unity. The results indicate that for the data considered in this study, only 2 principal components are required to summarize the bulk of genetic variation among the 10 traits.     

Formation of Transparent Gels of Sesame 13S Globulin: Effects of Fatty Acid Salts

The physicochemical properties of sesame 13S globulin gels containing fatty acid salts (FAS) were investigated. The softness, water-holding ability and transparency of the gels markedly increased in the presence of sodium oleate (NaCl_18:1) or sodium linoleate (NaC_18:2) at a 75 molar ratio to a molecule of the 13S globulin. The effect was not observed by the addition of sodium caprylate (NaC_8:0). Scanning electron microscopy showed that a very fine network structure was formed in the gel with 75 molar ratio of NaC_18:2. The addition of a suitable amount of FAS having a moderate length and an unsaturated carbon chain could improve the water-holding ability and transparency of the 13S globulin gel.     

酿酒酵母关键节点基因缺损对法尼烯合成的影响

以法尼烯为评价效应物,研究了缺损乙醇合成途径、甘油合成途径、胞质乙酰辅酶A转运途径和法尼基焦磷酸消耗支路关键基因对酿酒酵母WHE4菌株合成法尼烯的影响。通过CRISPR-cas9基因编辑技术,获得8株关键基因缺损菌株。结果表明,与WHE4菌株相比,缺损乙醇脱氢酶基因ADH3-6对乙醇和法尼烯产量没有影响;单独缺损甘油三磷酸脱氢酶基因GPD1和GPD2使甘油积累量分别降低了15%和34%,缺损半乳糖激酶基因GAL1、GAL7、GAL10下调了甲羟戊酸途径所有基因转录水平,它们的缺损均不能提高菌株的法尼烯产量;缺损香叶基香叶基焦磷酸合酶基因BTS1和二酰基甘油二磷酸磷酸酶基因DPP1,法尼烯产量提高了29%,在5 L发酵罐补料分批发酵,菌株WHE4-33(WHE4 Δbts1,Δdpp1)的法尼烯产量达到1 578.91 mg/L。该研究对甲羟戊酸途径上游和下游关键节点基因进行了缺损影响法尼烯合成研究,为构建酿酒酵母萜类化合物高效平台提供了参考价值。