Efficiency of genetically engineered yeast in the production of ethanol from dextrinized cassava starch

Summary The ability of a polyploid/aneuploidSaccharomyces diastaticus spheroplast fusion product and a diploidSaccharomyces diastaticus hybridization product, to produce ethanol from dextrinized cassava starch with varying amounts of supplemented glucoamylase (amyloglucosidase), was investigated. It was found that the added glucoamylase could be reduced by over 50% using these glucoamylase producing strains as compared to a commercially availableSaccharomyces cerevisiae strain commonly used in ethanol producing industries.     

Alcoholic fermentation of starch containing media using yeast protoplast fusion products

Summary Fermentation of starch based industrial media was tested with yeast fusion products previously described, from a Baker's yeastSaccharomyces cerevisiae and Saccharomyces diastaticus and from a highly flocculentSaccharomyces cerevisiae andSaccharomyces diastaticus. The (somatic) fusion products were capable to produce more ethanol than parental strains after 96 h of batch fermentation. The aim of this work was to reduce the amount of enzyme used in saccharification by using good fermenting amylolytic yeast strains.     

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.     

Interspecific protoplast fusion of the Baker's yeast Saccharomyces cerevisiae and Saccharomyces diastaticus

Summary The protoplast fusion technique provides a useful method for improving industrial yeasts and agglutinant agents like polyethylene glycol (PEG) MW 4000 and Ca⁺⁺ ions are widely used to stimulate the fusion process. Commercial Baker's yeast Saccharomyces cerevisiae and Saccharomyces diastaticus were selected as parental strains for somatic fusion. The Saccharomyces diastaticus carried a spontaneous petite mutation and could not metabolize starch unlike respiratory competent Saccharomyces diastaticus from which it was derived, that readily could.A medium containing soluble starch as a carbon source and 3 % agar was used as fusion products selection medium. Respiratory competent fusion products were capable of using dextrins and starch as carbon sources.     

Protoplast fusion between a petite strain ofCandida utilis andSaccharomyces cerevisiae respiratory-competent cells

Prototrophic RD mutant cells ofCandida utilis NRRL-Y-1084 and auxotrophic mutant respiratory-competent cells ofSaccharomyces cerevisiae 4003-5Ba his ₄ leu ₂ can ^S meth ₂ trp ₅ ade ₁ ura ₃ gal were turned into protoplasts to be further fused with the aid of polyethylene glycol (PEG) and Ca²⁺ ions. Minimal medium containing glycerol as the carbon source was employed for fusion product selection. The respiratory-competent fusion products, mainly oval cells, resembledCandida utilis and had the fermentative abilities of this strain (dextrose, sucrose, raffinose). Five fusion products were analyzed as to their ability to metabolize dextrose, xylose, cellobiose, trehalose, glycerol, succinic acid, citric acid, salycin, and maltose. Fusion products partially restored the respiratory-competentCandida utilis capacity to grow by use of these carbon compounds, and none of theSaccharomyces cerevisiae fermenting abilities were found. Our results would suggest either a partial recombination between parental mitochondria or some occurring phenomenon affecting the cell, membrane function after somatic fusion without concomitant nuclear fusion.     

Spheroplast fusion of a brewing yeast strain with Saccharomyces diastaticus

Summary One haploid and one diploid strain of Saccharomyces diastaticus carrying genes responsible for glucoamylase synthesis were fused with a brewing polyploid Saccharomyces uvarum lager strain. With the spheroplast fusion technique, the ability to use dextrin and starch was introduced in the brewing yeast. Spheroplasts of the strains to be used were obtained by enzymatic digestion of the cell walls. Fusion took place in polyethylene glycol; complete cells were then regenerated in hypertonic medium containing 3% agar at 37°C. In the first fusion experiment melibiose was used as carbon source; in the second fusion experiment glycerol was employed as carbon source, for the parental Saccharomyces diastaticus diploid strain was a petite mutant. Fusion products were capable of utilizing melibiose and dextrin as carbon sources.     

An unexpected response of Torulopsis glabrata fusion products to x-irradiation

Intra-species fusion products of Saccharomyces cerevisiae, Saccharomyces unisporus and Torulopsis glabrata have been isolated following polyethylene glycol-induced fusion of protoplasts and selection for prototrophic colonies. Staining with lomofungin showed that all fusion products were uninucleate. Measurement of DNA content mostly gave values between haploid and diploid levels indicating that the majority of fusion products were aneuploid. Nevertheless fusion products of S. cerevisiae and S. unisporus were, as expected, more resistant to X-irradiation than their haploid parents. By contrast, the X-ray doze—response curve of all T. glabrata fusion products was indistinguishable from their progenitors despite the fact that mitotic segregants could be recovered amongst the survivors to X-rays. A possible explanation for the behaviour towards X-rays of T. glabrata fusion products is that this species lacks a DNA repair pathway involving recombination between homologous chromosomes. We conclude from this study that the shape of the X-ray dose—response curve should not be taken to indicate the ploidy of new yeast isolates without supporting data.     

Glycerol and arabitol production by an intergeneric hybrid,PB2, obtained by protoplast fusion between Saccharomyces cerevisiae and Torulaspora delbrueckii

An intergeneric osmotolerant hybrid yeast, PB2, was used together with the parental strains to study glycerol and arabitol production in batch culture. This fusion product was previously obtained by protoplast fusion between Torulaspora delbrueckii and Saccharomyces cerevisiae. Polyols and biomass production were determined in batch culture under aerobic conditions. Under the conditions tested, using PB2 hybrid and both parental strains, the best results were obtained with the hybrid. Arabitol reached a final concentration of 70 g/l and glycerol was increased to up to 50 g/l. Electronic Publication     

Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion

Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency of cell fusion was severely reduced as ploidy increased.     

Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA

A functional strain Fhhh was constructed through protoplast fusion of three parental strains (Phanerochaete chrysosporium, Saccharomyces cerevisiae and native bacterium YZ) to improve the degradation efficiency of purified terephthalic acid wastewater. Randomly amplified polymorphic DNA (RAPD) and scanning electron microscope (SEM) analysis were applied to identify and confirm the fusant Fhhh through phenotypic and genetic relationship. The result of SEM analysis demonstrated that the cell shape of fusant Fhhh differed from all three parental strains. RAPD analysis of 40 arbitrary primers generated a total of 1,135 bands. The genetic similarity indices between Fhhh and parental strains Phanerochaete chrysosporium (PC), Saccharomyces cerevisiae (SC) and native bacterium (YZ) were 34.01%, 33.16%, and 35.97%, respectively. The targeted-gene PCR results showed that Fhhh inherited the DNA fragments of mnp and lip genes from parental strain PC and FLO1 gene fragment from parental strain SC. Our results suggested protoplast fusion technique may be considered as a promising technique in environmental pollution control.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.     

Construction of Lactose-Assimilating and High-Ethanol-Producing Yeasts by Protoplast Fusion

The availability of a yeast strain which is capable of fermenting lactose and at the same time is tolerant to high concentrations of ethanol would be useful for the production of ethanol from lactose. Kluyveromyces fragilis is capable of fermenting lactose, but it is not as tolerant as Saccharomyces cerevisiae to high concentrations of ethanol. In this study, we have used the protoplast fusion technique to construct hybrids between auxotrophic strains of S. cerevisiae having high ethanol tolerance and an auxotrophic strain of lactose-fermenting K. fragilis isolated by ethyl methanesulfonate mutagenesis. The fusants obtained were prototrophic and capable of assimilating lactose and producing ethanol in excess of 13% (vol/vol). The complementation frequency of fusion was about 0.7%. Formation of fusants was confirmed by the increased amount of chromosomal DNA per cell. Fusants contained 8 × 10⁻⁸ to 16 × 10⁻⁸ μg of DNA per cell as compared with about 4 × 10⁻⁸ μg of DNA per cell for the parental strains, suggesting that multiple fusions had taken place.     

Intergeneric protoplast fusion between Kluyveromyces and Saccharomyces cerevisiae – to produce sorbitol from Jerusalem artichokes

The construction of inulin-assimilating and sorbitol-producing fusants was achieved by intergeneric protoplast fusion between Kluyveromyces sp. Y-85 and Saccharomyces cerevisiae E-15. The cells of parental strains were pretreated with 0.1% EDTA (w/v) and 2-mercaptoethanol (0.1%, v/v) and then exposed to 2.0% (w/v) Zymolase at 30 °C for 30–40 min. The optimized fusion condition demonstrated that with the presence of 30% (w/v) polyethylene glycol 6000 (PEG-6000) and 10 mM CaCl₂ for 30 min, the fusion frequency reached 2.64 fusants/10⁶ parental cells. The fusants were screened by different characters between two parental strains and further identified by DNA contents, inulinase activity and sorbitol productivity. One of the genetically stable fusants, Strain F27, reached a maximal sorbitol production of 4.87 g/100 ml under optimal fermentation condition.     

Rapid identification and isolation of zygotes and the kinetics of mating in Saccharomyces cerevisiae

Summary Flow cytometry has been used in the selection of fusion products of the yeast Saccharomyces cerevisiae. Parental cells of opposite mating type were stained with different fluorescent dyes, permitting rapid identification of zygotes from natural matings based on dual-color flow cytometry. This procedure was then used to study the kinetics of mating in yeast and the physical and biological parameters that affect these kinetics, such as cell concentration, parental ratios, and parental strain growth rate.     

Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.     

Intergeneric yeast fusants with efficient ethanol production from cheese whey powder solution: Construction of a Kluyveromyces marxianus and Saccharomyces cerevisiae AY‐5 hybrid

Due to its high content of lactose and abundant availability, cheese whey powder (CWP) has received much attention for ethanol production in fermentation processes. However, lactose‐fermenting yeast strains including Kluyveromyces marxianus can only produce alcohol at a relatively low level, while the most commonly used distiller yeast strain Saccharomyces cerevisiae cannot ferment lactose since it lacks both β‐galactosidase and the lactose permease system. To combine the unique aspects of these two yeast strains, hybrids of K. marxianus TY‐22 and S. cerevisiae AY‐5 were constructed by protoplast fusion. The fusants were screened and characterized by DNA content, β‐galactosidase activity, ethanol tolerance, and ethanol productivity. Among the genetically stable fusants, the DNA content of strain R‐1 was 6.94%, close to the sum of the DNA contents of TY‐22 (3.99%) and AY‐5 (3.51%). The results obtained by random‐amplified polymorphic DNA analysis suggested that R‐1 was a fusant between AY‐5 and TY‐22. During the fermentation process with CWP, the hybrid strain R‐1 produced 3.8% v/v ethanol in 72 h, while the parental strain TY‐22 only produced 3.1% v/v ethanol in 84 h under the same conditions.     

Brewing up a storm: The genomes of lager yeasts and how they evolved

Yeasts used in the production of lager beers belong to the species Saccharomyces pastorianus, an interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus. The hybridisation event happened approximately 500–600 years ago and therefore S. pastorianus may be considered as a newly evolving species. The happenstance of the hybridisation event created a novel species, with unique genetic characteristics, ideal for the fermentation of sugars to produce flavoursome beer. Lager yeast strains retain the chromosomes of both parental species and also have sets of novel hybrid chromosomes that arose by recombination between the homeologous parental chromosomes. The lager yeasts are subdivided into two groups (I and II) based on the S. cerevisiae: S. eubayanus gene content and the types and numbers of hybrid chromosomes. Recently, whole genome sequences for several Group I and II lager yeasts and for many S. cerevisiae and S. eubayanus isolates have become available. Here we review the available genome data and discuss the likely origins of the parental species that gave rise to S. pastorianus. We review the compiled data on the composition of the lager yeast genomes and consider several evolutionary models to account for the emergence of the two distinct types of lager yeasts.     

Extracellular production of palmitoleic triglycerides by a yeast,Trichosporon

A yeast belonging to Trichosporon which produces triglycerides extracellularly was isolated. This strain accumulated palmitoleic triglycerides from ethyl palmitate used as a sole carbon source. To increase the level of extracellular palmitoleic triglycerides, mutant strains which supported growth of unsaturated-fatty-acid-auxotrophic cells (Saccharomyces cerevisiae KD115) layered on the mutant colonies were screened. The mutant strain excreted palmitoleic acid as triglyceride form at a significantly high level, corresponding to about double level of the parental strain.     

Inherent G418-resistance in hybridization of industrial yeasts

Eight strains of sake yeast exhibited inherent-resistance to 100 μg/ml of Geneticin (G418). Fourteen wine yeasts and 1 shochu yeast (Saccharomyces cerevisiae) and 1 miso yeast (Zygosaccharomyces rouxii) were inherent G418-sensitive. The petites converted from inherent G418-resistants by treatment with ethidium bromide retained G418-resistance (ϱ⁻ G418R), and thus were hybridized by electrofusion with the wine yeast W3 (ϱ⁺ G418S, wild type). A lag phase of 12–18 h was required prior to administration of the drug in glycerol medium when selecting G418-resistant hybridization products. Colonies were formed in the regeneration medium at a frequency of about 1 × 10⁻⁵ per used protoplasts. No growth of any parental strain (10⁶/_~10⁷ protoplasts) separately subjected to electrofusion and regeneration was observed. The hybridization products were G418-resistant “grande” strains (ϱ⁻ G418R) in which the genetic traits of parental strains had been complemented. Uninucleate cells (DAPI staining) of the hybridization products showed CHEF electrophoretic karyotypes similar to that of wine yeast, but possessed a single chromosome (approx. 320 kb) presumably from sake yeast.