Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii

Abstract An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii , which is resitant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The phospholipase B in T. delbrueckii cells is active in both acidic and alkaline conditions. However, activity of phospholipase B gene ( PLB1 ) in cells of disruption mutant ( plbI : : URA3 ) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delbrueckii cells showed ∼ 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells. Cells of plb1 Δ mutant showed increased survival when cells of plb1 Δ mutant and wild-type strain were incubated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.     

Improved gene disruption method for Torulaspora delbrueckii

PCR-based disruption cassettes are one of the most commonly used strategies for gene targeting in Saccharomyces cerevisiae . The efficiencies of gene disruption using this conventional method are highly variable among species, and often quite low with nonconventional yeasts. Here we describe an improved strategy to obtain deletion mutants in baker's yeast Torulaspora delbrueckii , one of the most abundant non- Saccharomyces species, present in home-made corn and rye bread dough.     

Cloning and characterization of the MAL11 gene encoding a high-affinity maltose transporter from Torulaspora delbrueckii

The transport and regulation of maltose utilization by Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species present in home-made corn and rye bread dough, has been investigated. A DNA fragment containing the MAL11 gene from T. delbrueckii (TdMAL11) was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 1884 bp, encoding a 627-amino acid membrane protein, which displays high homology to other yeast maltose transporters. Upstream of TdMAL11, the DNA insert contained a partial ORF (TdMAL12) on the opposite strand, which showed high similarity to the S. cerevisiae MAL12 gene. Sequence analysis, Northern blot and transport measurements indicated that TdMAL11 expression is regulated by the carbon source. Attempts to disrupt TdMAL11 revealed the presence of two functional MAL loci. Disruption of a single copy decreased the V(max) of maltose transport, but not the K(m), whereas the double disruption abolished the uptake of this sugar in T. delbrueckii.     

Osmotolerance and leavening ability in sweet and frozen sweet dough. Comparative analysis between Torulaspora delbrueckii and Saccharomyces cerevisiae baker's yeast strains

The response of Saccharomyces cerevisiae and freeze-tolerant Torulaspora delbrueckii strains to osmotic stress and their CO₂ production capacity in sweet and frozen-sweet dough has been examined. T. delbrueckii strains, IGC5321 and IGC5323 showed higher leavening ability than Saccharomyces, specially after exposure to hyperosmotic stress of bread dough containing 20% sucrose and 2% salt added. In addition, Torulaspora and especially T. delbrueckii IGC5321 exhibited no loss of CO₂ production capacity during freeze-thaw stress. Overall, these results appeared to indicate that Torulaspora cells are more tolerant than Saccharomyces to osmotic stress of bread dough. This trait correlated with a low invertase activity, a slow rate of trehalose mobilisation and the ability to respond rapidly to osmotic stress. Growth behaviour on high osmotic synthetic media was also examined. Cells of the IGC5321 strain showed intrinsic osmotolerance and ion toxicity resistance. However,T. delbrueckii IGC5323 exhibited a clear phenotype of osmosensitivity. Hence, this characteristic may not be essential or the only determinant for leavening ability in salted high-sugar dough. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterisation of osmotolerant hybrids obtained by fusion between protoplasts of Saccharomyces cerevisiae and heat treated protoplasts of Torulaspora delbrueckii

Saccharomyces cerevisiae was fused with heat-treated protoplasts of an osmotolerant yeast, Torulaspora delbrueckii, to obtain hybrids having increased tolerance to increased glucose concentrations (up to 700 gl–1). The production of glycerol and arabitol by the hybrids was within the range of those of the parental strains, but the production of ethanol was higher.     

Influence of oxygen on the biosynthesis of cellular fatty acids,sterols and phospholipids during alcoholic fermentation by Saccharomyces cerevisiae and Torulaspora delbrueckii

Saccharomyces cerevisiae and Torulaspora delbrueckii were grown under different O₂ availabilities on grape must. Oxygen requirements for the two yeasts were different: under anaerobic conditions, S. cerevisiae produced a higher percentage of unsaturated fatty acids, and had a greater cell yield and fermentation activity than T. delbrueckii. Addition of ergosterol (25mg/l) and oleic acid (31mg/l) caused total recovery of cellular growth and the fermentation activity of S. cerevisiae in anaerobiosis, but not of T. delbrueckii. However a short period of aeration to a 48 h culture in anaerobiosis, led to total recovery of the cellular growth and fermentation activity in both yeasts. Likewise, the effect of a short aeration period on unsaturated fatty acid biosynthesis was similar for both species.     

Purification and some properties of membrane-bound phospholipase B from Torulaspora delbrueckii

Membrane-bound phospholipase B was purified to a homogeneous state from Torulaspora delbrueckii cell homogenate. Cell homogenate was extracted with Triton X-100, and the enzyme was precipitated with acetone. The acetone powder was washed repeatedly with Tris-HCl buffer (pH 8.0) until no phospholipae B activity was detected in the soluble fraction. The enzyme was extracted with Triton X-100 from the final residue and purified about 1,390-fold by sequential chromatofocusing, Sepharose 6B, and DEAE-Sephadex A-50 column chromatography. The final preparation showed a single broad protein band on SDS-polyacrylamide gel electrophoresis when stained with silver stain reagent and PAS-reagent. The molecular weight of phospholipase B was about 390,000 and 140,000-190,000 as estimated by gel filtration on Sepharose 6B and SDS-polyacrylamide gel electrophoresis, respectively, suggesting that phospholipase B is an oligomeric protein. The isoelectric point was at pH 4.5. Phospholipase B has two pH optima, one acidic (pH 2.5-3.0) and the other alkaline (pH 7.2-8.0). At acidic pH the phospholipase B activity was greatly increased in the presence of divalent metal ions, although metal ions are not a factor for enzyme activity. On the other hand, at alkaline pH the enzyme required Ca2+ or Mn2+ for activity. The pH- and thermal-stabilities at both pHs were similar. The phospholipase B hydrolyzed all diacylphospholipids tested at acidic pH, but hydrolyzed only phosphatidylcholine at alkaline pH. The hydrolysis rates of lysophospholipids were much higher (about 10-fold) than those of diacylphospholipids at both pHs.     

Torulaspora maleeae sp. nov., a novel ascomycetous yeast species from Japan and Thailand

Nine strains of a new Torulaspora species were isolated from natural samples collected in Japan and Thailand including one strain obtained from a leaf of Rhizophora stylosa (NBRC 11061T), one strain from soil (NBRC 11062), six strains from mosses (ST-14, ST-266, ST-510, ST-511, ST-513 and ST-581) and one strain from sediment in mangrove forest (RV-51). On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) (ITS1-5.8S rRNA gene-ITS2) region, the nine strains were found to represent a single novel species of the genus Torulaspora, which were named Torulaspora maleeae sp. nov. The type strain is NBRC 11061T (BCC 25515T=CBS 10694T). In the phylogenetic trees based on the sequences of the D1/D2 domain of the LSU rRNA gene, T. maleeae showed a close relationship with the five recognized species of the genus Torulaspora, Torulaspora delbrueckii, Torulaspora franciscae, Torulaspora globosa, Torulaspora microellipsoides and Torulaspora pretoriensis. Torulaspora maleeae differed from the five recognized species of the genus Torulaspora by six to 12 nucleotide substitutions (1.1-2.1%) in the D1/D2 domain of the LSU rRNA gene and by 6.4-11.7% nucleotide substitutions in the ITS (ITS1-5.8S rRNA gene-ITS2) region.     

人成骨蛋白成熟肽基因在毕赤酵母中的克隆及其序列分析

采用PCR方法,根据献报道的人成骨蛋白-1（OP-1）成熟肽基因序列,设计并合一对引物,从含人成骨蛋白基因的质粒中扩增获得大小的420bp的DNA片段,连接到pGEM-T载体进行测序,证明获得人成骨蛋白成熟肽基因片段,继之以pPIC3.5k为表达载体构建重组表达质粒,并经PCR及酶切鉴定。     

Isolation and fundamental characteristics of a phospholipase B inhibitor from autolyzed Torulaspora delbrueckii.

Phospholipase B inhibitor was found in the autolyzate of yeast cells, Torulaspora delbrueckii. The inhibitor was purified to homogeneity by ethanol precipitation, gel filtration with Sephadex G-10, ion-exchange chromatography with DEAE-Sephacel, and gel filtration with Asahipak GS-320. On thin-layer chromatography the purified inhibitor was detected with the Hanes-Isherwood reagent, which is used to detect phosphorus. The activity of the inhibitor was not affected by heat treatment at 100 degrees C for 1 h. Heating at 100 degrees C for 1 h in 1 M HCl and 1 M NaOH lowered activity to 76 and 80% of the original values, respectively, but heating at 110 degrees C for 24 h in 6 M HCl completely abolished activity. The inhibitor was highly soluble in water, but practically insoluble in alcohol, acetone, ether, and chloroform. The degree of inhibition of enzyme activity was not proportional to the concentration of inhibitor. The inhibitor inhibited both membrane-bound and water-soluble phospholipase B activity from T. delbrueckii at the same level; however, the inhibitor did not inhibit the activity of phospholipase A2 from snake venom (Naja naja).     

Isolation and characterisation of the phospholipase B gene of Cryptococcus neoformans var. gattii

Cryptococcus neoformans var. gattii (serotypes B and C) is a human pathogen, ecologically, biochemically, clinically and genetically different from C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D). The phospholipase B (PLB1) gene from serotypes B and C was isolated and characterised. It resembled the serotype A and D genes, with an overall sequence homology of more than 85%. The respective open reading frames were 2236 bp (serotype B) and 2239 bp (serotype C) in length. Each contained six introns and encoded a 68-kDa protein destined for secretion. PLB1 was located on the second smallest chromosome in both serotypes. Gene expression, measured as mRNA, was not regulated by temperature, pH or exogenous nutrients.     

H.ylori尿素酶B亚单位在保加利亚乳杆菌的表达与鉴定

目的构建表达幽门螺杆菌（Helicobacter pylori,H、pylori）尿素酶B亚单位（UreB）的基因工程乳杆菌,并对其进行初步的安全性评估。方法采用高保真PCR从H.pylori标准菌株NCTC 11637中扩增ureB基因,插入乳酸菌表达质粒pMG36e,将重组质粒电转入保加利亚乳杆菌L6032中,获得表达ureB的基因工程乳杆菌。在含乳糖的MRS培养基诱导目的蛋白表达,Western blot鉴定其免疫原性。连续传代培养60代,检测基因工程乳杆菌的稳定性、形态学与生理生化特性以进行初步的安全性评估。结果特异PCR、酶切和测序鉴定均证实ureB基因克隆入表达载体pMG36e,SDS-PAGE结果显示,重组质粒pMG36e-ureB电转入保加利亚乳杆菌所构建的基因工程乳杆菌能表达约64KD的蛋白,Western blot证明该蛋白能与抗H.priori ureB的兔血清反应。稳定性、形态学与生理生化特性检测结果表明,基因工程乳杆菌与原始菌株保加利亚乳杆菌完全一致。结论成功构建能表达H.pylori UreB的保加利亚乳杆菌L6032-UreB,该基因工程菌在形态与生理生化特性上未发生任何变异,从而为探索幽门螺杆菌感染的益生菌制剂调理疗法奠定了坚实的基础。     

H.pylori尿素酶B亚单位在保加利亚乳杆菌的表达与鉴定

目的构建表达幽门螺杆菌（Helicobacter pylori,H、pylori）尿素酶B亚单位（UreB）的基因工程乳杆菌,并对其进行初步的安全性评估。方法采用高保真PCR从H.pylori标准菌株NCTC 11637中扩增ureB基因,插入乳酸菌表达质粒pMG36e,将重组质粒电转入保加利亚乳杆菌L6032中,获得表达ureB的基因工程乳杆菌。在含乳糖的MRS培养基诱导目的蛋白表达,Western blot鉴定其免疫原性。连续传代培养60代,检测基因工程乳杆菌的稳定性、形态学与生理生化特性以进行初步的安全性评估。结果特异PCR、酶切和测序鉴定均证实ureB基因克隆入表达载体pMG36e,SDS-PAGE结果显示,重组质粒pMG36e-ureB电转入保加利亚乳杆菌所构建的基因工程乳杆菌能表达约64KD的蛋白,Western blot证明该蛋白能与抗H.priori ureB的兔血清反应。稳定性、形态学与生理生化特性检测结果表明,基因工程乳杆菌与原始菌株保加利亚乳杆菌完全一致。结论成功构建能表达H.pylori UreB的保加利亚乳杆菌L6032-UreB,该基因工程菌在形态与生理生化特性上未发生任何变异,从而为探索幽门螺杆菌感染的益生菌制剂调理疗法奠定了坚实的基础。     

Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli

A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive clone showed LDH activity and production of D(−)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).     

Construction of an efficient amylolytic industrial yeast strain containing DNA exclusively derived from yeast

An amylolytic industrial yeast strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis SWA2 amylase gene was generated. The new strain contains DNA derived exclusively from yeast and expresses a high starch hydrolyzing activity. Yeast transformation was carried out by an integrative process targeted to a dispensable upstream region of the ILV2 locus, which determines sulfometuron resistance. The SWA2 enzyme was constitutively expressed under the ADH1 promoter. The growth, substrate utilization and fermentative capacity of this organism are described.     

An Activator Stimulating the Hydrolysis of Phospholipids by Phospholipase B from Torulaspora delbrueckii

An activator stimulating the enzymatic hydrolysis of phospholipids was purified to a homogeneous state from autolyzed Torulaspora delbrueckii cell washings. Autolyzed cell washings were extracted with chloroform and ethanol, and the activator was purified about 130-fold by sequential column chromatographies on DEAE-Sephacel, Sephacryl S-300, and TSK gel G 3000 SW (high performance liquid chromatography, HPLC). The molecular weight of the activator was about 175,000 as estimated by gel filtration on HPLC. However, the purified activator gave two protein bands corresponding to molecular weights from 102,000 to 129,000 and from 71,000 to 88,000, respectively, on SDS- polyacrylamide gel electrophoresis, when stained with silver stain reagent and periodic acid-Shiff (PAS) reagent. The activator was sensitive to heat treatment at 70°C for lOmin. The purified activator had no enzymatic activity, but stimulated the hydrolysis of phospholipids by water-soluble and membrane-bound phospholipases B if the substrates were pre-incubated with the activator. No stimulation of hydrolysis by the enzyme was observed when the activator was pre-incubated with the enzyme. The hydrolytic rate of phosphatidylcholine by the enzyme at acidic pH (pH 2.6) depended upon the amount of activator added. On the other hand, the hydrolytic rate at alkaline pH (pH 7.6) was stimulated greatly by more than 0.04 nmol of the activator.     

Bimodal regulatory effect of melittin and phospholipase A2-activating protein on human type II secretory phospholipase A2

Melittin and phospholipase A2-activating protein (PLAP) are known as efficient activators of secretory phospholipase A2(sPLA2) types I, II, and III when phospholipid liposomes are used as substrate. The present study demonstrates that both peptides can either inhibit or activate sPLA2 depending on the peptide/phospholipid ratio when erythrocyte membranes serve as a biologically relevant substrate. Low concentrations of melittin and PLAP were observed to inhibit sPLA2-triggered release of fatty acids from erythrocyte membranes. The inhibition was reversed at melittin concentrations above 1 microM. PLAP-induced inhibition of sPLA2 persisted steadily throughout the used concentration range (0-150 nM). The two peptides induced a dose-dependent activation of sPLA2 at low concentrations, followed by inhibition when model membranes were used as substrate. This opposite modulatory effect on biological membranes and model membranes is discussed with respect to different mechanisms the interaction of the regulatory peptides with the enzyme molecules and the substrate vesicles.     

Process parameters and reusability of the free cell mass of Torulaspora delbrueckii for the production of L-phenylacetylcarbinol (L-PAC)

The effect of process parameters on the biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) using a yeast isolate identified as Torulaspora delbrueckii was studied. The maximum yield of L-PAC obtained was (331 mg) per 100 ml biotransformation medium (glucose 3%, peptone 0.6% and at pH 4.5) from 600 mg of benzaldehyde with 8 h of reaction at 30 ± 2 °C. Growing the organism in presence of 3% glucose reduced the biotransformation time to 120 min. Addition of 0.6% acetaldehyde (30–35%) lead to an increase in L-PAC yield to 450 mg%. Semi-continuous feeding of benzaldehyde (200 mg) and acetaldehyde (200 l) four times at 30 min intervals could produce 683 mg of L-PAC/100 ml biotransformation medium. Chiral HPLC analysis of purified L-PAC and PAC-diol showed 99% enantiomeric purity. The cell mass was found to be reusable for biotransformation up to nine times when benzaldehyde and acetaldehyde levels were maintained at (350 mg and 350 l)–(400 mg and 400 l). At concentrations from 450 mg and 450 l to 600 mg and 600 l, however the cell mass could give efficient biotransformation only during one use.     

A new mannoheptaose containing alpha and beta-(1-->2) linkages isolated from the mannan of Torulaspora delbrueckii: ELISA inhibition studies

Torulaspora delbrueckii starin IFO 0955 was examined with respect to its structural and serological properties of the cell wall mannan (Td-0955-M). Td-0955-M revealed significant reactivities with sera from a commercially available factor serum kit (Candida Check) in ELISA. Td-0955-M was investigated for its chemical structure by acetolysis under conventional and mild conditions. NMR and GC techniques were used as analytical techniques. The mannooligosaccharide fractions eluted from a Bio-Gel P-2 column were found to consist of Man(alpha1-2)Man, M2, Man(alpha1-2)Man(alpha1-2)Man and Man(beta1-2)Man(alpha1-2)Man, M3, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M5, and a new mannoheptaose, which possesses the structure, Man(alpha1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(beta1-2)Man(alpha1-2)Man, M7. The results of the inhibition ELISA showed that the M7 oligosaccharide significantly inhibited the reactivities in the Td-0955-M-factor serum systems.