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In an era of economic globalization, the competition among wine businesses is likely to get tougher. Biotechnological innovation permeates the entire world and intensifies the severity of the competition of the wine industry. Moreover, modern consumers preferred individualized, tailored, and healthy and top quality wine products. Consequently, these two facts induce large gaps between wine production and wine consumption. Market-orientated yeast strains are presently being selected or developed for enhancing the core competitiveness of wine enterprises. Reasonable biological acidity is critical to warrant a high-quality wine. Many wild-type acidity adjustment yeast strains have been selected all over the world. Moreover, mutation breeding, metabolic engineering, genetic engineering, and protoplast fusion methods are used to construct new acidity adjustment yeast strains to meet the demands of the market. In this paper, strategies and concepts for strain selection or improvement methods were discussed, and many examples based upon selected studies involving acidity adjustment yeast strains were reviewed. Furthermore, the development of acidity adjustment yeast strains with minimized resource inputs, improved fermentation, and enological capabilities for an environmentally friendly production of healthy, top quality wine is presented. [ABSTRACT FROM AUTHOR]

The replacement of chemical synthesis by environmentally friendly energy-efficient technologies for production of valuable metabolites is a principal strategy of developing biotechnological industry all over the world. In the present study, we develop a method for α-ketoglutaric acid (KGA) production from rapeseed oil with the use of Yarrowia lipolytica yeast. Sixty strains of Y. lipolytica yeasts were tested for their ability to produce KGA, and the strain Y. lipolytica 212 ( Y. lipolytica VKM Y-2412) was selected as a promising KGA producer. Using a three-stage pH controlling, in which pH was 4.5 in the growth phase, then since 72 to 144 h, pH was maintained at 3.5 and in the later phase of acid production, the titration by KOH was switch off, selected strain produced 106.5 g l of KGA with mass yield of 0.95 g g. KGA in the form of monopotassium salt was isolated from the culture broth and purified. The isolation procedure involved separation of biomass, extraction of residual triglycerides, filtrate bleaching, and acidification with mineral acid (to pH 2.8-3.4), concentration, precipitation of mineral salts, and crystallization of the product. The purity of KGA isolated from the culture filtrate reached 99.1 %. [ABSTRACT FROM AUTHOR]

Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica JCM10317 (PaE) was analyzed by surface plasmon resonance (SPR). The adsorption of PaE and the degradation rate for polyester films were quantitatively monitored by a positive and negative SPR signal shifts, respectively. The decrease in SPR signal and the erosion depth of amorphous poly( l-lactide) (a-PLLA) film measured by atomic force microscopy (AFM) had a linear relationship, and the weight loss was estimated from the AFM data combined with a density of a-PLLA film. Furthermore, SPR sensorgrams for various polyester films showed that degradation rate of poly(ε-caprolactone) and poly(butylene succinate- co-adipate) which contain C6 units was higher than that of other polyesters such as poly(butylene succinate) and a-PLLA. These results suggest that C6 is the preferred chain length as substrates for PaE. [ABSTRACT FROM AUTHOR]

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins. [ABSTRACT FROM AUTHOR]

The introduction of multilocus sequence typing (MLST) for strain characterization provided the first sequence-based approach for genotyping many fungi, leading to reproducible, reliable, and exchangeable data. A MLST scheme based on the analysis of six housekeeping genes was developed for genotyping Geotrichum candidum. The scheme was first developed using 18 isolates for which the complete sequences of the alanyl-tRNA synthetase ( ALA1), pyruvate kinase ( CDC19), acetyl-coA acetyltransferase ( ERG10), glutaminyl-tRNA synthase ( GLN4), phosphoglucoisomerase ( PGI1), and phosphoglucomutase ( PGM2) housekeeping genes were determined. Multiple sequence alignments of these genes were used to define a set of loci showing, as closely as possible, the same phylogenetic resolution level as complete gene sequences. This scheme was subsequently validated with 22 additional isolates from dairy and non-dairy sources. Overall, 58 polymorphic sites were indexed among 3,009 nucleotides analyzed. Depending on the loci, four to eight alleles were detected, generating 17 different sequence types, of which ten were represented by a single strain. MLST analysis suggested a predominantly clonal population for the 40 G. candidum isolates. Phylogenetic analysis of the concatenated sequences revealed a distantly related group of four isolates. Interestingly, this group diverged with respect to internal transcribed spacers 1 (ITS1), 5.8S, and ITS2 analysis. The reproducibility of the MLST approach was compared to random amplification of microsatellites by PCR (RAM-PCR), a gel profiling method previously proposed for G. candidum strain typing. Our results found MLST differentiation to be more efficient than RAM-PCR, and MLST also offered a non-ambiguous, unique language, permitting data exchange and evolutionary inference. [ABSTRACT FROM AUTHOR]

Cysteine-conjugated volatile thiols are powerful aromatic compounds that contribute to the fruity notes of many white wines and especially Sauvignon Blanc. Genetic selection programs of wine yeast starters able to produce more volatile thiols constitute, therefore, an important goal for the wine industry. Recent investigations on yeast metabolism suggested that the ß-lyase Irc7p and the control of its gene expression by nitrogen catabolite repression constitute a rational way for yeast genetic improvement. This work demonstrates that the use of a natural ure2 mutation can be used to design wine starters with an enhanced capacity of volatile thiols production. By applying backcrosses driven by molecular markers, this allelic form was introduced in different starter backgrounds. Our investigations demonstrate that the ure2 inheritance is able to enhance the production of 4MMP (recently renamed 4MSP) and 3MH (recently renamed 3SH). For 4MMP, this effect depends of the presence of the allele IRC7 encoding a long form of the Irc7 protein. Moreover, a correlation in between the expression level of this allelic form and 4MMP production was found within industrial starters. All together, these results emphasised the use of molecular breeding for improving quantitative traits of industrial strains without the use of genetically modifying strategies. [ABSTRACT FROM AUTHOR]

The possibility of using rapeseed oil as a carbon source for microbiological production of α-ketoglutaric acid (KGA) has been studied. Acid formation on the selective media has been tested in 26 strains of Yarrowia lipolytica yeast, and the strain Y. lipolytica VKM Y-2412 was selected as a prospective producer of KGA from rapeseed oil. KGA production by the selected strain was studied in dependence on thiamine concentration, medium pH, temperature, aeration, and concentration of oil. Under optimal conditions (thiamine concentration of 0.063 μg g cells, pH 3.5, 30 °C, high dissolved oxygen concentration (pO) of 50 % (of air saturation), and oil concentration in a range from 20 to 60 g l), Y. lipolytica VKM Y-2412 produced up to 102.5 g l of KGA with the mass yield coefficient of 0.95 g g and the volumetric KGA productivity ( Q) of 0.8 g l h. [ABSTRACT FROM AUTHOR]

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form. [ABSTRACT FROM AUTHOR]

The methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. revealed enhanced biodegradation capability of exogenously applied formaldehyde (Fd) upon biostimulation achieved by the presence of methanol, as compared to glucose. Upon growth on either of the above substrates, the strains proved to produce the activity of glutathione-dependent formaldehyde dehydrogenase-the enzyme known to control the biooxidative step of Fd detoxification. However, in the absence of methanol, the yeasts' tolerance to Fd was decreased, and the elevated sensitivity was especially pronounced for Trichosporon sp. Both strains responded to the methanol and/or Fd treatment by increasing their unsaturation index (UI) at xenobiotic levels below minimal inhibitory concentrations. This indicated that the UI changes effected from the de novo synthesis of (poly) unsaturated fatty acids carried out by viable cells. It is concluded that the yeast cell response to Fd intoxication involves stress reaction at the level of membranes. Fluidization of the lipid bilayer as promoted by methanol is suggested as a significant adaptive mechanism increasing the overall fitness enabling to cope with the formaldehyde xenobiotic via biodegradative pathway of C-compound metabolism. [ABSTRACT FROM AUTHOR]

Recombinant strains of the oleaginous yeast Yarrowia lipolytica expressing the PHA synthase gene ( PhaC) from Pseudomonas aeruginosa in the peroxisome were found able to produce polyhydroxyalkanoates (PHA). PHA production yield, but not the monomer composition, was dependent on POX genotype ( POX genes encoding acyl-CoA oxidases) (Haddouche et al. FEMS Yeast Res 10:917-927, ). In this study of variants of the Y. lipolytica β-oxidation multifunctional enzyme, with deletions or inactivations of the R-3-hydroxyacyl-CoA dehydrogenase domain, we were able to produce hetero-polymers (functional MFE enzyme) or homo-polymers (with no 3-hydroxyacyl-CoA dehydrogenase activity) of PHA consisting principally of 3-hydroxyacid monomers (>80%) of the same length as the external fatty acid used for growth. The redirection of fatty acid flux towards β-oxidation, by deletion of the neutral lipid synthesis pathway (mutant strain Q4 devoid of the acyltransferases encoded by the LRO1, DGA1, DGA2 and ARE1 genes), in combination with variant expressing only the enoyl-CoA hydratase 2 domain, led to a significant increase in PHA levels, to 7.3% of cell dry weight. Finally, the presence of shorter monomers (up to 20% of the monomers) in a mutant strain lacking the peroxisomal 3-hydroxyacyl-CoA dehydrogenase domain provided evidence for the occurrence of partial mitochondrial β-oxidation in Y. lipolytica. [ABSTRACT FROM AUTHOR]

High energy prices, depletion of crude oil supplies, and price imbalance created by the increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives such as lubricants, adhesives, and plastics have given rise to heated debates on land-use practices and to environmental concerns about oil production strategies. However, commercialization of microbial oils with similar composition and energy value to plant and animal oils could have many advantages, such as being non-competitive with food, having shorter process cycle and being independent of season and climate factors. This review focuses on the ongoing research on different oleaginous yeasts producing high added value lipids and on the prospects of such microbial oils to be used in different biotechnological processes and applications. It covers the basic biochemical mechanisms of lipid synthesis and accumulation in these organisms, along with the latest insights on the metabolic processes involved. The key elements of lipid accumulation, the mechanisms suspected to confer the oleaginous character of the cell, and the potential metabolic routes enhancing lipid production are also extensively discussed. [ABSTRACT FROM AUTHOR]

Recombinant yeast strains highly tolerant to formic acid during xylose fermentation were constructed. Microarray analysis of xylose-fermenting Saccharomyces cerevisiae strain overexpressing endogenous xylulokinase in addition to xylose reductase and xylitol dehydrogenase from Pichia stipitis revealed that upregulation of formate dehydrogenase genes ( FDH1 and FDH2) was one of the most prominent transcriptional events against excess formic acid. The quantification of formic acid in medium indicated that the innate activity of FDH was too weak to detoxify formic acid. To reinforce the capability for formic acid breakdown, the FDH1 gene was additionally overexpressed in the xylose-metabolizing recombinant yeast. This modification allowed the yeast to rapidly decompose excess formic acid. The yield and final ethanol concentration in the presence of 20 mM formic acid is as essentially same as that of control. The fermentation profile also indicated that the production of xylitol and glycerol, major by-products in xylose fermentation, was not affected by the upregulation of FDH activity. [ABSTRACT FROM AUTHOR]

Biological synthesis of therapeutic drugs beneficial for human health using microbes offers an alternative production strategy to the methods that are commonly employed such as direct extraction from source organisms or chemical synthesis. In this study, we evaluated the potential for yeast ( Saccharomyces cerevisiae) to be used as a catalyst for the synthesis of tranilast and various tranilast analogs (cinnamoyl anthranilates). Several studies have demonstrated that these phenolic amides have antioxidant properties and potential therapeutic benefits including antiinflammatory, antiproliferative, and antigenotoxic effects. The few cinnamoyl anthranilates naturally produced in plants such as oats and carnations result from the coupling of various hydroxycinnamoyl-CoAs to anthranilic acid. In order to achieve the microbial production of tranilast and several of its analogs, we engineered a yeast strain to co-express a 4-coumarate/CoA ligase (4CL, EC 6.2.1.12) from Arabidopsis thaliana and a hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, EC 2.3.1.144) from Dianthus caryophyllus. This modified yeast strain allowed us to produce tranilast and 26 different cinnamoyl anthranilate molecules within a few hours after exogenous supply of various combinations of cinnamic acids and anthranilate derivatives. Our data demonstrate the feasibility of rapidly producing a wide range of defined cinnamoyl anthranilates in yeast and underline a potential for the biological designed synthesis of naturally and non-naturally occurring molecules. [ABSTRACT FROM AUTHOR]

The yeast Cryptococcus humicola has several attractive properties for practical applications such as in bioremediation and as a source of industrially useful enzymes and compounds. We have developed an autonomously replicating vector of C. humicola to improve its properties. We initially tried to isolate an autonomously replicating sequence (ARS) from genomic DNA by transformation using a genomic DNA library. We obtained a candidate plasmid vector harboring an ARS that gave high transformation efficiency. Southern blot analysis of transformants revealed the autonomous replication of the introduced vector in some transformants. However, the vector was not only variously altered in length but also linearized. PCR analysis indicated that a telomere-like sequence repeat (TTAGGGGG) was added to the termini of linearized vector. Thus, we constructed an autonomously replicating linear vector having ten repeats of the telomere-like sequence at both ends. The vector transformed the yeast cells with high transformation efficiency (3230 CFU/μg of DNA), which was approximately 25-fold higher than that of a control vector lacking the repeats, and was autonomously replicated at a roughly constant size. The copy number was estimated to be less than one copy, and Ura mitotic stability varied widely among the transformants and was related to plasmid segregation efficiency. [ABSTRACT FROM AUTHOR]

There are remarkably few reports on d-arabitol production from lactose. Previous studies in our laboratory have shown that the osmophilic yeast Kluyveromyces lactis NBRC 1903 convert lactose to extracellular d-arabitol. The present study was undertaken to determine the participation of osmotic stress caused by lactose on d-arabitol production by K. lactis NBRC 1903 and to provide the information on the kinetics of d-arabitol production from lactose by K. lactis NBRC 1903. It was confirmed that d-arabitol production was triggered when an initial lactose concentration was above 278 mmol L−1. d-Arabitol yield increased with an increase in initial lactose concentration. The highest d-arabitol concentration of 79.5 mmol L−1 was achieved in the cultivation of K. lactis NBRC 1903 in a medium containing 555 mmol L−1 lactose and 40 g L−1 yeast extract. Lactose was found to play two important roles in d-arabitol production by K. lactis NBRC 1903 grown on lactose. First, lactose was assimilated as the substrate both for cell growth and d-arabitol production. Second, a high lactose concentration induced cellular response to high osmotic stress and up-regulated the flow from d-glucose-6-phosphate to d-arabitol. The arrest of cell growth triggered d-arabitol production. [ABSTRACT FROM AUTHOR]

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining δ-integration and polyploidization through cell fusion. Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase/α-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The α-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts. [ABSTRACT FROM AUTHOR]

A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes. [ABSTRACT FROM AUTHOR]

Strains belonging to the yeast species Kluyveromyces marxianus have been isolated from a great variety of habitats, which results in a high metabolic diversity and a substantial degree of intraspecific polymorphism. As a consequence, several different biotechnological applications have been investigated with this yeast: production of enzymes (β-galactosidase, β-glucosidase, inulinase, and polygalacturonases, among others), of single-cell protein, of aroma compounds, and of ethanol (including high-temperature and simultaneous saccharification-fermentation processes); reduction of lactose content in food products; production of bioingredients from cheese-whey; bioremediation; as an anticholesterolemic agent; and as a host for heterologous protein production. Compared to its congener and model organism, Kluyveromyces lactis, the accumulated knowledge on K. marxianus is much smaller and spread over a number of different strains. Although there is no publicly available genome sequence for this species, 20% of the CBS 712 strain genome was randomly sequenced (Llorente et al. in FEBS Lett 487:71–75, 2000). In spite of these facts, K. marxianus can envisage a great biotechnological future because of some of its qualities, such as a broad substrate spectrum, thermotolerance, high growth rates, and less tendency to ferment when exposed to sugar excess, when compared to K. lactis. To increase our knowledge on the biology of this species and to enable the potential applications to be converted into industrial practice, a more systematic approach, including the careful choice of (a) reference strain(s) by the scientific community, would certainly be of great value. [ABSTRACT FROM AUTHOR]

The Aspergillus aculeatus β-glucosidase 1 ( bgl1) gene was expressed in a lactic-acid-producing Saccharomyces cerevisiae strain to enable lactic fermentation with cellobiose. The recombinant β-glucosidase enzyme was expressed on the yeast cell surface by fusing the mature protein to the C-terminal half region of the α-agglutinin. The β-glucosidase expression plasmids were integrated into the genome. Three strong promoters of S. cerevisiae, the TDH3, PGK1, and PDC1 promoters, were used for β-glucosidase expression. The specific β-glucosidase activity varied with the promoter used and the copy number of the bgl1 gene. The highest activity was obtained with strain PB2 that possessed two copies of the bgl1 gene driven by the PDC1 promoter. PB2 could grow on cellobiose and glucose minimal medium at the same rate. Fermentation experiments were conducted in non-selective-rich media containing 95 g l−1 cellobiose or 100 g l−1 glucose as a carbon source under microaerobic conditions. The maximum rate of l-lactate production by PB2 on cellobiose (2.8 g l−1 h−1) was similar to that on glucose (3.0 g l−1 h−1). This indicates that efficient fermentation of cellobiose to l-lactate can be accomplished using a yeast strain expressing β-glucosidase from a mitotically stable genomic integration plasmid. [ABSTRACT FROM AUTHOR]